DIAGNOSTIC IMMUNOHISTOCHEMISTRY THERANOSTIC AND GENOMIC APPLICATIONS
DIAGNOSTIC
IMMUNOHISTOCHEMISTRY THERANOSTIC AND GENOMIC APPLICATIONS
4TH EDITION DAVID J. DABBS, MD Professor and Chief of Pathology Department of Pathology University of Pittsburgh Pittsburgh, Pennsylvania
iii
1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899
DIAGNOSTIC IMMUNOHISTOCHEMISTRY: THERANOSTIC AND GENOMIC APPLICATIONS
ISBN: 978-1-4557-4461-9
Copyright © 2014, 2010, 2006, 2002 by Saunders, an imprint of Elsevier Inc. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).
Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data Diagnostic immunohistochemistry (2014) Diagnostic immunohistochemistry : theranostic and genomic applications / David J. Dabbs. — Fourth edition. p. ; cm. Includes bibliographical references and index. ISBN 978-1-4557-4461-9 (hardcover : alk. paper) I. Dabbs, David J., editor of compilation. II. Title. [DNLM: 1. Immunohistochemistry–methods. 2. Diagnostic Techniques and Procedures. 3. Neoplasms–diagnosis. QW 504.5] RB46.6 616.07′583—dc23 2013024051 Executive Content Strategist: William Schmitt Managing Editor: Kathryn DeFrancesco Publishing Services Manager: Patricia Tannian Project Manager: Carrie Stetz Design Direction: Ellen Zanolle Printed in the United States of America Last digit is the print number: 9 8 7 6 5 4 3 2 1
This book is dedicated to the patients we serve and to our colleagues in pathology and oncology.
CONTRIBUTORS
N. Volkan Adsay, MD Professor and Vice-Chair Department of Pathology and Laboratory Medicine Emory University Hospital Atlanta, Georgia Peter M. Banks, MD Clinical Professor of Pathology University of North Carolina–Chapel Hill Chapel Hill, North Carolina; Pathologist for Scientific Affairs Ventana-Roche Corporation Tucson, Arizona Olca Basturk, MD Assistant Attending Pathologist Department of Pathology Memorial Sloan Kettering Cancer Center New York, New York
Cheryl M. Coffin, MD Professor Department of Pathology, Microbiology, and Immunology Vanderbilt Children’s Hospital Nashville, Tennessee Hernan Correa, MD Associate Professor of Pediatric Pathology Chief, Division of Pediatric Pathology Vanderbilt Children’s Hospital Nashville, Tennessee David J. Dabbs, MD Professor and Chief of Pathology Department of Pathology University of Pittsburgh Pittsburgh, Pennsylvania
Parul Bhargava, MBBS Assistant Professor of Pathology Harvard Medical School Boston, Massachusetts
Sanja Dacic, MD, PhD Associate Professor of Pathology Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania
Rohit Bhargava, MBBS Associate Professor of Pathology University of Pittsburgh Director of Anatomic Pathology Magee Women’s Hospital of UPMC Pittsburgh, Pennsylvania
Ronald A. DeLellis, MD Professor Department of Pathology and Laboratory Medicine Warren Alpert Medical School of Brown University Rhode Island Hospital and The Miriam Hospital Providence, Rhode Island
Jennifer Black, MD Assistant Professor Department of Pathology, Microbiology, and Immunology Vanderbilt Children’s Hospital Nashville, Tennessee
Leona A. Doyle, MD Associate Pathologist Department of Pathology Brigham and Women’s Hospital Instructor of Pathology Harvard Medical School Boston, Massachusetts
Mariana Cajaiba, MD Assistant Professor Department of Pathology, Microbiology, and Immunology Vanderbilt Children’s Hospital Nashville, Tennessee
Jonathan I. Epstein, MD Professor of Pathology, Urology, and Oncology The Reinhard Professor of Urological Pathology Director of Surgical Pathology The Johns Hopkins Medical Institutions Baltimore, Maryland vii
viii
Contributors
Eduardo J. Eyzaguirre, MD Assistant Professor Department of Pathology Associate Director, Surgical Pathology Division Director, Immunohistochemistry Laboratory University of Texas Medical Branch Galveston, Texas Alton B. Farris III, MD Assistant Professor Department of Pathology Emory University Atlanta, Georgia Jeffrey D. Goldsmith, MD Director Surgical Pathology Laboratory and Gastrointestinal Pathology Fellowship Department of Pathology and Laboratory Medicine Beth Israel Deaconess Medical Center; Assistant Professor of Pathology Harvard Medical School; Consultant in Gastrointestinal Pathology Children’s Hospital Boston Boston, Massachusetts Kate E. Grimm, MD Clarient Inc./GE Healthcare Aliso Viejo, California; Clinical Assistant Professor of Pathology Keck School of Medicine University of Southern California Los Angeles, California Samuel P. Hammar, MD President Diagnostic Specialties Laboratory Inc. P.S. Bremerton, Washington Jason L. Hornick, MD, PhD Director of Surgical Pathology Director, Immunohistochemistry Laboratory Brigham and Women’s Hospital; Associate Professor of Pathology Harvard Medical School Boston, Massachusetts Marshall E. Kadin, MD Staff Physician Department of Dermatology & Skin Surgery Roger Williams Medical Center Providence, Rhode Island Alyssa M. Krasinskas, MD Director, Surgical Pathology Director, Gastrointestinal and Liver Pathology Department of Pathology and Laboratory Medicine Emory University Hospital Atlanta, Georgia
Teri A. Longacre, MD Professor of Pathology Department of Pathology Stanford University School of Medicine Stanford, California Pamela Lyle, MD Assistant Professor Department of Pathology, Microbiology, and Immunology Vanderbilt Children’s Hospital Nashville, Tennessee Paul E. McKeever, MD, PhD Professor of Pathology Department of Pathology University of Michigan Medical Center Ann Arbor, Michigan Sara E. Monaco, MD Associate Professor Program Director, UMPC Cytopathology Fellowship Program Director, FNA Biopsy Service at Children’s Hospital of Pittsburgh of UPMC Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania Cesar A. Moran, MD Professor of Pathology University of Texas MD Anderson Cancer Center Houston, Texas George J. Netto, MD Associate Professor Department of Pathology, Urology, and Oncology Director of Surgical Pathology Johns Hopkins University Baltimore, Maryland Yuri E. Nikiforov, MD, PhD Professor Director, Division of Molecular Anatomic Pathology UPMC Presbyterian Hospital Pittsburgh, Pennsylvania Marina N. Nikiforova, MD Assistant Professor Associate Director, Molecular Anatomic Pathology Laboratory Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania Dennis P. O’Malley, MD Hematopathologist Clarient Inc./GE Healthcare Aliso Viejo, California; Adjunct Associate Professor Department of Hematopathology University of Texas MD Anderson Cancer Center Houston, Texas
Contributors
Liron Pantanowitz, MD Associate Professor of Pathology and Biomedical Informatics Department of Pathology University of Pittsburgh Pittsburgh, Pennsylvania Victor G. Prieto, MD, PhD Professor Department of Pathology University of Texas MD Anderson Cancer Center Houston, Texas Joseph T. Rabban, MD, MPH Associate Professor Department of Pathology Yale University School of Medicine New Haven, Connecticut David L. Rimm, MD, PhD Professor Department of Pathology Yale University School of Medicine New Haven, Connecticut Shan-Rong Shi, MD Professor of Pathology Keck School of Medicine of the University of Southern California Los Angeles, California Sandra J. Shin, MD Associate Professor of Pathology and Laboratory Medicine Chief of Breast Pathology Weill Cornell Medical College; Associate Attending Pathologist New York–Presbyterian Hospital New York, New York
Clive R. Taylor, MD, DPhil Professor of Pathology Keck School of Medicine of the University of Southern California Los Angeles, California Lester D.R. Thompson, MD Consultant in Pathology Department of Pathology Southern California Permanente Medical Group Woodland Hills, California Diana O. Treaba, MD Assistant Professor Department of Pathology and Laboratory Medicine Warren Alpert Medical School of Brown University; Director Hematopathology Laboratories Rhode Island Hospital and The Miriam Hospital Providence, Rhode Island David H. Walker, MD Professor and Chairman Department of Pathology Executive Director Center for Biodefense and Emerging Infectious Diseases University of Texas Medical Branch Galveston, Texas Annikka Weissferdt, MD Asistant Professor of Pathology University of Texas MD Anderson Cancer Center Houston, Texas Sherif R. Zaki, MD Chief, Infectious Disease Pathology Branch Centers for Disease Control and Prevention Atlanta, Georgia
ix
FOREWORD
Many are the “special” techniques that pathologists have used over the years to confirm, complement, and refine the information they were able to obtain with their “old faithful” armamentarium; that is, formalin fixation, paraffin embedding, and hematoxylin-eosin staining. Most of these techniques have come and gone, their usual life cycle beginning with an initial period of unrestrained enthusiasm on the part of the pathologist, turning to a phase of disappointment, and finally leading to a more sober and realistic assessment. Many of these methods have left a permanent mark on the practice of the profession, even if often not as deep or wide ranging as initially hoped. These techniques include special stains, tissue culture, electron microscopy, immunohistochemistry, and molecular/genetic methods. Much was expected of the first three, and infinitely more is anticipated of the last, but it is fair to say that as of today no special technique has influenced the way that pathology is practiced as profoundly as has immunohistochemistry, or has come even close to it. I don’t think it would be an exaggeration to speak of a revolution, particularly in the field of tumor pathology. Those of us who have lived through it certainly feel that way. The newer generations of pathologists who order so glibly an HMB-45 or a CD31 stain to identify melanocytes and endothelial cells, respectively, have very little feeling of the efforts one had to make to achieve that identification in the past. The virtues of the technique are so apparent and numerous as to make it as close to ideal as any biologic method carried out in human tissue obtained under routine (i.e., less than ideal) conditions can be; it is compatible with standard fixation and embedding procedures, it can be performed retrospectively in material that has been archived for years, it is remarkably sensitive and specific, it can be applied to virtually any immunogenic molecule, and it can be evaluated against the morphologic backgrounds with which pathologists have long been familiar. As with many other breakthroughs in medicine, immunohistochemistry started with a brilliant yet disarmingly simple idea: to select antibodies that bind the specific antigens being sought, and to make those antibodies visible by hooking them to a fluorescent compound. All subsequent modifications, such as the use of nonfluorescent chromogens, amplification of the reaction, and unmasking of antigens, merely represented
technical improvements, although certainly not ones to be minimized. Because of these technical advances the procedure spread beyond the research laboratories and is now applied in pathology laboratories throughout the world. Alas, it has its drawbacks. Antigens once believed to be specific for a given cell type have later been found to be expressed by other tissues; cross-reactions may occur between unrelated antigens; nonspecific absorption of the antibody may take place; entrapped nonneoplastic cells reacting for a particular marker may be misinterpreted as part of the tumor; and, most treacherously, antigen may diffuse out of a normal cell and find its way inside an adjacent tumor cell. Any of these pitfalls may lead to a misinterpretation of the reaction and a misdiagnosis; worse, it may lead to a final mistaken diagnosis after an initially correct interpretation of the hematoxylin-stained slides. A good protection against this danger is a thorough knowledge of these pitfalls and how to avoid them. An even more important safeguard is a solid background in basic anatomic pathology that will allow the pathologist to question the validity of any unexpected immunohistochemical result, whether positive or negative. There is nothing more dangerous (or expensive) than a neophyte in pathology making diagnoses on the basis of immunohistochemical “profiles” in disregard of the cytoarchitectural features of the lesions. Alas, this is true of any other “special” technique applied for diagnostic purposes to human tissue, molecular biology being the latest and most blatant example. However, when applied selectively and judiciously, immunohistochemistry is a notably powerful tool, in addition to being refreshingly cost effective. As a matter of fact, pathologists can no longer afford to do without it, one of the reasons being that failure to make a diagnosis because of the omission of a key immunohistochemical reaction may be regarded as grounds for a malpractice action. Any listing of the virtues of immunohistochemistry would be incomplete if it did not include the visual pleasure derived from examination of this material. I am only half-kidding when making this remark. There is undoubtedly an aesthetic component to the practice of histology, as masters of the technique such as Pio del Rio Hortega and Pierre Masson used to say. It is sad that these superb artists of morphology left the scene without having had the opportunity to marvel at the beauty of xi
xii
Foreword
a well-done immunohistochemical preparation. As their more fortunate heirs, let’s enjoy the application of the immunohistochemical technique written by a superb group of contributors. Diagnostic Immunohistochemistry: Theranostic and Genomic Applications is a book that summarizes in a lucid and thorough fashion the current knowledge in the field in terms of both the technical aspects and the practical applications. The first edition of this book, published in 2002, rapidly became one of the standard works in the field. The second edition featured a more standardized format, a wider coverage of organ systems, and an extensive update of markers. It also incorporated a large number of useful tables listing the various antibody groups, an algorithmic approach to the differential diagnosis, and Key Diagnostic Points of all the major subjects. The third edition expanded considerably on these aspects and kept it remarkably up to date in this rapidly evolving field. This fourth edition has followed this tradition, thus keeping this book at the forefront of the specialty literature. This new edition has several important features. (1) Several excellent contributors have been recruited to write the more specialized chapters. (2) Inclusion of markers with an increasing degree of specificity has been greatly expanded. Among these, transcription factors stand out because of their remarkable degree of specificity and their tendency to remain
attached to the sought antigen with little or no tendency for diffusion. (3) Coverage of the increasing use of immunohistochemical and molecular/genetic markers for prognostic and therapeutic (predictive) purposes has been expanded. These are remarkably useful properties, but there is no question that the latter is the potentially most useful and exciting development from a medical standpoint. As an example, it is much more useful to determine whether a breast carcinoma is immunoreactive to estrogen and progesterone receptors (and therefore potentially sensitive to hormonal manipulation) than whether it is of ductal or lobular type. Similarly, knowledge of the presence and type of mutation pattern in a gastrointestinal stromal tumor by molecular probing will be medically more significant than whether it is differentiating in a neural or smooth muscle direction, in both, or in neither. In summary, the authors have once more brilliantly succeeded in producing an authoritative, comprehensive, and updated book that pathologists will find next to indispensable as a theoretical backbone for the method and as a practical aid for their daily diagnostic work. Juan Rosai, MD Milan, Italy
PREFACE
The fourth edition of this book keeps the title change from the third edition, Diagnostic Immunohistochemistry, Theranostic and Genomic Applications. The use of immunohistochemistry for accurate diagnosis remains the focal point of this work, and it is this diagnostic accuracy that we must seek to satisfy the patients and clinicians need for “personalized” therapies. Even more so today, personalized medicine is the buzzword that clinicians, patients, and the media have gravitated toward, expecting individualized therapy based on specific molecular pathways that are spawned by gene alterations in the disease process. Theranostics is a rapidly emerging field in oncology. Pathologists need to be prepared to serve oncologists and their patients, not only with accurate tissue diagnosis, but also with appropriate tissue theranostic testing that will drive therapies. Once again, this work begins with detailed information of techniques and standardization in immunohistochemistry, followed by an appropriate primer of molecular anatomic pathology for the surgical pathologist. The remaining chapters address specific issues in surgical pathology with an organ system approach. Once again, with few exceptions, each chapter is designed to be a standalone work, which means that there is some redundancy among chapters. This approach
is intended to relieve the reader of extensive crosschecking to other chapters. It is extremely exciting that there are eight new authors who address new issues in this edition. In addition, there is a new chapter that addresses imaging related to immunohistochemistry, which is a critical issue affecting this discipline and the demands on pathologists to quantitate tissue markers for theranostics. Lastly, the chapter on prostate, bladder, kidney, and testis has been divided into two separate chapters. Once again, the challenge of putting this work together has been to ensure that the base of knowledge in each chapter is relevant and robust long after the ink has dried. The contributions of expert authors in each discipline are unique works. This book is meant to serve as a diagnostic and theranostic reference for pathologists, oncologists, and those in training as well as researchers who require a basis of knowledge in tissue pathology. The positive feedback that I received on this work continues to grow, and I personally welcome any feedback regarding this book, no matter how small. Please feel free to email me at
[email protected] or
[email protected]. My special thanks go to the dedicated investigators and pathologists across the globe who have given me feedback on this work.
xiii
HOW
TO
USE THIS BOOK
The first chapter of this book details the techniques and development of immunohistochemistry and includes new information regarding standardization in immunohistochemistry. The second chapter focuses on diagnostic molecular anatomic pathology as used by the practicing surgical pathologist. Molecular anatomic pathology has grown exponentially over the past decade, and the discipline supplies critically important testing that supplements diagnostic, theranostic, and genomic applications. With instruments such as the Ion Torrent, tissue specimens can be examined for specific gene mutations that may affect patients and their drug-able targets. It is the goal of immunohistochemistry to closely correlate those mutational findings with protein expression in tissue. The same terminology is used as in previous editions and includes the use of immunohistograms, Key Diagnostic Points boxes, and diagnostic pitfalls, where appropriate.
To further the concept of theranostics and genomics, where particularly relevant, chapters provide information related to drug-able targets or potential drug-able targets. These are extraordinarily exciting times for the discipline of surgical pathology, and immunohistochemistry in particular. With molecular anatomic and mutational testing for patients’ tumors, we continue to receive input and correlation with molecular anatomic and protein expression by immunohistochemistry. Currently, one of the most exciting aspects of this discipline is the promise of imaging to help us semiquantitate or quantitate, with standardization, protein content that will drive patient therapies. Once again, this work should be viewed as a focal point, a punctuation mark in the continuous quality improvement of the knowledge base for immunohistochemistry for surgical pathologists.
xv
C H A P T E R 1
TECHNIQUES OF IMMUNOHISTOCHEMISTRY: PRINCIPLES, PITFALLS, AND STANDARDIZATION CLIVE R. TAYLOR, SHAN-RONG SHI
Overview 1 Basic Principles of Immunohistochemistry 2 Antibodies as Specific Staining Reagents 3 Blocking Nonspecific Background Staining 4 Detection Systems 5 Quality Control and Standardization 14 Tissue Fixation, Processing, and Antigen Retrieval Techniques 19 Techniques, Protocols, and Troubleshooting 22 Conclusion 38
Overview Immunohistochemistry (IHC), or immunocytochemistry, is a method for localizing specific antigens in tissues or cells based on antigen-antibody recognition; it seeks to exploit the specificity provided by the binding of an antibody with its antigen at a light-microscopy level. IHC has a long history that dates back more than 70 years, when Coons1 first developed an immunofluorescence technique to detect corresponding antigens in frozen tissue sections. However, only since the early 1990s has the method found general application in surgical pathology.2-5 A series of technical developments led eventually to the wide range of IHC applications in use today. The enzymatic label known as horseradish peroxidase (HRP), developed by Avrameas6 and by Nakane and colleagues,7 allowed visualization of the labeled antibody by light microscopy in the presence of a suitable colorogenic substrate system. At Oxford,
Taylor and Burns5 developed the first successful demonstration of antigens in routinely processed formalinfixed paraffin-embedded (FFPE) tissues. A critical issue in the early development of immunoperoxidase techniques was related to the need to achieve greater sensitivity, because greater sensitivity would facilitate staining of FFPE tissues. Methods evolved from a simple, onestep, direct-conjugate method to multistep detection techniques such as the peroxidase-antiperoxidase (PAP), avidin-biotin conjugate (ABC), and biotin-streptavidin (B-SA) methods. This evolution eventually led to amplification methods, such as tyramide, and highly sensitive polymer-based labeling systems.4,8-20 We will describe these methods in detail later in this chapter. As the IHC method has evolved, its role in diagnostic pathology has expanded such that the use of one or more IHC “stains” is routine in surgical pathology, especially with respect to tumor diagnosis and classification. Furthermore, IHC has been adapted to the identification and demonstration of both prognostic and predictive markers, with corresponding requirements for semiquantitative reporting of results. The widespread use of IHC and the demands for comparison of qualitative and semiquantitative findings among an increasing number of laboratories have resulted in a growing focus on reproducibility and a new emphasis on standardization. Standardization serves as an underlying theme of this chapter and is discussed in detail in the “Quality Control and Standardization” section. Another dramatic change in the last several years is the increasing adoption of automated staining instruments, with major implications with respect to choice of reagents, protocols, and controls. As a result, parts of this chapter have been revised to accommodate the majority of laboratories that perform all, or most, of their IHC work on automated platforms. Sufficient basic methodology has been retained to allow for intelligent and effective use of manual methods where necessary. The development of the technique known as hybridoma21 facilitated the development of IHC and the manufacture of abundant, highly specific monoclonal antibodies, many of which found early application in 1
2
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
staining of tissues.22 The critical significance of rendering the IHC technique suitable for routine paraffin sections was illustrated in 1974 by Taylor and Burns, who showed that it was possible to demonstrate at least some antigens in routinely processed tissue.5,23,24 These initial studies led to serious attempts by pathologists to improve the ability to perform IHC staining on FFPE sections.5,23-28 Although great effort has been expended in the search for alternative fixatives (formalin substitutes) to preserve antigenicity without compromising preservation of morphologic features, no ideal fixative has been found to date. Indeed, Larsson29 states “An ideal immunocytochemical fixative applicable to all antigens may never be found.” As for the near future, we agree. In addition, any new fixative must show preservation of morphologic features comparable with FFPE to avoid problems in interpretation and diagnosis. Enzyme digestion was introduced by Huang30 as a pretreatment to IHC staining to “unmask” some antigens that had been altered by formalin fixation. However, the enzyme digestion method, although widely applied, did not improve IHC staining of many antigens, a subject well reviewed by Leong and colleagues.31 One drawback of enzyme digestion is that it is difficult to control, as shown in external proficiency studies, which will be discussed later with reference to antigen retrieval (AR). These difficulties in standardization provided a powerful incentive for the development of a more powerful, more widely applicable method to enhance IHC staining of routine FFPE tissue sections. The AR technique—also known as heat-induced epitope retrieval (HIER), based on a series of biochemical studies by Fraenkel-Conrat and coworkers32-34—was developed by Shi and associates in 1991.35-40 The AR technique is a simple method that involves heating routinely processed paraffin sections at high temperature (e.g., in a microwave oven) before IHC staining procedures. An alternative method that does not use heating was developed for celloidin-embedded tissues.36-38 The intensity of IHC staining for many antigens was increased dramatically after AR pretreatment.39-43 Subsequently, various modifications of the AR technique have been described that serve to validate the feasibility of AR-IHC and to expand its use in molecular morphology.39,40,43-54 At the same time, some basic questions and practical issues were raised with respect to standardization2,3,33,40,55-61; these questions will be discussed in more detail in the “Antigen Retrieval” section. It is the authors’ view that there is no immediate prospect of replacing formalin in routine surgical pathology. Even if consensus were to be reached in regard to a superior fixative, the logistics of converting all laboratories nationwide, yet alone worldwide, would be formidable. Formalin is thus what we have to work with for the foreseeable future. For this reason, this chapter will focus on IHC as applied to archival FFPE tissue sections for diagnostic pathology. In addition to basic principles and practical technical issues, the limitations and pitfalls of IHC are discussed with the intention of providing food for thought in the further development of IHC, particularly with respect to standardization and, ultimately, quantitative IHC applications.
The principles and practices described are derived from the long experience of the authors and are supported by certain core reference materials, particularly the Revised Guidelines of the Clinical and Laboratory Standards Institute62; the published materials of two major proficiency testing programs, United Kingdom (UK) National External Quality Assessment (NEQAS)63 and NordiQC64; plus select ad hoc committees that have issued recommendations in areas of difficulty.65-70
Basic Principles of Immunohistochemistry Surgical pathologists have long recognized their fallibility, although they have not always publicized it.2,3,4,26 They have, however, sought more certain means of validating morphologic judgments. A variety of special stains were developed to facilitate cell recognition and diagnosis, and most of these early stains were based on chemical reactions of cell and tissue components in frozen sections (histochemistry). In certain circumstances, these histochemical stains proved to be of critical value in specific cell identification. More often, they served merely to highlight or emphasize cellular or histologic features that supported a particular interpretation without providing truly specific confirmation. When the new field of IHC was created by combining immunology with histochemistry, the result was an enormous variety of truly specific special stains, potentially as many stains as there are specific antibodies— literally thousands. IHC has been the subject of thousands of papers, and today the vast majority of publications in anatomic pathology incorporate some IHC findings. The aims of IHC are akin to those of histochemistry. Indeed, IHC builds on the foundations of histochemistry but greatly extends the variety of tissue components that can be demonstrated specifically within tissue sections or other cell preparations. As emphasized by pioneers in this field of functional morphology, “the object of all staining is to recognize microchemically the existence and distribution of substances which we have been made aware of macrochemically.”71 The basic critical principle of IHC, as with any other special staining method, is a sharp visual localization of target components in the cells and tissue based on a satisfactory signal-to-noise ratio. Amplifying the signal while reducing nonspecific background staining (noise) has been a major strategy to achieve a satisfactory result that is useful in daily practice. In the past 40 years, advances in IHC have provided a feasible approach to performing immunostaining on routinely processed FFPE tissues, such that this method is now routine for the performance of IHC special stains in surgical pathology laboratories worldwide. Recently, demands for improved reproducibility and quantification have led to a growing recognition that IHC has the potential to be more than just a special stain. If properly controlled in all aspects of its performance, IHC can provide a tissue-based immunoassay with the
Antibodies as Specific Staining Reagents
reproducibility and quantitative characteristics of an enzyme-linked immunosorbent assay (ELISA) test, which not only detects the presence of an analyte, a protein or antigen, in relation to tissue and cell architecture, but also provides an accurate and reliable measure of its relative or real amount; this critical topic will be discussed further in the “Quality Control and Standardization” section.
Antibodies as Specific Staining Reagents An antibody is a molecule that has the property of combining specifically with a second molecule, termed the antigen. Further, the production of antibody by an animal is induced specifically by the presence of the corresponding antigen, which constitutes the basic immune response. Antigen-antibody recognition is based on the three-dimensional (3D) structure of protein or some other antigen, which may be compromised by formalin-induced modification of protein conformation (“masking”) but is restored in part by AR. We will discuss this process later in this chapter. Antibodies are immunoglobulin molecules that consist of two basic units: a pair of light chains, either a kappa or a lambda pair, and a pair of heavy chains— gamma, alpha, mu, delta, or epsilon. An antigen is any molecule sufficiently complex such that it maintains a relatively rigid 3D profile and is foreign to the animal into which it is introduced. Good antigens are proteins and carbohydrates that possess a unique 3D chargeshape profile. In fact, such molecules may bear more than one unique 3D structure capable of inducing antibody formation (Fig. 1-1). Each of these individual sites on a molecule may be termed an antigenic determinant, or epitope—the exact site on the molecule with which the antibody combines. For a protein, the term epitope corresponds to a cluster of amino acid residues that binds specifically to the paratope of an antibody.72 Although it is part of the protein, an epitope cannot be recognized independently of its paratope partner.72 Antigenic determinants (epitopes) may be classified as continuous or discontinuous; the former are composed of a continuum of residues in a polypeptide chain, whereas
Like determinants
Unlike determinants (diverse)
Figure 1-1 Antigens and antigenic determinants. An antigenic molecule may be considered to consist of an immunologically “inert” carrier component and one or more antigenic determinants of like type (left) or diverse types (right). From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier, p 6.
3
Anti-B B Anti-A
Figure 1-2 Antibodies as antigens. Anti-A antibody binds specifically to antigen A in the tissue section. Antigen B (B) is depicted as a second antigenic determinant that is part of the anti-A molecule; anti-B antibody, made in a second species, will bind to this determinant. Thus anti-B, the so-called secondary antibody, can be used to locate the site of binding of anti-A, the primary antibody, in a tissue section. From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier, p 9.
the latter consist of residues from different parts of a polypeptide chain, brought together by the folding of the protein conformation.73 This interesting issue may reflect the variable influence of formalin fixation on “antigenicity” and variations in the effectiveness of AR. Antibody molecules are proteins, thus any rigid part of an antibody molecule may itself serve as the antigenic determinant to induce an antibody. IHC techniques exploit the fact that immunoglobulin molecules can serve both as antibodies, binding specifically to tissue antigens, and as antigens, providing antigenic determinants to which secondary antibodies may be attached (Fig. 1-2). Evaluation of an antibody for use in IHC is based on two main points: the sensitivity and the specificity of the antibody-antigen reaction. The development of the hybridoma technique provided an almost limitless source of highly specific antibodies.21 However, monoclonal antibodies do not guarantee absolute antigen specificity, because different antigens may share similar or cross-reactive epitopes. Nonetheless, the “practical” specificity reflected by IHC is excellent for most monoclonal antibodies. In contrast, a polyclonal antibody is an antiserum that contains many different molecular species of antibody with varying specificities against different parts of the antigen, or antigenic determinants, used to immunize the animal. It is important to remember that polyclonal antibodies may also include varying amounts of antibodies to a whole range of other antigens, including bacteria and viruses, that the immunized animal encountered before its use as a source of antibody. As a result, polyclonal antibodies often give more nonspecific background staining in slides than is encountered using monoclonal antibodies. By the same token, however, the presence of a mixture of different antibodies against an antigen may on occasion confer an advantage to the use of polyclonal antibodies in the staining of certain hard-to-detect antigens in fixed tissues. For these reasons, the use of highly purified antigen preparations to produce high-affinity conventional polyclonal antibodies (antisera), which are then subjected to
4
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
multiple absorption procedures to maximize specificity, is of value for certain applications. It is important to note that immunodiffusion and ELISA assays typically used by manufacturers in the assessment of the specificity of antisera often fail to detect trace unwanted antibody specificities that may become apparent only when the antiserum is applied to tissue sections that contain many different antigens. Comparison of sensitivity and specificity between polyclonal and monoclonal antibodies indicates that polyclonal antibody may be more sensitive but is often less specific than monoclonal antibody. The reason may be that polyclonal antibody, actually a composite of many antibodies, may recognize several different binding sites (epitopes) on a single protein (antigen), whereas a monoclonal antibody recognizes only a single epitope. Sophisticated amplification techniques, coupled with the use of the AR technique, have reduced the practical importance of this distinction. Although the specificity of monoclonal antibody is, as noted, not absolute because of cross-reactivity with nontarget molecules,74 most commercially available monoclonal antibodies are highly reliable for IHC. Most monoclonal antibodies in current use are derived from murine clones. More recently, a number of rabbit-derived monoclonal antibodies have appeared on the market, and manufacturers may sell both, or one or the other, for a single antigen. Some rabbit monoclonal antibodies appear to offer advantages over murine clones for detection of certain antigens by IHC, but this phenomenon may be more a function of differences in relative immunogenicity of different proteins in the two species, rather than an absolute superiority of rabbit monoclonal antibodies over murine monoclonal antibodies, or vice versa. In addition, recombinant DNA techniques have been used to develop antibodies that may demonstrate improved practical specificity following stringent affinity purification, exemplified by the use of recombinant protein epitope signature tags (PrESTs).75 Selection of the best antibody is described briefly in the following paragraphs, with additional detail in the sections on detection systems and quality control (QC) and standardization. It must be emphasized that the ultimate specificity control for both monoclonal and polyclonal antibodies should be the observation of the expected pattern of staining in control tissue sections and a corresponding lack of unexpected or inexplicable staining reactions (see the “Quality Control and Standardization” section). Correlation of the staining result of a new antibody with the literature for antigen distribution is strongly encouraged. Comparison of the staining of the test antibody with that of a second antibody known to bind to the same antigen but to a different epitope is also valuable in establishing useful specificity, a process that may be accomplished by comparing reagents from different manufacturers, with careful attention to clone designation in the package insert. Lastly, Web sites such as www.proteinatlas.org offer a wealth of information with respect to the patterns of reactivity of numerous antibodies and clones on normal and abnormal (cancer) tissues. Such information is useful as a guide, but it is important to remember it will
not necessarily reflect the findings of another laboratory with differing fixation methods and protocols.
Blocking Nonspecific Background Staining Two aspects of the blocking of background staining of tissues may be attributable either to nonspecific antibody binding or to the presence of endogenous enzymes. Nonspecific antibody binding is generally more of a problem with polyclonal antibodies, because multiple unwanted antibodies may exist in the antiserum. The greater the optimal working dilution, the less significant the problem. Another form of nonspecific binding may result from the fact that antibodies are highly charged molecules that may bind nonspecifically to tissue components that bear reciprocal charges, such as collagen. Such nonspecific binding may lead to localization of either the primary antibody or the labeled moiety, producing false-positive staining of collagen and other tissue components of a degree sufficient to obscure specific staining (Fig. 1-3). Preincubation with normal serum usually reduces these kinds of nonspecific binding. In theory, proteins in the normal serum occupy the charged sites within the tissue section, excluding or at least reducing nonspecific attachment of antibodies added subsequently. In practice, it is customary to use normal serum or purified immunoglobulin of the same species as the secondary antibody (in conjugate methods), because this normal serum neither interferes with nor participates in the immunologic reactions that occur as part of the IHC procedure. Blocking endogenous enzyme activity is also important. The degree of susceptibility of an enzyme to denaturation and inactivation during fixation varies. Some enzymes, such as peroxidase, are preserved in both paraffin and frozen sections; others, such as alkaline phosphatase, are completely inactivated by routine fixation and paraffin-embedding procedures. Any residual activity of these endogenous enzymes must be abolished during immunostaining to avoid false-positive reactions when using the same or similar enzymes as labels. Peroxidase activity is present in a number of normal and neoplastic cells that include erythrocytes, neutrophils, eosinophils, and hepatocytes. When performing an IHC study in tissues rich in blood cells, such as bone marrow, it is recommended that a peroxidase-blocking step be used, coupled with a substrate control (i.e., a section treated only with the hydrogen peroxide–chromogen mixture to visualize the extent of endogenous peroxidase activity). Otherwise, alternative methods such as alkaline phosphatase, glucose oxidase, or immunogold labeling may be used to avoid the possibility of confusion with any endogenous peroxidase activity. To risk stating the obvious, the blocking of endogenous enzymatic activity must be carried out before the addition of enzyme-labeled secondary reagent; otherwise, the enzyme label is also inactivated by the blocking procedure, resulting in a false-negative result. Various approaches have been devised to inhibit peroxidase
Detection Systems
A
5
B
Figure 1-3 Example of the effectiveness of blocking nonspecific binding of primary and secondary antibodies. A, A section of spleen stained for immunoglobulin (Ig) G by the peroxidase-antiperoxidase method; scattered positive plasma cells (small dots) are seen, but staining of collagen bands is heavy. B, The adjacent parallel section was treated in an identical fashion, except that normal serum from the same species as the linking antibody (in this case normal swine serum to match the swine antirabbit Ig-linking antibody) was added before the primary antibody. In this instance, the plasma cells are seen even more clearly, because the heavy, nonspecific staining of collagen is markedly diminished. Paraffin sections, diaminobenzidine with hematoxylin counterstain (×60). From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
activity, primarily by using solutions of hydrogen peroxide (H2O2).76-78 For general purposes, we have obtained satisfactory results with a 15-minute incubation in a methanol-H2O2 combination. Many manufacturers also include proprietary reagents and protocols that effectively neutralize endogenous peroxidase in both manual and automated methods; in these instances, the recommendations of the manufacturer should be validated for satisfactory performance and then followed strictly. For those who encounter difficulty or wish to explore other approaches, a more detailed discussion follows. Some investigators79-81 believe that the methanol-H2O2 approach is too drastic and that it may cause some denaturation of antigen. Straus80,81 advocated the use of a combination of phenylhydrazine, nascent H2O2 (freshly produced by a glucose oxidase–glucose mixture), and sodium azide.82 Robinson and Dawson83 adopted a different approach, first developing the endogenous peroxidase with 4-chloro-1-naphthol, giving it a bluegray color, then performing the IHC staining procedure with a peroxidase label and diaminobenzidine (DAB), giving a contrasting brown reaction product. Taylor used a similar strategy 30 years ago in the initial reports to describe the feasibility of demonstrating immunoglobulin antigens in paraffin sections; α-naphthol pyronine was used for endogenous peroxidase (pink), followed by DAB (brown) for the HRP label.5,23
Detection Systems Antibody molecules cannot be seen with the light microscope, or even with the electron microscope, unless they are labeled or flagged by some method that permits their visualization. Essentially, detection systems attach labels or flags to primary or secondary antibodies in order to visualize the target antibody-antigen localization in the tissue sections. A variety of labels or flags have been used, including fluorescent compounds and active enzymes that can be visualized by virtue of their property of inducing the formation of a colored reaction product from a suitable substrate system. Such methods can be adapted to electron microscopy if the products are rendered electron dense by suitable treatment. Alternatively, labels directly visible by electron microscopy may be used, such as gold, ferritin, or virus particles. Fluorescent labels are also making a comeback, based upon a variety of new fluorescent labels and “Q dots” that do not fade. The focus of this chapter is upon methods that are interpreted by the usual bright-field microscopy used in diagnostic surgical pathology; other approaches are discussed only briefly. Another important goal of various detection systems is the enhancement of sensitivity through amplification of the signal, and we will discuss this later in the chapter.
6
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Direct-Conjugate–labeled Antibody Method
Px
The method of attaching a label to an antibody by chemical means and then directly applying this labeled conjugate to tissue sections (Fig. 1-4) has been used widely in immunohistology. In preparing a labeled antibody conjugate, the aim is to attach the maximal number of molecules of label to each individual antibody molecule. It is desirable to label 100% of antibody molecules and to render none of them immunologically inactive by the labeling process. The final labeled reagent should not contain free molecules of unlabeled antibody or molecules of antibody linked to an inactivated label. These are exacting requirements that are difficult for individual scientists to meet in-house. However, conjugation methods have improved immensely since the early 1980s, and quality labeled reagents that include peroxidase, glucose oxidase, and alkaline phosphatase labels are available from a number of commercial sources. The direct-conjugate procedure has the advantages of rapidity and ease of performance. With this method, the purity (i.e., monospecificity) of the primary antibody or antiserum (polyclonal antibody) is of critical importance. As noted previously, an antiserum contains a range of antibody molecules of differing specificity in addition to the antibody having the desired specificity; all these antibodies are labeled during the conjugation procedure, and any or all may produce staining in tissue sections that may lead to erroneous interpretation. One practical disadvantage of the direct-conjugate procedure is that it is necessary to conjugate each of the appropriate primary antibodies separately to detect different antigens. Also, with regard to precious (scarce) antibodies, the direct-conjugate procedure usually demands that the primary antibody be used at a relatively high concentration in comparison with indirect and unlabeled antibody methods.
Indirect, or Sandwich, Procedure The indirect, or sandwich, conjugate procedure (Fig. 1-5) is a relatively simple modification of the directconjugate method. It has the following advantages: 1. Versatility is increased, because a single conjugated antibody can be used with several different primary antibodies. Px
F
F
* A
* B
Figure 1-5 Indirect-conjugate (sandwich) method. The primary antibody is unlabeled (asterisk). The method uses a labeled secondary antibody with specificity against the primary antibody (boxed antigen determinant on primary antibody.) Px, Peroxidase label; F, fluorescein label. From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
2. The labeling process is applied only to the secondary antibody. 3. The primary antibody can usually be used at a higher working dilution than in the direct method to achieve successful staining. 4. The secondary antibody, which is produced against immunoglobulin of the species from which the primary antibody is derived, is readily prepared with a high order of specificity and affinity. Many commercial sources are available for labeled secondary reagents, including polymer-based reagents. 5. The method lends itself to additional specificity controls in that the primary specific antibody may be omitted, or another antibody of irrelevant specificity may be substituted, providing a valuable assessment of the validity of any staining pattern observed. All labeled antibody methods performed by the indirect procedure are analogous in principle; peroxidase and fluorescent indirect-conjugate methods are illustrated in Figure 1-5, A and B, respectively. The primary antibody that has specificity against the antigen in question (e.g., rabbit antitriangle) is added to the section, and the excess is washed off. The labeled secondary antibody, which has specificity against an antigenic determinant present on the primary rabbit antibody (e.g., swine antibody vs. rabbit immunoglobulin), is then added; it serves to label the sites of tissue localization of the primary antibody, which, in turn, is bound to the antigen.
Unlabeled Antibody Methods ENZYME BRIDGE TECHNIQUE
A
B
Figure 1-4 Direct-conjugate method. The label, or flag, is attached directly to the antibody with specificity for the antigen under study. A, Peroxide labeled. B, Fluorescence labeled. Px, Peroxidase; F, fluorescein. From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
The disadvantages of the chemical conjugation procedure may be entirely avoided by devising techniques whereby the labeled moiety is linked to the antigen solely by immunologic binding. To achieve this end, Mason and colleagues84 developed a technique that has become known as the enzyme bridge method (Fig. 1-6). This method is rarely used today but is included for its value in research applications in which chemical conjugation is undesirable.
Detection Systems
Px
Px
Px
Free peroxidase Px
Anti-Px antibody
*
A
A
B
Secondary (bridge) antibody
*
7
*
B Primary antibody Tissue section
Figure 1-6 Enzyme bridge method. A second antibody is used to link (bridge) the primary antibody to an antiperoxidase antibody, which in turn binds to free peroxidase. Boxed asterisk represents antigen determinant on primary and secondary antibodies. Px, Peroxidase label. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
PEROXIDASE-ANTIPEROXIDASE METHOD
The peroxidase-antiperoxidase (PAP) method (Fig. 1-7) also avoids the problems inherent in chemical conjugation. First used by Sternberger and colleagues for the detection of anti-Treponema antibodies,85 the PAP system was reported to have a sensitivity a hundredfold to a thousandfold greater than that of comparable conjugate procedures. The principle of the PAP method is similar to that of the enzyme bridge method (see Fig. 1-6). The acronym denotes the peroxidaseantiperoxidase reagent that consists of antibody against HRP and HRP antigen in the form of a small,
Px Px Px Px
Px
Px
Rabbit PAP
Bridge antibody R
* *
Secondary (bridge) antibody
Rabbit-antibody vs mouse Ig M
Primary antibody Mouse primary antibody
Tissue section
A
B
Figure 1-8 Biotin-avidin methods. A, Direct biotin-avidin method. The primary antibody is linked to biotin (the boxed B), and an avidin-peroxidase conjugate (A-Px) is then added. B, Indirect biotin-avidin method. Used for monoclonal antibodies, the primary antibody is not conjugated; its localization is detected by a biotinylated secondary antibody. Boxed asterisk represents antigen determinant on primary antibody. A, Avidin; B, biotin; Px, peroxidase label. From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
stable immune complex. Available evidence suggests that this immune complex typically consists of two antibody molecules and three HRP molecules in the configuration (see Fig. 1-7). The PAP reagent and the primary antibody must be from the same species, or from closely related species with common antigenic determinants, whereas the bridge or linking antibody is derived from a second species and has specificity against the primary antibody (e.g., rabbit antitriangle) and the immunoglobulin incorporated into the PAP complex (e.g., rabbit antiperoxidase). This method enjoyed extensive use in routinely processed paraffin sections because of its high degree of sensitivity, but it has been replaced by streptavidin- and polymer-based systems.
Biotin-Avidin Procedure
R
PAP
A
B
Figure 1-7 Peroxidase-antiperoxidase (PAP) methods. A, Threestage PAP method. PAP reagent (dashed lines) is a preformed stable immune complex linked to the primary antibody by a “bridging” antibody. B, Four-stage PAP method. PAP reagent (dashed line) is a preformed stable immune complex. Primary antibody in this example is murine (mouse immunoglobulin [Ig], as in a monoclonal antibody [M]); this antibody is followed by a rabbit antimouse Ig (R), a bridge antibody (e.g., swine antirabbit Ig), and rabbit PAP. Px, Peroxidase label. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
The biotin-avidin procedure (Fig. 1-8) exploits the high-affinity binding between biotin and avidin. Biotin can be linked chemically to the primary antibody (see Fig. 1-8, A) to produce a biotinylated conjugate that localizes to the sites of antigen within the section. Subsequently, avidin, which is chemically conjugated to HRP, is added; the avidin binds tightly to the biotinylated antibody, thus localizing the peroxidase moiety at the site of antigen in the tissue section. This method is rapid and has been used particularly in indirect procedures (see Fig. 1-8, B). Two significant disadvantages exist. First, different batches of biotin and avidin have differing affinities for one other, which affects the sensitivity and reproducibility of the procedure. Second, some tissues contain significant amounts of endogenous biotin that may bind the avidin-peroxidase complex directly, thus producing nonspecific (false-positive) staining. This effect can be combated by suitable blocking techniques, described in the following paragraphs.
8
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
antibodies. Either peroxidase or alkaline phosphatase may be used as the enzyme label.
Px B Px
B
A
B
Px
B
*
Figure 1-9 Avidin-biotin conjugate (ABC) method. A biotinylated secondary antibody serves to link the primary antibody to a large, preformed complex of avidin, biotin, and peroxidase. Boxed asterisk represents antigen determinant on primary antibody. A, Avidin; B, biotin; Px, peroxidase label. From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
Avidin-Biotin Conjugate Procedure Hsu and colleagues14,15 developed a further modification of the biotin-avidin system that greatly enhanced its sensitivity (Fig. 1-9). The avidin-biotin conjugate (ABC) procedure serves to localize several molecules of HRP at the site of the antigen. Binding to endogenous biotin remains a problem.
Biotin-Streptavidin Systems The biotin-streptavidin (B-SA) method overcomes several of the problems associated with the ABC systems by substituting streptavidin for avidin and directly conjugating the streptavidin to the enzyme molecule. Streptavidin, a tetrameric 60-kD avidin analog isolated from the bacterium Streptomyces avidinii, is capable of binding biotin with a very high affinity—approximately 10 times higher than that of most antibodies for their antigens. The use of streptavidin is preferred to avidin for several reasons: 1. Streptavidin contains no carbohydrates, which if present can bind nonspecifically to lectin-like substances found in normal tissue from kidney, liver, brain, and mast cells. 2. The isoelectric point of streptavidin is close to neutrality, whereas avidin has an isoelectric point of 10; thus streptavidin conjugates exhibit less nonspecific electrostatic binding than avidin conjugates. 3. Because the enzyme is directly conjugated to streptavidin in the B-SA system, it is a highly stable reagent that can be diluted and stored for long periods in a ready-to-use (RTU) form. Secondary and labeling reagents based on these principles are available commercially that can provide substantial increases in sensitivity and also allow for increased dilution of expensive primary
Alkaline Phosphatase Labels, Double (Multiplex) Stains, and Polyvalent Detection Systems Increasingly, pathologists are seeking to demonstrate more than one antigen in a single tissue section (slide). The reasons for this approach are manifold: first, it reduces the number of slides stained, which makes the most efficient use of tissue biopsies that are progressively smaller in size, including fine needle aspirates (FNAs); and second, it facilitates interpretation of complex staining patterns in mixed cell populations. Many automated platforms (discussed later) now provide the capability for multiplex staining, thereby rendering this approach more useful and more widely available. Double stains must produce contrasting colors to be effective in routine pathology. The simplest way to accomplish this has been to use a second enzymatic label, alkaline phosphatase, which has its own distinct range of chromogens (this approach is discussed and illustrated later). Many commercial suppliers now offer polyvalent detection systems (Biocare, Leica, Agilent/ Dako, Roche/Ventana) that facilitate the detection of primary antibodies from two or more different species. Initially, double stains—or those with greater multiples, so-called multiplex stains—traditionally were performed sequentially, but recently the use of these newer approaches has allowed for concurrent performance. Some manufacturers provide primary and secondary reagents in “cocktails” of antibodies, typically raised in different species (e.g., mouse and rabbit monoclonal antibodies) to avoid troublesome cross-reactions. Each component of any double or multiplex stain must be separately validated for performance. Multiple stains are discussed more extensively later in this chapter. Alkaline phosphatase–labeled reagents may be introduced in several ways, essentially paralleling those methods used with HRP. Ongoing improvements in polymer-based methods (discussed in the following section) are so dramatic, it appears likely that these methods will supersede PAP and B-SA methods, as well as double stains, as the primary method. A brief description of special alkaline phosphatase applications follows as the basis for working with this useful method. ALKALINE PHOSPHATASE ANTIALKALINE PHOSPHATASE METHOD
The principles of the alkaline phosphatase/antialkaline phosphatase (APAAP) technique are the same as those described for the PAP method (Fig. 1-10).86 As with PAP, the APAAP method has largely been replaced by new polymer-based systems, and APAAP reagent is not widely available today. The method is included here for historic and research interest. Alkaline phosphatase labeling is not only useful as a second, “double” stain but also may be preferred for tissues rich in endogenous peroxidase, such as bone
Detection Systems
9
Px AP Figure 1-10 Alkaline phosphatase/antialkaline phosphatase (APAAP) and peroxidase-antiperoxidase (PAP) methods. The feasibility of double staining is shown by the use of different primary and secondary antibodies; for example, mouse antivimentin, horse antimouse immunoglobulin G (IgG), mouse APAAP (left); rabbit antikeratin (polyclonal goat antirabbit IgG), rabbit PAP (right). AP, Alkaline phosphatase; Px, peroxidase. From Taylor CR, Cote RJ, eds: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
marrow or lymphoid tissue that contains infiltrating myeloid cells, particularly with frozen sections. Because complete blocking of endogenous peroxidase in blood and bone marrow smears may be difficult, and because blocking procedures may denature some of the antigenic determinants, alkaline phosphatase–based methods have proved useful in staining bone marrow. The study by Erber and McLachlan87 provides an excellent resource for those wishing to adopt this method. For double immunostaining, it is convenient to use an alkaline phosphatase method in conjunction with an immunoperoxidase. The use of alkaline phosphatase as the second label has the advantage of avoiding the crossreactivity that can occur when two immunoperoxidase procedures are used together (see discussion below). In addition, a simultaneous double immunostaining procedure may be carried out by using heterospecific antibodies, such as polyclonal and monoclonal antibodies, or mouse and rabbit monoclonals as the two primary antibodies under investigation (see Fig. 1-10). We will discuss this procedure in the context of cocktail methods. Sequential double immunostaining with the alkaline phosphatase method may produce excellent contrasting colors by using fast-red and fast-blue stains.88 In sequential double stains, care must be taken to avoid mixedcolor staining (i.e., having the initial red color change to purple); to this end, the weaker staining antigen is usually stained first, and the second label applied may be developed for a shorter time (10 to 15 minutes, monitored by microscopy). These general principles apply equally to double stains with polymer-based systems, discussed below. In some cases, the bright red color produced by alkaline phosphatase substrates (fast red or new fuchsin) may provide more distinct staining than the conventional peroxidase chromogens. Most automated platforms now offer reagents and protocols for double or multiplex staining based on the polymer-type labels instead of PAP methods. With these automated approaches, the protocols should be followed exactly and only modified as necessary to obtain results with new antibody pairings, whereupon careful revalidation is then required. POLYMER-BASED LABELING METHODS
The demand for more sensitive, more reliable, and simpler methods for IHC continues to escalate.
AP
* *
APAAP
Px
Secondary (bridge) antibody
Px
* *
PAP
Secondary (bridge) antibody
Primary antibody
Primary antibody
Tissue section
Tissue section
Traditional multistep detection systems have several drawbacks: these include complex, time-consuming protocols; difficulties in standardization; and difficulties with sensitivity and ability to demonstrate hard-todetect antigens. In practice, the reduction of the number of steps has, unfortunately, always been accompanied by a reduction in sensitivity. Approaches such as catalyzed reporter deposition or tyramine signal amplification (TSA),9 immunopolymerase chain reaction (immunoPCR),89 and end-product amplification90 have improved sensitivity; however, these techniques are accompanied by more complicated protocols, nonspecific staining, and, often, poor reproducibility. The development of polymer-based amplification methods has circumvented some of these issues. These methods use linking antibodies and marker enzymes attached to a backbone of synthetic polymers or to polymerized proteins (Fig. 1-11, A and B). The natural or synthetic polymers increase the number of enzymes or ligands that are coupled to the linking antibodies.91-96 Examples of such carriers include dextran, polypeptides, dendrimers, and DNA branches. The trend is also to manufacture polymers of smaller overall molecular size, often called micropolymers, to reduce the possibility of steric interference. One big advantage of these polymerbased detection systems is that they avoid the use of biotinylated secondary antibody. Blocking of endogenous biotin is therefore not required, and the falsepositive staining as a result of endogenous biotin is eliminated. In practical terms, these advances have been greatly facilitated by automation, and polymer-based detection systems are now widely available for automated platforms. This outcome is consistent with the overall philosophy that, all other things being equal, simple techniques are better than complicated ones (see Fig. 1-11, A and B). Again, it must be emphasized that in the purchase and use of such methods, the instructions provided by the manufacturer must be followed rigorously. Departure from the provided protocol invalidates any implied performance warranty and may lead to false-positive or false-negative results or to slides that cannot be interpreted. As will be described later, the resulting colored products of some multiplex stains cannot be reliably separated by the naked eye and thereby require the use of digital images and sophisticated software for proper interpretation (see Fig. 1-21).
10
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Primary antibody Secondary antibody Peroxidase Dextran polymer
A Tissue antigen Primary antibody Secondary antibody Peroxidase
B Figure 1-11 Schematic of polymer-based detection systems, which allow the attachment of multiple molecules of enzyme, either peroxidase or alkaline phosphatase, to the secondary labeling antibody. A, This structure shows several molecules of secondary antibody and multiple enzymes attached to a dextran polymer “backbone.” B, This configuration has a more compact molecular shape and allows the attachment of multiple conjugates in close proximity to each other. In both approaches the net effect is to attach multiple enzyme molecules at the antigen site, thereby enhancing sensitivity. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
As with IHC labeling methods in general, polymerbased systems may be one-step, direct, or multistep methods. The enhanced polymer one-step staining (EPOS) system (Dako, Carpenteria, CA) was introduced in 1993 by Bisgaard.95 In this system, the monoclonal primary antibody and HRP are covalently bound to an inert, high-molecular-weight dextran polymer. This technique offers the advantage of combining the link antibody step and the detection complex incubation step into a single step, giving a more rapid staining process. However, utility of this system is restricted by limited availability of labeled primary antibodies. The one-step method may be performed on paraffinembedded tissue sections, archival frozen material, and intraoperative frozen sections to yield sensitive and reproducible results. Turnaround time can be reduced to approximately 1 hour for routinely processed paraffinembedded material and to less than 7 minutes for frozen-section material.96 Today, two-step indirect polymer strategies are available from many manufacturers: Agilent/Dako, Roche/Ventana, Novocastra/Leica, Thermo/Lab Vision, BioCare, and others. In general, reagents and protocols are available both for manual use and for automated platforms, and the principle is similar in all: the tissue is first incubated with the primary antibody and is then
incubated with a polymeric conjugate in sequential steps. The polymeric conjugate may be relatively large, with as many as 100 peroxidase enzyme molecules and as many as 20 secondary antibody molecules (e.g., goat antimouse or goat antirabbit) bound to the polymer backbone. As noted, the trend is toward progressively more compact polymers. Because it is a two-step process, this method offers the laboratory the flexibility of choosing different primary antibodies, the concentration and incubation times of which may be individually optimized. Today, these polymer-based reagents continue to improve and offer many advantages to previous methods. Higher sensitivity allows detection of minute amounts of antigen, increased dilution of the primary antibody, plus absence of false-positive staining as a result of endogenous biotin. In addition, these reagents are stable and offer robust reproducible protocols, particularly in an automated environment. Polymer-based methods also may be adapted to rapid performance for frozen sections, for assessment of margins (e.g., in Mohs surgery)97 and micrometastases, as well as to signal amplification for chromogenic in situ hybridization (CISH), with an increase in sensitivity and shorter assay times.98 Note that successful application of any ultrasensitive detection system raises the possibility that it may be necessary to retitrate primary reagents to a higher working dilution to avoid nonspecific staining.91,99,100 Polymer-based methods also lend themselves well to double and multiplex staining, which will be discussed later. TYRAMINE SIGNAL AMPLIFICATION
Based on the principle of enzyme amplification for immunoassays adopted in the 1980s, Bobrow and associates9,99 developed a catalyzed reporter deposition technique (CARD) to achieve amplified signal for solidphase immunoassay systems and membrane immunoassays. In this method, signal amplification is based on biotinylated tyramine (tyramide) deposition through free radical formation, which is catalyzed by the oxidizing action of HRP. It is postulated that radicalized biotinylated tyramine covalently attaches to electron-rich moieties—tyrosine, phenylalanine, tryptophan, and so on—resulting in aggregation of additional biotinylated molecules at the site of antigen-antibody reaction (i.e., amplification of the signal; Fig. 1-12).99-101 So-called TSA for IHC has achieved positive immunostaining for some hard-to-detect antigens in archival paraffin-embedded tissue sections.100 Several commercial TSA reagents are available in kit form. With use of these TSA kits, additional steps are performed after use of a regular HRP-conjugated detection system; namely, incubating the slides with a biotinylated tyramine reagent, washing them thoroughly, and then incubating the slides with more HRP-conjugated streptavidin. Finally, chromogen is used—DAB, amino-ethyl carbazole (AEC), and so on—to visualize the amplified signal in the tissue sections. Although the TSA method has achieved satisfactory results in terms of significantly increasing intensity of
Detection Systems Tyramine-based Enhancement
11
Px
Protein A
Primary antibody
* Tissue section
*covalent bond = Avidin-biotin-peroxidase complex Figure 1-12 Tyramine amplification, an example of catalyzed signal amplification, relies on the effect of peroxidase enzyme to catalyze the deposition of multiple biotin-tyramine molecules (depicted as a halo) at the site of an initial immunoperoxidase reaction (shown as an indirect ABC reaction). The deposited biotin-tyramine in turn serves as a target for localization of additional molecules of streptavidin-peroxidase, with an incremental increase in the generation of colored reaction product with a chromogen, giving the method great sensitivity. Control of the end point may be difficult. Asterisk denotes covalent bond. Modified from Taylor and Cote: Immunomicroscopy: a diagnostic tool for the surgical pathologist, Philadelphia, 2005, Elsevier.
IHC and ISH, it has not been widely applied in diagnostic pathology for several reasons: 1) additional steps make the method more time consuming; 2) nonspecific background staining may increase as the signal increases; 3) reproducibility is more difficult to achieve; 4) secondgeneration polymer-based methods are simpler and also give high sensitivity sufficient for many requirements; and 5) automated platforms provide the possibility of much more reproducible performance of this powerful method, achieving high sensitivity and short assay time.
Figure 1-13 Protein A conjugate method. Protein A, labeled with peroxidase, binds to the constant (Fc) component of the primary antibody. Px, Peroxidase label. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 2. Philadelphia, 1994, WB Saunders.
The enzyme-labeled antigen method (Fig. 1-15) was devised as perhaps the ultimate in specificity among immunoperoxidase techniques. Only one antibody is used, and the method exploits the fact that an antibody molecule possesses two valences, one of which may be bound to the antigen under study, with the second valency left free to bind with additional molecules of antigen added subsequently. The additional antigen is presented in a form that is directly conjugated with HRP, thus this is considered a labeled antigen procedure. The primary antibody is generally used at a relatively high concentration, therefore this method is not economical in its use of the primary antibody. However, one major advantage is that the primary antibody need not be particularly pure, because antibodies of irrelevant specificity will not be detected by this technique, even if they bind to the tissue section; lacking specificity for
Px
OTHER METHODS WITH LIMITED OR RESEARCH APPLICATIONS
A variety of other methods exist that allow for labeling of antigen (protein) in tissue sections. Most are restricted to research applications, but the pace is such that further development and increased availability of new, more sensitive methods will continue. Two approaches are described briefly for specific research advantages. Protein A, derived from Staphylococcus, has the remarkable ability to bind with the constant (Fc) portion of immunoglobulin (Ig) molecules from several different species. The only absolute requirement is that the primary antibody binds with protein A; most IgG molecules bind protein A, although affinity varies among different IgG subclasses and among different species (Figs. 1-13 and 1-14). Protein A methods generally do not match the sensitivity of corresponding streptavidin or polymer-based techniques, but they have advantages that may warrant their use in specific circumstances.
Px
Px
PAP
Protein A
Primary antibody Tissue section Figure 1-14 Protein A–peroxidase-antiperoxidase (PAP) method. Protein A is used to link the primary antibody (Fc) to the antibody (Fc) within the PAP complex. Px, Peroxidase label. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 2. Philadelphia, 1994, WB Saunders, p 14.
12
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Px
Px
AP
Figure 1-15 Labeled antigen method. The antibody is added in excess so that one valency is bound to the antigen in the section, leaving the second valency free to bind the labeled antigen that is added subsequently. Px, Peroxidase label. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 2. Philadelphia, 1994, WB Saunders, p 14.
Figure 1-16 Labeled antigen double stain. Two different antibodies recognize their respective antigens in the tissue section and subsequently bind only the corresponding labeled antigen (labeled with peroxidase [Px] or alkaline phosphatase [AP]). From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 2. Philadelphia, 1994, WB Saunders, p 14.
the target antigen, they will not bind the same antigen presented in the antigen-peroxidase conjugate, and thus they will not be visualized. This method has proved particularly suitable for high-specificity double-staining techniques, whereby the goal is to stain two antigens simultaneously within the same section (Fig. 1-16) by using two labeled antigens (e.g., κ-peroxidase and λ– alkaline phosphatase) together.
antibody) with several different dilutions of the primary antibody; comparison is achieved by checkerboard titration (Table 1-2). Multistep procedures require more complex checkerboard titrations of each of these separate steps. The reader is invited to refer to previous editions of this book or the 2006 work of Taylor and Cote, Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist,91 for further details of more complex checkerboard titrations for research use. Once optimal dilutions have been determined, the stock of undiluted reagent should be divided into convenient aliquots for the preparation of working dilutions immediately before use. Generally speaking, it is not wise to store reagents in a highly diluted form unless additional protein or stabilizers are added to conserve activity, because reactivity may decline unpredictably. That stability of highly diluted reagents can be achieved is evidenced by the availability of commercial immunostaining kits that contain prediluted RTU reagents and have a defined but often limited shelf life (Table 1-3). A principal reason for the use of reagents freshly prepared from aliquots is the need to avoid repeated sampling of a single reagent tube or bottle, as with a Pasteur pipette, because this practice almost invariably results in bacterial contamination and loss of reactivity that is both unpredictable and aggravating. Here the “pure” scientists might learn a lesson from their often
Titration of Primary Antibody and Detection System The optimal dilution for an antibody in immunohistology is defined as the dilution at which the greatest contrast is achieved between the desired (specific) positive staining and any unwanted (nonspecific) background staining. Selection is subjective and is based not simply on the greatest intensity but rather on the greatest useful contrast. Titration is relatively straightforward in the direct method, with only a single antibody (Table 1-1). In two-layer methods, exemplified by the indirect and polymer-based labels, each of the separate immune reagents must be applied at optimal dilution. In addition, the dilutions of the primary and secondary antibodies, or labels, are interdependent in terms of contrast developed by the procedure as a whole. This fact necessitates comparison of the results obtained by using several dilutions of the labeling reagent (secondary
TABLE 1-1 Representative Dilution Titration to Determine Optimal Titer of Antibody for Use in Direct Conjugate Method Serial Dilutions of Primary Antibody 1/5
1/20
1/80
1/320
1/1280
1/2560
Unwanted background†
++
+
±
±
±
±
Specific antigens‡
+++
++++
++++
+++
++
+
Intensity of staining*
Modified from Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier. *Intensity of staining scored on a semiquantitative scale from 0 to ++++; ± indicates faint positivity of uncertain significance. † Unwanted background staining may result from several different mechanisms. ‡ Intensity of staining of specific antigen (e.g., with titration of anti-κ antibody, specific staining would be present in plasma cells).
Detection Systems
13
TABLE 1-2 Determination of Optimal Titers for Indirect Immunoperoxidase Method: Checkerboard Titration Dilutions of Primary Antibody
Dilutions of secondary antibody (conjugate)
1/5
1/20
1/80
1/320
1/1280
Negative Control*
1/10
Slide 1 +++† (++)
Slide 2 ++++ (++)
Slide 3 +++ (++)
Slide 4 ++ (++)
Slide 5 + (++)
Slide 16 ± (++)
1/40
Slide 6 +++ (+)
Slide 7 +++ (+)
Slide 8 ++++ (+)
Slide 9 ++++ (±)
Slide 10 ++ (±)
Slide 17 − (±)
1/160
Slide 11 ++ (+)
Slide 12 ++ (±)
Slide 13 + (±)
Slide 14 ± (−)
Slide 15 ± (−)
Slide 18 − (−)
Modified from Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier. Note: In this example of an 18-slide titration, the optimal result is achieved with slide 9. *Negative control (omit primary antibody, replace with preimmune serum or serum with irrelevant specificity; see negative controls). † Intensity of specific staining is indicated on a scale of 0 to ++++. Nonspecific background is given on the same scale but is shown in parentheses; for example, +++ (+) indicates strong specific staining (+++) with moderate background (+).
maligned cousins in the commercial sector, who have to a large extent overcome the contamination problem by providing diluted reagents in sealed dropper bottles so that the reagent can be dropped directly from the reagent bottle. We have unashamedly borrowed this technique in our laboratory, and whenever we make up new dilutions of reagents for use over a period of several days, we use these small plastic dropper bottles.
Establishing a New Immunohistochemistry Stain in the Laboratory In general, two approaches may be used to establish a new IHC stain in the laboratory or, in fact, to perform IHC stains in general. The first is to work from basic principles, purchasing all reagents separately and building the protocol in-house. This approach often is referred to as home brewing, and it requires extensive validation of reagents and protocol plus an obvious
ability to perform detailed checkerboard titrations reliably. The second approach is to purchase all of the IHC stain components in an optimized kit, in its simplest form, the so-called RTU kit. Validation of the performance of the overall kit against appropriate controls (see below) is still required, but the need to perform multiple complex titrations is obviated (see Table 1-3). Both of these approaches have their different advantages and disadvantages, as summarized in Table 1-3. If performance is not adequate, the “home brew” has the advantage of greater flexibility in choice of reagents and concentrations, but it requires in-house expertise to make such adjustments and to validate performance. In practice, the need for such adjustments is mostly a result of different, often inadequate methods of sample preparation. In general, it is better to correct poor or variable sample preparation than to try to compensate for such by resorting to overly vigorous retrieval or increased concentrations of antibodies or labels. Note that the use of RTUs does enforce some uniformity upon different laboratories, which is a good
TABLE 1-3 “Home Brewing” vs. Ready-to-Use Approaches Home Brew
Ready-to-Use
Select primary antibodies and purchase Purchase suitable labeling antibodies Purchase chromogens, etc.
Purchase kit that includes all reagents, matched and tested for performance
Titrate primary antibodies Identify appropriate labeling method Select chromogen Establish time and concentration
Prediluted primary antibodies included Includes labeling method, pretested Includes chromogen Includes protocol
Obtain serial dilutions of labeling reagent Establish incubation times
Prediluted reagents included Recommended times provided
Establish protocol Select controls
Protocol included Recommended controls
14
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
thing, as discussed below. Also, automated platforms usually are accompanied by a menu of RTU reagents optimized for each specific platform. As noted elsewhere, deviation from the recommended reagents or protocol places the full burden of validation on the laboratory.
Quality Control and Standardization Standardization to achieve reproducibility of results within a lab, and among different labs, has become increasingly important as IHC has been adapted to tests for prognostic and predictive markers. Such tests require some degree of quantification, and greater stringency of performance is therefore necessary.91,102 As defined by the College of American Pathologists (CAP) quality control (QC) is “the aggregate of processes and techniques so derived to detect, reduce and correct deficiencies in an analytic process.”103 It is an integral component of a laboratory’s quality assurance program, focusing mainly on procedural and technical aspects of the test under question. As it pertains to IHC, quality control standards should address and define each step of the “total test” (Table 1-4), including tissue procurement, fixation, processing, sectioning, staining, and, finally, the interpretation and reporting of the staining results. As part of a laboratory’s QC program, all steps of the test should be described separately, and parameters of each step must be established and monitored to ensure consistency of performance and reproducibility of results. Daily records of control results are maintained, and corrective actions are undertaken and documented when results are unacceptable. In practice, the surgical pathology histology laboratory typically falls short of the ideal standards for IHC, especially with regard to
tissue preparation. In this section, we will discuss QC issues as they pertain to the validation of antibodies and the use of controls. We will also address measures that may be adopted to minimize variability that derives from inconsistent tissue preparation, including fixation. Following the last edition of this book, the longawaited guidelines of the Clinical and Laboratory Standards Institute (CLSI) have been published.62 This document is the most comprehensive treatise of quality assurance and performance aspects of IHC in existence, and it devotes 139 pages to the design and implementation of IHC assays. Every laboratory that performs IHC should have a copy at hand for reference and guidance. The CLSI guidelines incorporate many of the tenets put forward previously in the “total test approach,”104 borrowed, in essence, from rigorous clinical laboratory practice. Sample preparation, validation of all reagents and procedures, and proper use of controls are given particular emphasis. This chapter presents the essentials; for further detail, the text Immunomicroscopy91 or the CLSI guidelines62 should be consulted directly.
Sample Preparation Sample preparation has emerged as a key issue in recent years,62-70 in large part because of use of IHC assays for HER2 to qualify patients for Herceptin therapy in clinical trials, during which unacceptably large variations were noted in tests performed by different laboratories. Since the 1990s, several conferences have been convened on the topic of standardization, with participation by representatives of the National Cancer Institute (NCI); CAP; National Institute of Standards and Technology (NIST); Centers for Medicare and Medicaid Services (CMS), which pays the bills in the United States; the Biological Stain Commission (BSC); and various
TABLE 1-4 Components of the “Total Test” in Immunohistochemistry Elements of the Testing Process
Quality Assurance Issues
Responsibility
Clinical question, test selection
Indications for immunohistochemistry Selection of stains
Surgical pathologist, sometimes the clinician
Specimen acquisition and management
Specimen collection, fixation, processing, and sectioning
Pathologist/technologist
Analytic issues
Qualifications of staff Intralaboratory and interlaboratory proficiency testing of procedures
Pathologist/technologist
Results validation and reporting
Criteria for positivity/negativity in relation to controls Content and organization of report Turnaround time
Pathologist/technologist
Interpretation, significance
Experience/qualifications of pathologist Proficiency testing of interpretational aspects Diagnostic and prognostic significance Appropriateness/correlation
Surgical pathologist, clinician, or both
Modified from Taylor CR: Report of the Immunohistochemistry Steering Committee of the Biological Stain Commission. Proposed format: Package insert for immunohistochemistry products. Biotech Histochem. 1992;67:110-117.
Quality Control and Standardization
experts in IHC. These meetings of the minds serve to focus attention on the need for improved reproducibility of IHC and particularly on the enormous and unknown variability in specimen handling and preparation (fixation). The NCI even issued requests for proposals in the area of sample preparation, but overall, few tangible practical results were seen. Searches for new, improved, and molecularly friendly fixatives similarly have not yielded satisfactory results, and the logistics of introducing such a fixative, even if universally accepted, are formidable. As already noted, no such fixative is likely to replace formalin in the next decade, at least to a significant degree. Thus FFPE tissues are what we have, and we must learn to work with them as best we can to extract the maximum information available in as consistent and reliable a manner as possible. One pragmatic approach has been the development of recommendations for all phases of the total test, including the definition of fixation, and more rigid and uniform adherence to common protocols (Box 1-1). The American Society of Clinical Oncologists (ASCO) in conjunction with CAP issued such guidelines with respect to HER2 testing.65,67,69,105 These guidelines recognized the key importance of fixation time, assigning both minimum and maximum limits—6 to 48 hours for excised specimens—although the authors of the guidelines succumbed to clinical pressures for speed in allowing a 1-hour fixation time for
Box 1-1 SAMPLE RECOMMENDATIONS FOR IMPROVED STANDARDIZATION OF IMMUNOHISTOCHEMISTRY BY THE AD HOC COMMITTEE68,70: ESTROGEN RECEPTOR IN BREAST CANCER 1. Fix all specimens promptly in 10% neutral buffered formalin. 2. Fix and process resections and core biopsies in an identical manner. 3. Only use 10% neutral phosphate-buffered formalin. 4. Note that fixation is 8 to 72 hours for both core biopsies and resections. 5. Use formalin, not alcohol, to fix cytology specimens for estrogen receptor assay. 6. Use conventional tissue processors to process breast tissue. 7. Ensure that the first formalin containers on the tissue processor are always newly replenished. 8. Ensure that tissue processor fluids do not exceed 37° C. 9. Be sure that paraffin in the tissue processor does not exceed 60° C. 10. Record and document fixation times in your report. 11. Use in vitro diagnostic kits that use clone, 6F11, 1D5, or SP1. 12. Include positive and negative controls with each batch run. 13. Use a threshold for a positive result of 1% positive cells. 14/15. Report semiquantitation and tabulate the intensity (0, 1+, 2+, 3+) and percent of positive cells.
15
core biopsies. This concession appears to be unwise and without scientific basis. Although penetration may have occurred in 1 hour, fixation, which is a “clock reaction,” is unlikely to be complete. Yaziji and Taylor67 also supported the idea that all practitioners should adopt these guidelines, stating that in performance of an IHC test one should “begin at the beginning, with the tissue.” The members of the Ad Hoc Committee on Immunohistochemistry Standardization published a separate, very detailed set of guidelines with the goal of improving the reproducibility of testing for estrogen receptor (ER),68 following principles previously set forth by the same group for standardization of IHC in general.66 These recommendations also began with the “total test” premise and are summarized in Table 1-4 and Box 1-1. The ultimate expression of this effort has been the publication of the CLSI guidelines, which include a detailed discussion of the various phases of sample preparation. Factors that have a potential impact upon the outcome of the IHC test are summarized in Table 1-5. In addition to the use of detailed guidelines, marked improvement in standardization may also be achieved by the use of reference standards, against which the impact, adverse or otherwise, of sample preparation (fixation) can be measured. This vital aspect is discussed in the following section.
Validation of Reagents, Protocols, Controls, and Staining Results In a simplistic sense, it would be best in terms of reproducibility if one manufacturer established an absolute monopoly in IHC, forcing uniformity of reagents and protocols upon us all. However, such uniformity is unlikely to happen. Therefore the use of proper control systems (reference standards; Table 1-6) is vital in ensuring quality and some degree of effective standardization. External proficiency testing programs have been important in driving the validation and standardization process. For example, as part of the UK NEQAS program,63 Rhodes and colleagues106,107 demonstrated improved reproducibility of IHC for ER and Her2 detection through stringent QC, an ongoing quality assurance program, and the use of standard reference materials based on a comparative study among laboratories in many different countries. The UK NEQAS program has now expanded on a worldwide basis, and NordiQC64 runs a similar highly effective program in Europe. In the United States, the ASCO/CAP65,67,69 and CLSI guidelines62 have made it a requirement that the HER2 detection test be done in a qualified accredited laboratory by proficiency standards. Data from these proficiency programs have also served to underline the extent of the problem with respect to standardization and to identify those areas in which improvement can be made. Table 1-7, from UK NEQAS published data, shows the enormous variation in reagents and protocols among different laboratories. Vani and colleagues108 performed an elegant study that compared the proficiency of different laboratories and included peptide slides as standards. They found
16
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
TABLE 1-5 Major Factors that Affect Outcome for Tissues Preanalytic Variables
Process
Effects Unknown for Individual Specific Analytes (Proteins)
Warm ischemia
Vessels clamped at surgery
Beginnings of anoxic damage are apparent.
Cold ischemia
Time before fixation; transport; saline or other transport media
Anoxic damage occurs; proteins, RNA, and DNA are degraded.
Grossing (in pathology lab)
Time to grossing of specimen; block size/ thickness, 2 to 3 mm maximum
Variable fixation is found within large specimen or block.
Fixation
Total time in fixative*; type of fixative, freshness, pH; penetration varies with block size and tissue type
Cross-linking of proteins leads to “loss of antigenicity.”
Processing; dehydration, clearing, impregnation in paraffin wax
Varying times in alcohols, xylol, and paraffin; temperature of wax
Parts of tissue block poorly fixed in formalin will be alcohol fixed.
Storage as formalin-fixed paraffin-embedded block
Time and conditions of block storage
Effects are unknown.
Cutting
Thickness of section; avoidance of tears; time from cutting to staining
Thick sections show apparent increase in intensity; tears may give artifacts, and loss of antigenicity occurs for some proteins.
Antigen (epitope) retrieval
Great variation in solution, time, and temperature
Recovery of detectable protein is variable.
Cytopathology specimens are often processed in a similar manner but may use different fixation. Blocks used for frozen sections may show diffusion of proteins and ischemic change before being fixed. *Total time in fixative extends from the time tissue is first placed in fixative and includes all time in the fixative on the tissue processor.
TABLE 1-6 Types and Purposes of Daily Quality Control Materials for Immunohistochemistry Type of Control
Antigen (Analyte)
Antibody (Reagent)
Purpose
Positive
Nonpatient tissue or cells containing antigen to be detected and quantified Known expected result, ideally low and moderate intensities Fixed-processed in same way as a patient sample Fixed-processed in manner shown to preserve antigen under analysis
Antibody reagent (of the kit) is constituted in the same way as that intended for the patient sample.
Control of all steps of the analysis Training user for appearance of positive reaction; comparison for semiquantitation of reaction Validates all steps of analysis, including fixation and processing Validates all steps of analysis except fixation or processing used by individual laboratory
Negative (specific)
Tissues or cells expected to be negative by antibody (of the kit) Processed in same way as patient sample May be portion of patient sample
Antibody reagent (of the kit) is constituted in the same way as that intended for the patient sample.
Detection of unintended antibody cross-reactivity to cells or cellular components
Negative (nonspecific)
Patient tissue with components that are the same as tissue to be studied Processed in same way as patient sample
Diluent (as used with antibody) is without antibody, or antibody (immunoglobulin) is not specific for antigen of interest in same diluents as used with kit antibody.
Detection of unintended background staining
Modified from Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
Quality Control and Standardization
17
TABLE 1-7 UK NEQAS Data Showing the Enormous Variation in Practice Among Participating Laboratories Run 96: 365 Participants
Run 97: 365 Participants
Markers: SMA, CK
Markers: SMA, CD34/CD31
Retrieval Heat: 297 labs; 76% acceptable results Enzymatic: 146 labs; 32% acceptable Retrieval reagents Mostly pH 6 or 9
Retrieval Heat: 336 labs; 83% acceptable results Enzymatic: 32 labs; 29% acceptable Retrieval reagents Mostly pH 6 or 9
Primary Antibodies SMA: 18 antibodies from 10 suppliers CK: 26 antibodies, 16 suppliers
Primary Antibodies SMA: 20 antibodies from 9 suppliers CD34/CD31: 25 antibodies, 11 suppliers
Detection Reagents 26 different detection reagents from 13 suppliers
Detection Reagents 23 different detection reagents from 11 suppliers
Autostainers 17 different instruments from 7 suppliers
Autostainers 17 different instruments from 7 suppliers
Chromogen+ Great majority used DAB from 19 suppliers
Chromogen+ Great majority used DAB from 11 suppliers
In these studies, UK NEQAS distributes paired slides and requests that the performing laboratory stain with designated antibodies, report the results, and return one slide for central review. CK, Cytokeratin; DAB, diaminobenzidine; SMA, smooth muscle actin.
that almost 100% of the “failures” resulted from errors in AR, antibody titration/protocol, or both. This result clearly identified where the focus must be for performing laboratory tests, and it showed the value of reference standards that can be compared across laboratories. Today, automated platforms increasingly dictate the choice of reagents and protocols, but control tissues are not provided, and at present cannot be provided, for reasons discussed below. Thus the selection and proper use of controls is the most important single step for a laboratory in achieving quality reproducible results for both IHC and in situ hybridization (ISH). A discussion of the principles of such controls follows. The use of reference standards for the QC of reagents in the clinical laboratory is well established. For example, serum assay results can be validated by large, standardized serum pools, such as those established by CAP’s Check Sample program. More recently, the development of reference-standard controls for IHC has been under consideration; however, this undertaking is not simple. Unlike serum samples, pathologic tissues cannot be pooled, and the supply of control tissue samples is not infinite. Moreover, morphologically similar tumors are not necessarily similar in terms of antigenicity. The use of tissue microarrays (TMAs) does not alleviate these problems, for these also contain only a limited amount of tissue, which will eventually become depleted. To overcome this problem, the development of “infinite” standard reference controls, composed of artificial tumors or human tumor cell lines, and the use of defined peptide deposits have been proposed.109,110 Internal reference standards would be of special value in assessing the effectiveness of sample preparation and fixation, giving an indication of the suitability of the tissue for IHC studies; we will address this new
area, following the discussion of reagent and protocol validation and again at the end of this chapter. Reagent and protocol controls are essential in the validation of reagents, the evaluation of performance of the IHC protocol, and the staining result.62,91 We will discuss these basic types of controls first (see Table 1-6). Validation studies are required for each test and antibody used by a laboratory, including each new lot of antibody. Such testing is also required for RTU pretested reagents, including those that are part of a kit; however, this is less extensive, because optimal dilutions and protocols have been established by the manufacturer on tissues available to the manufacturer. In this instance, the performing laboratory has only to show satisfactory performance on in-house tissues. With reagents purchased as concentrates, for which manufacturer data may be very limited, detailed validation studies are imperative at each step (see Tables 1-3 and 1-4 and Box 1-1), because the reagents may be of uncertain origin, composition, concentration, or specificity— or all of these.91,111 Performance parameters to be addressed by these studies include the sensitivity, specificity, precision, accuracy, and reproducibility of the results. It is recommended that these initial studies be carried out on known tissues or multitissue control blocks (TMAs) that contain both known-positive and known-negative normal and tumor tissues. The latter approach allows comparison of findings in many cells and tissues subjected to identical staining protocol on a single slide; it is also convenient and economical in time and use of tissue. The results obtained are reviewed and attest to the specificity and precision of the antibody under study. The specificity of antibody staining is shown by the expected absence of staining in certain cells, tissues, and tumors within the multitissue control blocks known not
18
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
to contain the antigen (protein) in question. In contrast, precision attests to the validity of the entire procedure and is shown by the presence of both positive and negative elements as expected on the same control slide. A sensitive test is one that detects a small amount of antigen; this is shown by positive results with tissue with known low expression. Accuracy is determined by the evaluation of nonspecific background staining; a negative reagent control can be used in place of the primary antibody. Finally, if there is no run-to-run variation in the results obtained, the test is considered reproducible. The BSC, in conjunction with the Food and Drug Administration (FDA), published a set of guidelines for reagent package inserts. These guidelines include recommendations to manufacturers for the testing and marketing of reagents as well as for the use and purpose of positive and negative controls.112 Because of the variability in tissue fixation, processing, and embedding— which are inherent aspects of the IHC test—it is impossible to establish a single, universal staining protocol. Proper controls ensure proper technique and specificity of the staining method used and are essential for correct interpretation of IHC results. A more detailed discussion is included in the book by Taylor and Cote, Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist,91 and in the CLSI guidelines.62 As a minimum both positive and negative controls should be used. A positive control (see Table 1-6) is one that is known to contain the antigen under question, ideally at a level of expression comparable to that being assayed in the test section; by default, a low or mediate expression level is preferred to high expression. These controls should be fixed and processed in a manner that is analogous, ideally identical, to the tissue being tested. A falsenegative test result can occur if the test tissue is overfixed, which results in diminished or absent antigenicity, especially if the control tissue is optimally fixed and the test was optimized to the control tissue that was fixed more ideally. For this reason, the manufacturers’ positive control slides cannot be used as a substitute for positive control slides made in-house, because they are not processed in the same manner as the tissue being tested. They merely validate reagent performance but cannot verify proper tissue fixation and processing. Identical processing would be optimal in routine surgical pathology, but usually it is only achievable in laboratory experiments, if it can be achieved at all. Later in this chapter, we will discuss the possibility of using internal reference standards to correct for variation in fixation. For positive controls it is most cost-effective to use surgical tissues that have been fixed and processed along with the regular workday’s surgical specimens. Tissue (containing cells) with a level of expression comparable to the test tissue (cells) is preferred. As noted earlier, optimizing the test by using normal tissue with a high level of expression may result in false-negative results on tumor tissue, in which expression may be low. The ideal positive control should therefore show a range of intensity of staining in order to detect subtle changes in primary antibody sensitivity. Surgical material is
preferable to autopsy tissue, because the latter may contain areas of autolysis that can affect staining results. For immunocytologic testing of body fluids and FNA material, cytospins can be prepared and stored unfixed, or cell blocks can be fixed and embedded, to be used as cytologic control material. The most important considerations in the selection of the appropriate control tissue are that it must contain the antigen under question and that it must be fixed and processed by using the same protocol as the test specimen. Negative controls (see Table 1-6) are used to confirm the specificity of the method used and to exclude the presence of nonspecific background staining, defined by staining the test tissue in the absence of the primary antibody, for which there are multiple causes (described later). Absorption controls are negative reagent controls produced by absorbing the primary antibody, polyclonal or monoclonal, with the highly purified antigen that was used to generate the antibody. The objective is to eliminate staining; however, the findings can be misleading because of impurities in the antigen or the presence of unsuspected cross-reactive “epitopes.”113-115 The usual negative controls include substitution of the primary antibody with antibody diluent (buffer plus bovine serum albumin carrier protein) or with nonimmune immunoglobulin, derived from the same species and used at the same concentration. The substitution of an irrelevant antibody can also be used as a negative control. In practice, when a panel of different antibodies is being tested on the same tissue, the results obtained from the different primary antibodies may be used as negative controls for each other. Finally, with regard to negative controls, if more than one protocol is used on a particular day—microwave AR pretreatment or protease-trypsin digestion, for example—separate negative control slides should be run for each in accordance with each protocol used. TMAs have particular utility when a new antibody is first assessed. Each TMA slide contains samples of tissues arranged in either a checkerboard or a “sausage” pattern. TMA slides typically require “maps” that designate the types of tissues present and their specific locations. Although particularly useful for validation studies of new reagents, these TMA slides may also be used for routine QC purposes; their disadvantage is a relatively high cost and an inability to control how different specimens that constitute the “array” were fixed and processed. Some of these disadvantages have been addressed by improvements in the preparation of TMAs, in particular the commercial availability of instruments that allow for the production of microarray blocks that consist of multiple fine-tissue cores (e.g., Beecham Instruments, Hackensack, NJ). The TMA method provides a means of incorporating 200 to 300 cores of internally processed tissues into a template. Because each core is small (generally 0.6 to 1.5 mm), a single initial biopsy tissue block can serve as a source for multiple core samples, while still preserving much of the original block for the archival files. The pros and cons of this technology are further discussed in Applied Immunohistochemistry and Molecular Morphology by Skacel, Mengel, and others.116,117
Tissue Fixation, Processing, and Antigen Retrieval Techniques
Internal controls are present when the tissue being tested contains the antigen under question in adjacent normal cells or tissue. The presence of positive “internal control staining” in the expected cells indicates appropriate immunoreactivity. For ubiquitous antigens, such as vimentin, positive internal control staining may be used as a positive control. Moreover, because of its ubiquity, staining for vimentin is also helpful as a reporter molecule, that is, to give a general idea as to the adequacy of fixation and processing of the tissue.118 The intensity of staining of vimentin with monoclonal antibody V9 provides a crude indication of overfixation and can also be used to monitor the recovery from formalin overfixation by AR. Taylor and Burns5 used internal controls for kappa and lambda in their first publication of the immunoperoxidase method applied to FFPE in 1974. INTERNAL REFERENCE STANDARDS FOR QUANTIFICATION AND THE EVALUATION OF SAMPLE PREPARATION
The human eye and the human mind have proved remarkably proficient in creating the art of surgical pathology and in sustaining it for more than 100 years as the gold standard for cancer diagnosis. Remarkable though this may be, some areas of significant limitation remain. One is the lack of reproducibility of a morphologic interpretation, not only among different observers but also for the same observer over time.3,91 The second is lack of consistency in counting events, such as numbers of positively stained cells, and especially in the judgment of degree of intensity of a stain reaction: Is it strongly, moderately, or weakly positive, or is it negative or weakly negative (whatever that might be)? In both aspects, computer-assisted image analysis may hold significant advantages. Previously hampered by cumbersome software and the limitations of digitizing, storing, and transmitting data, image analysis had a reputation for being time consuming and expensive, an embellishment at best and no substitute for the tutored eye. These limitations are quickly receding. Integrated hardware/software systems now permit scanning at ×40 to produce a whole-slide image (WSI) in less than 1 minute. The concurrent development of FDA-approved scoring algorithms (e.g., for HER2 and for ER and PR), together with sophisticated programs that can simultaneously analyze and compare multiple colored signals, both IHC and immunofluorescent,119-121 makes it certain that digital pathology will play an increasing role in IHC and quantification (see Chapter 23). It therefore becomes possible to perform an IHC stain for a target protein (antigen) in conjunction with a double stain for an internal control protein (reference analyte) and then to compare the intensity of one with the other by image analysis for relative quantification, or even for absolute measurement by weight, once the staining protocol has been calibrated.120,121 Calibration is possible by experimental determination of the loss of antigenicity of the reference analyte, patented as a Quantifiable Internal Reference Standard (QIRS),120 under different conditions of fixation and AR. Following
19
calibration, the observed intensity of staining of the reference analyte can be used to make corrections for observed loss of antigenicity as a result of sample preparation. This approach provides a potential method for correcting for variable fixation or processing; it also provides a method of improving reproducibility of IHC results that does not require the standardization of fixation per se—a task already described as difficult or impossible! Further study of QIRS is being pursued, to follow the advice of Sherlock Holmes: “Whenever one has excluded the impossible, whatever remains, however improbable, must be the truth.”122 When the objective measure of surgical pathology was morphologic quality, this degree of rigor was not necessary. However, the increasing numbers of critical prognostic/predictive markers being introduced into anatomic pathology adds to the pressures for improved results and strict quantification. In principle, the IHC test is a close relative of the ELISA test, but it is performed on a tissue section as opposed to in a test tube.120,121 If similar rigor to that used with ELISA is applied to the performance of an IHC test, similar accuracy and reproducibility may be achieved. For the results of any prognostic/predictive test to be clinically meaningful, rigorous QC measures must be applied and followed. If for practical reasons we cannot “begin at the beginning,” with proper specimen acquisition and handling protocols, then we can “begin at the end,” by instituting a system of defined internal reference standards.120,121 This approach is further discussed later in this chapter with the topic of quantification. From this discussion, it is evident that the understanding and application of appropriate positive and negative controls is one of the most important yet often misunderstood aspects of IHC. The proper use of these controls is essential for correct interpretation of IHC results.
Tissue Fixation, Processing, and Antigen Retrieval Techniques The following section reflects current practice, with formalin as the common fixative. Recommendations for increased documentation of total time in fixative have already been emphasized. Tissue preparation consists of fixation, subsequent dehydration, and embedding in paraffin wax to provide a rigid matrix for sectioning. Tissues to be embedded in paraffin wax are first fixed to optimize preservation, a process that profoundly affects the morphologic and immunohistologic results. The ideal fixative for IHC studies should not only be readily available but should also be in widespread use to maximize the range and number of samples available for IHC studies across different institutions. The fixative should preserve antigenic integrity and should limit extraction, diffusion, or displacement of antigen during subsequent processing. Also, it should show good preservation of morphologic details after embedding in a support medium (e.g., paraffin).
20
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Common fixatives used in histopathology are divided into two groups: coagulant fixatives, such as ethanol, and cross-linking fixatives, such as formaldehyde. Both types of fixative can cause changes in the steric configuration of proteins, which may mask antigenic sites (epitopes), and they adversely affect binding with antibody. It is well recognized that cross-linking fixatives alter the IHC results for a significant number of antigens, whereas coagulant fixatives, especially ethanol, have been reported to produce fewer changes, although some controversy remains regarding this.40,123-126 In most surgical pathology laboratories, the fixative used is 10% neutral buffered formalin (NBF), a cross-linking fixative. Subsequent processing usually includes a period in 100% ethanol, thus tissues are effectively “double fixed” in both formalin and ethanol: if formalin fixation is inadequate, tissues will be alcohol fixed in part, with resultant “in section” variation in IHC staining (see illustrations under “Troubleshooting”). For tissues fixed in formalin, the intensity of staining is known to be fixation time– dependent for many antigens.40,123-126 A long history of using formalin as a standard tissue fixative has revealed the following advantages, which suggest it will neither be replaced easily nor soon: 1. Preservation of morphology is good, even after prolonged fixation. In this case, “good” is a somewhat subjective term that encompasses the various artifactual changes that result in the morphologic features that “please” the pathologist, based on his or her previous experience and the manner of fixation and processing to which the pathologist has become accustomed. 2. Formalin is economical and is much cheaper than most alternatives. 3. Formalin fixation sterilizes tissue specimens in a more reliable way than precipitating fixatives, particularly for viruses. 4. Carbohydrate antigens are well preserved.127 5. Cross-linking of protein in situ avoids leaching out of proteins that may diffuse in water or alcohol. Many low-molecular-weight antigens (peptides) are extracted by non–cross-linking fixatives, such as alcohol- or methanol-based solutions, but they are well preserved in tissue by formalin.29 Today, experience shows that formalin may be regarded as a satisfactory fixative for both morphology and IHC, provided that an effective AR technique is available to recover those antigens that are diminished or modified.
Antigen Retrieval A simple heat-induced AR technique is now widely applied in pathology. The current status of AR methods has been reviewed by the authors elsewhere,39,40,57,59 to which reference should be made to the detailed discussion of the effect of different fixatives, heating methods, and retrieval solution. A brief summary of the salient points follows for routine use. Successful application of the AR technique for routine IHC staining of formalin-fixed tissues in diagnostic pathology has rendered the search for alternative
fixatives to replace formalin less urgent. In 1997, Prento and Lyon128 compared the performance of six commercial fixatives offered as formalin substitutes and concluded that the best IHC staining was obtained by combining formalin fixation with AR technique. Williams and coworkers129 investigated the effect of tissue preparation on IHC staining by using tonsil tissues subjected to variations in fixation, processing, section preparation, and storage. They reported that 10% neutral buffered formalin, 10% zinc formalin, and 10% formal saline gave the most consistent results overall and showed excellent antigen preservation. In contrast, 10% formal acetic acid, B5, and Bouin’s fixative all showed poor antigen preservation even after AR treatment. Reduced effectiveness of AR when used with other fixatives has been documented by others.130-132 Although storage of cut tissue sections was not an issue in the study of Williams and associates,129 others have reported that decreased intensity of staining may occur for some antigens in slides stored for protracted periods.133-139 It is our experience that storage-induced decreases in IHC staining are relatively uncommon, and most of these adverse effects also can be recovered by AR treatment.18,91 The AR technique not only has utility for enhancement of IHC staining on archival tissue sections but also may contribute to standardization of routine IHC, conditional upon the AR method itself being optimized and standardized.18,39,40,91,140 That AR methods are not optimized and standardized is evident in the proficiency testing programs of UK NEQAS (see Table 1-7), thereby undermining the otherwise positive contribution of AR to overall improvement in IHC staining. Recently, AR has also been adopted successfully in formalin-fixed frozen cell and/or tissue sections.141,142 A key element in the appropriate use and standardization of the AR technique for IHC is understanding major factors that influence the effectiveness of AR, as described in the following paragraphs (Fig. 1-17). HEATING CONDITIONS
An optimal result for AR-IHC is correlated with the mathematical product of the heating temperature multiplied by the duration of the AR heating treatment: T (temperature of heating procedure) × t (heating time). As noted previously, the AR-IHC heating method is based on biochemical studies by Fraenkel-Conrat and coworkers,32-34 who documented that the chemical reactions that occur between protein and formalin may be reversed, at least in part, by high-temperature heating or strong alkaline hydrolysis. Over the past two decades, we have demonstrated that a variety of heating methods and various AR “solutions” may give closely equivalent results, but optimal conditions must be defined for each antibody in the context of the overall total test protocol.143 Subsequently, several publications have reported similar results,143-146 although the chemical reactions that occur during the formalin fixation process remain obscure. Mason and O’Leary144 demonstrated that the process of cross-linking does not result in discernible alteration of protein secondary structure. They also noted that significant denaturation of unfixed purified
Tissue Fixation, Processing, and Antigen Retrieval Techniques
A
B
C
D
E
F
G
H
I
J
21
Figure 1-17 Comparisons of the intensity of antigen retrieval (AR) immunostaining in routinely formalin-fixed, paraffin-embedded tissue sections with a monoclonal antibody (MAb) to estrogen receptor (ER) in breast tissue (A to E) and MAb MT1 in lymph node (F to J). Sodium diethylbarbiturate-HCl (SDH) buffer was used as the AR solution for both antibodies. A to E, The pH values of the AR solution were 2, 3, 4, 6, and 8, which correspond to staining intensity of ++++, +++, +, ++, and +++, respectively, for ER with a type B pattern. F to J, Shown here is a type C pattern with SDH as the AR solution with a pH of 2, 3, 4, 6, and 8 and intensity of staining −, +, ++, +++, and ++++, respectively, for MT1. Some nuclei showed very weak false nuclear staining (F). Diaminobenzidine was the chromogen, and hematoxylin was the counterstain (original magnification ×100; bar = 20 mm). From Shi S-R, Imam SA, Young L, et al: Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 1995;43:193-201.
proteins occurred at temperature ranges of 70° to 90° C, whereas similar temperatures had virtually no adverse effect on formalin-fixed proteins (i.e., formalin-fixed proteins are more heat stable). Subsequently, this same group studied the mechanism of AR using a purified protein, RNase A, and demonstrated that heat treatment may reverse formalin-induced cross-links for restoration of immunoreactivity.147,148 Support for this conclusion followed from Sompuram and coworkers.149,150 Thus the AR heating technique appears to take advantage of the fact that the cross-linkage of protein produced by formalin fixation may protect the primary and secondary structure of formalin-modified protein from denaturation during the heating phase, while allowing reduction of cross-linkages at the surface of the molecule, thereby restoring antigenicity. Other theories, including that of protein denaturation,143 do exist; therefore at the present time, the mechanism must be considered unresolved. In general, the heating conditions appear to be the most important factor in the effectiveness of AR.38-42,143,151,152 Evidence shows that significant enhancement of IHC staining can be achieved by using high-temperature heating in pure distilled water, and higher temperatures in general yield superior results. Equivalent performance of AR-IHC can be obtained by using different buffers as AR solutions, if the pH value
of AR solutions is monitored in a comparable manner, thereby demonstrating that specific individual chemical constituents may not be critical factors in yielding a satisfactory result. Our early experience that even prolonged exposure of paraffin sections in citrate buffer solution, or indeed any buffer, without heating gave reduced or no noticeable AR effect has subsequently been confirmed by numerous studies.53,55,143,153
Chemical Composition and pH of the Antigen Retrieval Solution The pH value of the AR solution is important for some antigens. From a comparative study,50,51,143 we concluded that antigens fell into three broad categories with respect to the importance of pH on AR: 1. Most antigens showed similar levels of retrieval using AR solutions with pH values that ranged from 1.0 to 10.0. 2. Certain other antigens, especially nuclear antigens (e.g., MIB1, ER), showed a dramatic decrease in the intensity of the AR-IHC at middle-range pH values but showed optimal results at low pH. 3. A small group of antigens (MT1, HMB-45) showed negative or very weak focally positive immunostaining with a low pH (1.0 to 2.0) but showed excellent results in the high-pH range.
22
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Evers and Uylings152 also found that AR-IHC is both pH and temperature dependent. They concluded that it is not important what kind of solution is used as long as the pH is at an appropriate level. The chemical composition and molarity of the AR solution are cofactors that should be explored when satisfactory results cannot be obtained with the solutions usually used.40,41,56-59,154,155 Namimatsu and colleagues155 reported a novel AR solution of 0.05% citraconic anhydride solution at pH 7.4, heating at 98° C for 45 minutes, which they advocated as a universal AR method. Using their approach, we found that more than 90% of antibodies, but not all, showed a stronger or equivalent signal, so the search for the “universal” AR continues. Commercial AR or epitope retrieval solutions are widely available, some of a proprietary nature, with compositions held as trade secrets. Many of these produce good results when used according to the manufacturers’ specifications, with the disadvantage of the user not knowing the detailed composition of the solution. In conclusion, major factors that influence the results of AR-IHC staining include heating temperature and heating time (heating condition T × t) and the pH value of the AR solution. TEST-BATTERY APPROACH FOR ANTIGEN RETRIEVAL TECHNIQUE
A test battery may be defined as a preliminary test of AR technique that examines the two major factors, heating condition (T × t) and pH value, and it is performed to establish an optimal protocol for the antigen being tested.143,156 Typically, three levels of heating conditions and pH values—low, moderate, and high—may be applied to screen for a potential optimal protocol of AR-IHC for a particular antigen of interest, as indicated in Table 1-8. We have demonstrated that different heating methods— including microwave, microwave and pressure cooker, steam, and autoclave heating methods39,40—can be evaluated in a similar fashion. The test battery serves as a rapid screening approach to identify an optimal protocol for each antibody-antigen to be tested. The goal is to establish the maximal retrieval level for formalin-masked antigens after undefined fixation times to standardize immunostaining results.48-51 The use of “on-platform” AR methods—that is, those methods included in the automated stainer process—with commercial “retrieval solutions” should always be used in accordance with the manufacturer’s protocol. When results are unsatisfactory, as with setting up a new antibody, a test battery approach may be adopted. In this respect, automated platforms allow for less flexibility in adjustment of the AR protocol; and in some circumstances, to achieve satisfactory results, it may be necessary to resort to retrieval “off-platform,” with different heating methods and different solutions. Validation of such new protocols is of course required. Newly available antibodies or highly sensitive detection systems with improved staining characteristics may in the future obviate the need for special AR methods, and laboratories should pay particular attention to the claims of
TABLE 1-8 Test Battery Suggested for Screening an Optimal Antigen Retrieval Protocol TRIS-HCl Buffer
pH 1-2
pH 7-8
pH 10-11
Super high (120° C)*
Slide 1
Slide 4
Slide 7
High (100° C for 10 minutes)
Slide 2
Slide 5
Slide 8
Mid-high (90° C for 10 minutes)†
Slide 3
Slide 6
Slide 9
Modified from Shi S-R, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem. 1997;45:327-343. One more slide may be used for control without antigen retrieval treatment. The citrate buffer (pH 6.0) may be used to replace TRIS-HCl buffer (pH 7 to 8), because the results are the same. *The super-high temperature at 120° C may be obtained by either autoclaving or microwave heating for a longer time. † The mid-high 90° C temperature may be obtained by using either a water bath or a microwave oven monitored with a thermometer. TRIS-HCl, Tris-(hydroxymethyl)-aminomethane hydrochloride.
manufacturers in this regard, again always validating such claims in-house.
Techniques, Protocols, and Troubleshooting The following descriptions and protocols are focused on IHC techniques for archival paraffin-embedded tissue sections. The basic principles and protocols for freshfrozen tissue sections are the same as those for paraffin sections, except that the AR and dewaxing procedures are not required for frozen tissue sections. Titrations (dilutions) may also differ and must be separately optimized. Because the success of the IHC staining method depends on the correct application of both histologic and immunologic techniques, it is recommended that the user be familiar with the literature concerning the antigen under investigation before performing IHC staining. In particular, it is important to know the cellular localization of the antigen and the specificity of the primary antibody. Also important are the results of previous IHC staining tests, from the literature and especially from the experience of the performing laboratory with respect to any adverse influence on the antigen from tissue-fixation processing, and the value of any pretreatment procedure such as heat-induced AR. In addition, detailed information regarding the reagents, particularly the primary antibody and detection system—such as the manufacturer, clone number of monoclonal antibody, and recommended concentration—is helpful in achieving a successful result. It is advisable to read the package insert provided by the manufacturer as well as key related literature. This discussion continues to address manual methods, recognizing that the adoption of automated
Techniques, Protocols, and Troubleshooting
platforms requires that the protocols and troubleshooting procedures advocated by the manufacturers must take precedence. As noted previously, any deviation by the performing laboratory from the recommendations of the manufacturer in reagents or protocols places the burden of validation upon the performing laboratory.
Antigen Retrieval by the Microwave Heating Method Microwave ovens have been advocated by some as an alternative to regular fixation or for use in rapid fixation, and they are also commonly available in labs as a convenient and widely used method of applying heating for AR.35,157-159 A brief protocol follows: 1. Deparaffinized slides are placed in plastic Coplin jars containing AR solution; it is recommended that the same number of slides be used every time, using “blanks” if necessary to ensure consistent heating. 2. Jars are covered with loose-fitting screw caps and are heated in the microwave oven for 10 minutes. The 10-minute heating time is divided into two 5-minute cycles with an interval of 1 minute between cycles to check the fluid level in the jars. If necessary, more AR solution is added after the first 5 minutes to avoid drying out the tissue sections. It is recommended that the heating time be standardized by beginning to count the time only after the solution has reached boiling in order to avoid discrepancies among laboratories when using various microwave ovens. 3. After completion of the heating phase, the Coplin jars are removed from the oven and are allowed to cool for 15 minutes. 4. Slides are rinsed twice in distilled water and in phosphate-buffered saline (PBS) for 5 minutes and are then ready for IHC staining. Various heating methods include conventional heating in a hot water bath, steamer, pressure cooker, and autoclave; these may be used, along with different AR solutions, which contributes to lack of uniformity of results across laboratories (see Table 1-7). As noted, external QC programs have shown that one of the largest causes of poor IHC results is improper AR (see sections on fixation and AR). BLOCKING NONSPECIFIC BINDING
Quenching endogenous peroxidase by an H2O2methanol solution is performed immediately after the dewaxing procedure. Blocking of endogenous biotin may be performed before the biotin-conjugated link is established by using the avidin-biotin blocking reagent or by using skim milk as an alternative blocking reagent (incubate for 10 minutes).131 Normal serum taken from the same species as the secondary (link) antibody should be used to block nonspecific binding sites, as discussed previously. Commercial reagents are available for both these purposes and should be used according to specifications.
23
WASHING STEPS
Thorough washing is critical for each step, except for the blocking step of normal serum. PBS (0.01 mol at pH 7.4) is widely used, with the washing procedure carried out in a jar containing PBS by immersing slides for two 5-minute periods. Alternatively, PBS may be flooded onto the slides in a humidity chamber for 10 minutes with one change. With automation of IHC staining technique, the washing procedure is also automated, and multiple washes are used. Some manufacturers provide proprietary diluents and washing solutions; if these are not purchased, the method must be carefully revalidated for performance. INCUBATION OF PRIMARY ANTIBODY
The incubation time depends on the sensitivity and concentration of the primary antibody used and the quality of the tissue section. Concentration of the primary antibody is based on a titration study as described previously, with reference to the manufacturer’s instructions. In general, frozen tissue sections require shorter incubation times than do archival paraffin tissue sections. With multistage methods, successive incubation periods and washings result in a lengthy overall procedure. Some attempt has been made to shorten the procedure by performing incubations at 37° C in a warmed humidity chamber. Some automated stainers also accelerate the process by operating at 37° or 42° C. Microwave acceleration of the IHC staining technique has also been described for rapid IHC staining,158,159 but generally it is not convenient. Incubation of slides in a humidity chamber is advisable whatever the temperature. In practice, if a slide treated with antibody is permitted to dry, excessive nonspecific background staining invariably occurs as a result of an effective increase in antibody concentration. Humidity chambers that contain level slide racks may be purchased or can be made rather easily from glass rods and Plexiglas glued together with waterresistant bonds. Staining racks should be level to avoid drainage of antibody off the section to other areas of the slide. Overnight or extended incubation may permit the use of a primary antibody at very high dilutions, giving excellent results with reduced nonspecific background staining (i.e., select an antibody that yields satisfactory results when incubated for 30 minutes, dilute it tenfold or even a hundredfold, and then incubate it for 12 or more hours). This approach conserves precious antibody and reduces background staining (the antibody is present at a much lower concentration, which favors binding by high-affinity immunologic reactions). With this method, only the primary antibody is given prolonged incubation; other reagents are used normally. Overnight incubation of primary antibody can fit well into the working schedule, initiating staining requests in the late afternoon, incubating the primary antibody overnight, and completing the labeling steps early the next morning for examination by the pathologist.
24
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
INCUBATION OF DETECTION REAGENTS
As noted previously, titration is necessary for optimum concentrations. A good place to begin is by following the manufacturer’s instructions for concentration of each reagent and the exact protocol for the detection system. Incubation is carried out carefully, usually at room temperature for 30 minutes for each step (link and label). Dropping reagents on each slide to cover the tissue section completely is a simple but critical procedure. Caution must be taken to confirm that the whole tissue section is immersed in the reagent, including the margins, and that no air bubbles exist, particularly when using automation (see the figures in the section below on troubleshooting). In the RTU approach (see Table 1-3), both the primary antibody and the labeling reagents usually are included in prediluted form.
TABLE 1-9 Commonly Used Chromogens Color
Solubility in Alcohol
DAB
Brown
Insoluble
DAB with enhancement
Black
Insoluble
Procedure Peroxidase
†
a
Red
Soluble
4-CN
Blue-black
Soluble
Hanker-Yates reagent
Blue
Insoluble
α-Naphthol pyronin
Red
Soluble
3,3′5,5′-tetramethylbenzidine
Blue
Insoluble
Barjoran purple
Purple
Insoluble
Vina green
Green
Insoluble
SUBSTRATE AND CHROMOGEN
Deep space black
Black
Insoluble
Several different chromogens, color-producing substrate systems, are available (Table 1-9). With HRP, diaminobenzidine (DAB) may be preferred, because the brown reaction product is alcohol fast and thus is suitable for use with a wide range of counterstains and mounting media. Note that if an alcohol-soluble chromogen is chosen (see Table 1-9), a “progressive” nonalcoholic hematoxylin should be used (e.g., Mayer’s, not Harris’s) to avoid removal of the colored product, which is alcohol soluble. At high concentrations of antigenantibody enzyme, amino-ethyl carbazole (AEC) may produce a brown-yellow color. (AEC has two reactive sites: when one is converted, it turns red; if both react with peroxidase, a green-brown product results.) It may be helpful to maintain the acetic acid buffer at pH 4.8 to minimize this effect. For alcohol-soluble chromogens, the sections must be mounted in an aqueous medium (e.g., 80% glycerol). Aquamount contains small amounts of organic solvent and may cause slow diffusion or loss of stain. Dehydration through alcohol must be avoided. Glycerol-mounted preparations may be made permanent by sealing the edges of the coverslip with nail varnish. A range of substrates is also available for alkaline phosphatase; fast red, fast blue, and several commercial variants give excellent contrast and good morphology. Some substrates contribute to the sensitivity of the alkaline phosphatase systems by continuing to convert at the enzyme site, resulting in a granular accumulation of the colored product. If carried to excess, this granularity may obscure morphologic definition. DAB is also valuable for the electron microscopist, because it is electron dense. For light microscopy, some investigators advocate osmication of the DAB reaction product, which gives a more intense color. We have not found this necessary for light microscopy; indeed, we feel it may be a disadvantage, because background staining is also enhanced, and the end result may be diminished contrast despite the more intense staining. A similar effect may be achieved by post treatment with nickel sulfate or cobalt chloride, which produces excellent contrast.160,161 If DAB is used, solutions should be
Alkaline Phosphatase Fast blue BBN
Blue
Soluble
Fast red TR
Red
Soluble
New fuchsin
Red
Insoluble
BCIP-NBT
Blue
Insoluble
Tetrazolium
Blue
Insoluble
TNBT
Black
Insoluble
Black
Insoluble
AEC
Glucose Oxidase
Immunogold With silver enhancement
Modified from Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier. *Other proprietary “chromogens” are for peroxidase and for alkaline phosphatase. These may be found in catalogs from several companies and usually have been formulated to be most effective when used with the same company’s reagents and automated stainers. Use outside of these proprietary systems requires careful validation of effectiveness. † AEC has two reaction sites: In the presence of enzyme excess, both react, with the color passing from red to green-brown. AEC, 3-Amino-9-ethyl carbazole; 4-CN, 4-chloro-1-naphthol; BCIP-NBT, 5-bromo-4-chloro-3-indolyl-phosphate nitro blue tetrazolium; DAB, Diaminobenzidine; TNBT, tetranitroblue tetrazolium.
prepared under a hood by a masked, gloved technician. Unused solution should be disposed of with an excess of water. At the working dilution used, the danger is considered minimal; it is the powdered form that evokes concern. Preweighed aliquots in sealed tubes are available from some manufacturers but are expensive. Ready-to-use liquid DAB is now provided commercially. It has the advantages of conveniencea and safety with less potential for environmental pollution. COUNTERSTAINING AND MOUNTING SLIDES
The final step in the process is counterstaining and mounting slides. Hematoxylin is used as the nuclear
Techniques, Protocols, and Troubleshooting
counterstain for most routine IHC staining. In the case of nuclear antigen immunolocalization, one must take caution to avoid overdevelopment of the hematoxylin stain. A light hematoxylin stain is critical to allow any nuclear localization of chromogen to be discerned. In our experience, the time of exposure to hematoxylin depends in part on how fresh the dye solution is: a freshly prepared solution of hematoxylin requires a much shorter time than does exposure in an old solution. It may be necessary to monitor the development of the stain by microscopy in order to determine the optimal time of counterstaining. For alcohol-soluble stains (e.g., AEC or fast-red variants), an aqueous mounting medium is used. Care should be taken to avoid trapping any air bubbles between the cover slip and the tissue section. For alcohol-insoluble stains such as DAB or new fuchsin, a permanent mounting medium, such as Permount, may be used. The tissue section is first dehydrated by immersing the slide in graded alcohols, 90% and 100%, twice for each, followed by clearing in xylene, twice for 3 minutes each. Note that xylene and Permount should be used in a fume hood.
Double or Multiplex Immunoenzymatic Techniques The identification of two or more antigens in FFPE tissues is usually accomplished by immunoperoxidase staining of adjacent serial sections (Fig. 1-18). Although this approach suffices for general use, on occasion it may be difficult to identify with certainty the pattern of staining of a particular cell population in adjacent sections, especially if the cells under study are small or are scattered among other cells. Double multiplex immunoenzymatic techniques (Figs. 1-19 through 1-22) permit the demonstration of two or more antigens concurrently within a single section. As described earlier with reference to alkaline phosphatase methods, new polymer-based methods and polyvalent detection systems have greatly simplified the
A
25
performance of concurrent double or multiplex staining, both by manual method and on automated platforms. The sequential method is now largely obsolete and of academic interest only. The first sequence of staining for one antigen is completed with the development of the peroxidase reaction by using DAB as the substrate. Staining for the second antigen is then carried out with a primary antibody of different specificity and a second, different substrate system for the peroxidase enzyme (for example, 4-chloro-1-naphthol); this produces a contrasting blue reaction product. The relative merits of elution of the first sequence of antibodies (after reaction with DAB) before titrating the second sequence of antibodies have been much debated. However, Sternberger and Joseph19 were able to demonstrate two antigens without elution of the first set of reagents, despite the fact that both primary antibodies were of the same species, and the same labeling reagents were used for both. The success of this system may be due to the fact that the polymerized product of DAB oxidation, used for the first antigen, blocks the catalytic site of peroxidase while also obscuring the antigenic reactivity of the first sequence of antibodies. Lan and colleagues162 developed a simple, reliable, and sensitive method for multiple IHC staining by adding a microwave heating procedure (10 minutes) between each sequence of IHC staining for elution of the previous sequence of IHC staining reagents. The possibility of cross-reaction of the second sequence of antibodies with the first sequence may be eliminated by the use of a double immunoenzymatic method in which two specific primary antibodies, produced in two different species, are used in combination with separate species-specific secondary antibodies coupled with different enzymes (e.g., peroxidase and glucose oxidase or peroxidase and alkaline phosphatase). This approach has been greatly enhanced by the availability of rabbit monoclonal antibodies used in conjunction with mouse monoclonals. It has the
B
Figure 1-18 B5-fixed section of bone marrow aspirate depicts moderate numbers of plasma cells, raising the question as to whether these are reactive or monoclonal. Many of the plasma cells reacted strongly with anti-kappa antibody (A). Anti-lambda reacted only with one or two cells (B), suggestive of a monoclonal (kappa) plasma cell population. Serologic studies should be performed to further evaluate the clinical condition. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
26
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
A
B
Figure 1-19 Double staining. A, Double staining for kappa (brown) and lambda (blue) in paraffin section of a reactive lymph node by using sequential horseradish peroxidase (brown) and alkaline phosphatase (blue) methods. B, Double staining for CD20 (red, alkaline phosphatase– fast red, micropolymer-based system), and CD3 (brown, peroxidase-diaminobenzidine) with mouse and rabbit monoclonal antibodies. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
advantage of expediency in that some of the reagents, such as the primary antibodies, can be added simultaneously. This advantage is particularly exploited in new polymer-based methods, in which primary antibodies and secondary labeled reagents may be added as cocktails. Considerable ingenuity has been manifested over the years in adopting and adapting these techniques. For example, more than 20 years ago, it was possible to demonstrate six different antigens on the same section.163 Combinations of immunogold-silver staining (black) with immunoperoxidase-AEC (red-brown) or immunoalkaline phosphatase fast red or fast blue produce excellent double or triple stains.164 A combination of immunoperoxidase-AEC (red) with immunoalkaline phosphatase fast blue provides excellent contrasting colors, and with the advent of both mouse and
A
rabbit monoclonals, plus numerous new chromogens, reliable triple and quadruple stains are now available commercially. In first establishing these methods in a laboratory, it is important to carefully control and check the development of the second (and third) colors by microscopy and to use detection systems of comparable sensitivity to obtain the best contrast of multiple colors in the same section. In the beginning it is also wise to check the number and distribution of cells stained positively in each “multiple stain step” against a separate adjacent section separately stained to detect any cross-reactivity or masking of reaction product by multiple colors. In practice, some double stains and most triple stains require computer-assisted image analysis methods for accurate interpretation of the intensity and localization of the double stain. This problem is particularly evident
B
Figure 1-20 Double staining for BCL6 (red) and BCL2 (brown) in paraffin section of a reactive follicle of a lymph node using mouse and rabbit monoclonals and polymer-based detection systems. A, Reactive center; the BCL6-positive follicular center cells (red nuclei) do not show membrane staining for BCL2 (brown rings). Those cells that are BCL2 positive are infiltrating T cells. B, Contrast with a neoplastic follicle in a case of follicular center cell (FCC) lymphoma, in which the FCC cells (BCL6-positive red nuclei) clearly also show membrane staining for BCL2 (brown). This double stain may be useful in separating reactive follicles from cases of B-cell FCC lymphoma. From Taylor CR: IHC and the WHO classification of lymphomas: cost-effective immunohistochemistry using a deductive reasoning “decision tree” approach. Appl Immunohistochem Mol Morphol. 2009;17:366-374.
Techniques, Protocols, and Troubleshooting
A
27
B
Figure 1-21 Double staining for BCL6 and MUM1, both of which give nuclear staining; the exact proportion of nuclei staining with either or both is not clear to the naked eye. A, In practice, identification of nuclei in which there is colocalization of differing amounts of the two products can only be distinguished with image-analysis methods such as spectral separation, in which the cells showing staining for both BCL6 and MUM1 show yellow (B); BCL6 alone is red; and MUM1 alone is brown. The hematoxylin counterstain has been “removed” in the spectral separation process (Nuance image analysis software, Perkin Elmer, Waltham, MA).
when antigens are colocalized and therefore colors overlay one another such that they are impossible to distinguish by the naked eye. In this respect, the use of digital images and color separation, such as by spectral image analysis, is virtually essential (see Fig. 1-21). The growing use of digital image analysis and WSIs renders double stains both practical and useful.119,165 The recently formed Digital Pathology Association
provides an excellent resource for those who seek further information; it may be found online at www.digitalpathologyassociation.org. The growing availability of image analysis systems has encouraged manufacturers to now offer a range of double or triple stains, often as RTU reagents for specific applications. Today these polymer- or micropolymerbased double and multiplex stains are widely available for automated platforms. In our experience, many of these reagents perform superbly; however, it is important to note that each part of the double or triple stain must itself be separately validated, and proper controls must be included that represent the first-choice approach for the performance of double or multiplex stains.
Automation
Figure 1-22 Immunohistochemistry (IHC) can be combined within situ hybridization (ISH) by using a variety of these multiplex staining methods. IHC and ISH “triple” stain shows high-molecular-weight (HMW) keratin 34βE12 (IHC, peroxidase-diaminobenzidine, brown), HMW keratin 35βH11 (sequential IHC, peroxidase–amino-ethyl carbazole, red), and ISH for human papilloma virus (HPV cocktail, alkaline phosphatase–Fast Blue BBN, blue). Paraffin section. From Taylor CR, Cote RJ: Immunomicroscopy: a diagnostic tool for the surgical pathologist, ed 3. Philadelphia, 2006, Elsevier.
Because of the great variability in specimen fixation and processing among laboratories, it has not been possible to establish a universal standard protocol for IHC. Instead, the goal has become the standardization of protocols within a single laboratory and across laboratories. As discussed earlier (the “total test”104), this goal can be approached by rigorously applying proper controls and strictly adhering to all aspects of the “total test.” Such in-house standardization serves to ensure run-to-run reproducibility of accurate results, and the newly recommended guidelines we have discussed are designed for this purpose. Adoption of automated platforms, which are designed in conformation with applicable guidelines, also leads to increased reproducibility across laboratories. Although many different autostainers are available from many
28
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
different manufacturers (see Table 1-7), a situation that at first glance would seem to encourage yet greater diversity, in practice the use of automated platforms has led to an increased focus on standardization and a general increase in QC across laboratories. The following discussion is not intended to champion the use of any particular instrument but rather to provide an understanding of the principles to assist a laboratory in selecting the best platform for their particular environment. Automated immunostaining devices started to appear in the early 1980s. Through pilot studies, improvement in software design, and innovations of hardware, this technology continues to evolve and today is in use in many IHC laboratories worldwide (see Table 1-7). Increasingly the systems are being integrated with other automated processes such as sample preparation, AR, staining, mounting, and automated cellular imaging systems. With regard to the staining procedure itself, most of the steps in manual staining methods are technician dependent, and focused care by a skilled technician is required during each step to ensure the quality of the results obtained. There are more than 20 separate steps; these include preparing slides and reagents, blocking steps, AR, applying reagents and antibodies, monitoring incubation times, washing and wiping slides, and so on. Moreover, many of these steps are tedious and repetitive. Because of the large number of operator-dependent steps, the likelihood of technical error is increased. For example, a step may be accidentally omitted, extended, or performed in the incorrect sequence. In addition, reagent variables must be considered that include the proper selection and use of antibodies, with appropriate avidities at the optimal dilutions, as well as the selection of different chromogens, buffers, and enzymes. All these variables must be considered when using a manual staining method. With the advent of automation, manufacturers have addressed many of these issues and have standardized procedures, and often reagents have been standardized in the form of RTU products (see Table 1-3). Automated instruments are designed to imitate manual staining methods. Steps that lend themselves to automation include application of reagents, incubation of tissues, and rinsing of slides. These manipulations can be accomplished in several different ways, as evidenced by the various platforms available and new derivatives that appear regularly. No general recommendation is possible other than to give a system, and the reagents designed for it, careful in-house trial before purchase. Price of the instrument is not necessarily a guide; over time, reagent use is often the major expense, best appreciated by developing a reagent budget based on real and anticipated slide numbers and comparing existing known costs with projected total costs for the automated platform and reagents. Efficient reagent use with an automated platform may yield overall savings, especially when technician time is budgeted. Knowledge and availability of service technicians are also vital for overall satisfaction. Remember, purchasing an autostainer is like buying a car—the vendor and technician are selling to you and
will present the instrument to its best advantage. Depending on the need of the individual laboratory, a variety of choices are available with different methods, analytic flexibility, and productivity; at least 17 instruments from seven vendors were included in the UK NEQAS study cited in Table 1-7, and in practice, this number is a significant underestimate. Before an automated immunostainer is put to use, parallel studies should be performed to confirm that the results obtained by automated technique are comparable to or better than those obtained previously by the manual method or prior automated system. Furthermore, staining results obtained from the automated instrument, with the same antibody and the same tissues—for example, the positive controls—should show intralaboratory run-to-run reproducibility. The ideal instrument is easy to use and requires minimal technician attention during a run. Should a problem occur, a machine error-tracking program should be able to report the problem. Regarding the dispensing of reagents, precise and reproducible microliter quantities of each reagent should be dispensed to result in complete tissue coverage. The evaporative loss and carryover of reagents should also be kept to a minimum. Some instruments, those in so-called open systems, permit the use of multiple antibodies and detection systems and provide random access to accommodate a diversity of antigens and recipes, with user-friendly software that allows flexible programming. As noted above, such machines are best used in larger, more sophisticated laboratories. Many manufacturers supply pro prietary reagents (RTUs), enzymes, chromogens, and counterstains (see Table 1-3) to be used in conjunction with their instrument. Protocols that require the strict use of proprietary reagents are not flexible and cannot be customized (closed systems). These RTU reagents tend to be more costly per test, but they may produce savings in efficient use over time. Moreover, the use of RTU reagents with bar codes permits computer-driven tracking and monitoring of reagent volume, lot number, and expiration date. These reagents, therefore, assist with matters pertaining to QC, and they may save costs by means of reduced technician time and lack of errors. As described, open automated systems, in contrast, are more flexible and allow for the use of other reagents and protocols. Generally, the more open the system, the more the individual laboratory must do in-house to qualify the instrument, reagents, and protocols and the greater the requirement for in-house expertise. Each laboratory should make this decision. It is worth noting that the highest level of reproducibility among different laboratories would likely be achieved by the use of totally closed systems, as is usual in the clinical laboratory, with increased attention to sample preparation. The application of automation for immunostaining offers several advantages. The true value of automation is that it offers a uniform and standardized microenvironment that results in greater intralaboratory run-to-run assay consistency. The costs and inconvenience of repeated procedures are, therefore, reduced. Moreover, small microliter amounts of expensive reagents can be dispensed accurately; this not only
Techniques, Protocols, and Troubleshooting
saves cost but also adds to accuracy. Moreover, automation allows for walk-away function and thereby frees not only the time but also the cost of skilled technicians. The use of automated instrumentation may also decrease the amount of technical training required through a user-friendly software interface. From a safety standpoint, the use of a consolidated and enclosed automated workstation increases biosafety by reducing exposure to, and facilitating the disposal of, hazardous chemicals. Other benefits of automation include increased throughput and decreased turnaround time as a result of increasing the speed of reactions with heat and mixing. Finally, most systems also offer computer-driven accountability and reporting capabilities for each step of the staining procedure.166-169 Automated AR systems have also recently been introduced, sometimes integrated into or coupled with the automated staining instrument. AR has been in use since the early 1990s. Nevertheless, because of the multiple variables inherent in this technique, standardization has been a challenge, as discussed elsewhere in this chapter. With automation, these variables are controlled within the protocol, giving greater reproducibility, although adjusting the conditions of AR to suit antigens that are difficult to stain may be more challenging. As mentioned earlier, with effective automation, quantitative IHC becomes possible, because the intrinsic stain procedure is more reliable. IHC tests that are useful for prognosis and therapy require some quantification; to date this has been achieved by crude semiquantitative scoring methods (+, ++, +++) that are unreliable. Automated IHC coupled with automated cellular imaging systems provide the promise of detection of molecular markers, hormone receptors, or occult micrometastases in a more reliable, more reproducible manner.170,171 As discussed below, automated microscopy may be used to evaluate target cells using morphometric parameters, as well as spectral imaging for multiple stains,119-121 with digital records of images and data. The results have the potential to be truly quantitative, when based on standardized scoring and reporting. Images of the cells, as well as the results, are stored permanently for later review or confirmation, or both if necessary. Despite the advent of automated technologies, not all problems are corrected by automation, and in some respects, the proliferation of automated staining platforms has aggravated problems of reproducibility, because now almost any laboratory can perform IHC staining, often without sufficient grounding or understanding of the principles. As discussed elsewhere, proper control systems and external proficiency programs are essential (see Table 1-7). Automation cannot overcome the improper selection of antibody (stain), poor quality of the tissue to be examined, faulty tissue fixation, or problems with processing or sectioning, all of which can compromise antigen detection and interpretation (see Box 1-1 and Table 1-5). Automated platforms cannot replace a pathologist’s expertise when selecting the appropriate tests (stains) to be performed or when interpreting tissue sections. Automation is, therefore, by no means a panacea for laboratories with poor QC standards. Only a laboratory with high
29
standards and proper controls can operate an automated machine effectively. However, under ideal conditions, automation can and does provide reproducible, standardized, and uniform results and serves as a prelude to quantitative IHC and computerized image analysis. QUANTIFICATION: DEVELOPMENT OF REFERENCE STANDARDS AND STANDARD CURVES FOR CALCULATION OF ANTIGEN CONTENT IN TISSUE SECTION
The use of current in-house controls has been discussed, together with the rationale for development of quantitative internal references standards119-121 that would provide a method for correcting for variations in sample preparation and would also provide a basis for accurate quantification. A number of investigators have reported improved control systems, which includes some with the potential for calibrating the IHC reaction. These systems will be discussed briefly in the expectation that improved control methods will soon be required by proficiency testing and certification programs. If properly used, controls enhance quality within any laboratory, but because they are “local” in production and availability, they do not ensure reproducibility among different laboratories. Universal reference standards, or controls, are therefore actively under investigation. Protein “Spots”
Theoretically, it is possible to develop preparations of purified protein (antigen), which can be diluted to produce a series of known reference standards for both Western blotting and, when suitably prepared, for IHC. The peptide or protein deposits described by Sompuram, Bogen, and colleagues represent one promising approach in the production of known reference standards.109,110,150 Faux Tissues and Histoids
The technique of matrix models172 has been used to create what is in effect an artificial control tissue for the protein (antigen) in question. In this approach, a conversion formula may be developed from a standard curve to determine the exact amount of antigen present in FFPE tissue sections under various conditions of immunostaining that include AR pretreatment, analogous to the method proposed for QIRS.119-121 These types of peptide or matrix preparations may also be used as a pretest to establish a standardized protocol of IHC and may also serve as practical reference standards for quality control of IHC staining. The matrix model has advantages over an alternative, Quicgel,173 essentially an artificial tissue control block that incorporates a breast cancer cell line, which is then added to the tissue cassette that contains the clinical biopsy specimen. Histoids174,175 (Fig. 1-23) are 3D faux-tissue pellets grown in centrifugal culture conditions. They provide another approach to total process control. Histoids may be constructed to consist of two, three, or more cell lines cocultured under standardized conditions to yield a faux-tissue pellet (e.g., of fibroblasts, breast cancer cells,
30
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
WI.38
associated analytic methods are expected to continue to improve and are far superior to the human eye for quantification, with the prerequisite that internal reference controls must be used. This ability to combine precise morphologic resolution on a cellular and subcellular level, combined with accurate measurement of minute amounts of protein, will create a new field of quantitative in situ proteomics (QISP) applied to the tissue section by surgical pathologists.
Technical Issues: Troubleshooting Figure 1-23 Faux-tissue breast histoid stained for Her2. Good positivity of the breast epithelial elements (MCF-7 cell line) are shown, but they lack staining in fibroblasts. Collaborative project, Dr. M. Ingram, Dr. A. Imam, Dr. C. Taylor, 2002, IMAT program grant, National Cancer Institute.
and endothelial cells), which may then serve as a reference standard, available in unlimited quantities for the control of many commonly used antibodies, including the control of tissue fixation and processing steps. Quantitative Internal Reference Standards
Although the methods outlined above are potentially widely available and will serve to control the staining method, these controls cannot serve as controls for sample preparation, including fixation, unless they are included in every step of the preparation process. In this regard, the use of internal reference standards that rely upon the demonstration of proteins intrinsic to each tissue under study provides the only practical method proposed to date for evaluating sample preparation.119-121 This method was described in greater detail earlier in this chapter in the section on quality control and standardization. Quantitative In Situ Proteomics
Certain manufacturers have begun to include control materials in test kits, especially when graded (semiquantitative) interpretation is required. Examples of such kits are Agilent/Dako’s Her2 Hercept test and Roche/ Ventana’s Pathway Her2 test). The control slides provided are useful for validating the IHC staining reagents and protocol, but as described, they do not speak to the adequacy of in-house sample preparation, including fixation. To accomplish this step requires additional control materials. It is anticipated that the trend of including graduated controls for semiquantitative testing will increase as IHC tests become available in the form of “companion diagnostics” for the many targeted therapies under development for precision or personalized therapeutics. But here again, the requirement for precise quantification will supersede current semiquantitative scoring with plus and minus marks. The ongoing development of improved, more sensitive IHC staining methods, coupled with the use of QIRS121 and calibrated image analysis will ultimately produce IHC methods that are precise and accurate. WSI and
IHC is a multistep diagnostic procedure that involves the proper selection, fixation, processing, and staining of tissue.62,176,177 The final interpretation of the results is the responsibility of an experienced pathologist based on the presence, pattern, and intensity of colored chromogen products deposited on the tissue as the result of specific antibody-antigen reactions in the cells. The resultant pattern of staining can be focal or diffuse, nuclear, cytoplasmic, or membranous. When the expected results are not obtained, the clinician must troubleshoot the problem in a systematic way, and each single variable of this multistep diagnostic procedure should be addressed separately, one at a time (Tables 1-10 and 1-11). Some common problems are illustrated in Figures 1-24 through 1-36. Technical problems can be classified into two main categories: those that occur before staining and those that are related to staining. Preanalytic effects (see Tables 1-4 and 1-5 and Box 1-1) include so-called warm and cold ischemia or delayed fixation, overfixation, underfixation, or uneven fixation; these problems are extremely common and have already been discussed. The subsequent effects of tissue processing are less studied and less well understood. One of the few problems in this area that has been described is due to inadequate tissue dehydration before paraffin embedding. This problem can be reduced by preparing fresh alcohol solutions on a more frequent and regular basis. Other processing problems include the use of incorrect slides, which can result in loss of tissue adherence. Imprecise sectioning can cause crinkling or folding of the tissue, which may result in reagent trapping and uneven staining patterns. Subsequent to processing, other non–staining-related problems pertain to pretreatment protocols: enzyme digestion, AR, or both. During pretreatment, technical problems are difficult to avoid because of the many variables involved. The recognition of inappropriate staining can be divided into five main categories based on the pattern of staining results on the test tissue as well as the pattern of staining results on the positive control.62-70,91,177 We will discuss troubleshooting considerations for each of the following five staining patterns: 1) absence of staining of both the test tissue and positive control; 2) absence of staining of the test tissue with appropriate positive staining of the positive control; 3) weak staining of the test tissue with appropriate staining of the positive control; 4) background staining on the test tissue, the positive control, or both; and 5) artifactual
Techniques, Protocols, and Troubleshooting
31
Figure 1-24 “Chromogen freckles” that result from undissolved precipitates of chromogen.
(unexpected) staining on the test tissue, positive control, or both. ABSENCE OF STAINING OF BOTH SPECIMEN AND CONTROL
When neither the specimen nor the control stains, it is necessary to check to ensure that 1) all the steps of the staining process were performed in the correct order, 2) the incubation times were sufficient, and 3) no reagents were accidentally omitted. If other cases were stained in the same run with the same reagents, check their performance. Antibody titrations and dilutions should also be reviewed. Also check the expiration dates and storage of the reagents, because using reagents beyond their expiration date can result in false-negative results. Moreover, antibodies stored in self-defrosting freezers are exposed to repeated freezing and thawing, and this can result in antibody breakdown. The rinse buffer should also be checked, because it may be incompatible with the reaction reagents. The buffer pH must be appropriate, and sodium azide should not be present in buffers used with peroxidase enzyme. Other possible causes of absence of staining include problems with the chromogen. One must confirm that the chromogen
solution was properly prepared and that it is working. This can be accomplished by adding the labeling reagent to a small amount of the prepared chromogen and confirming that a color change occurs. Keep in mind that some chromogen solutions tend to deteriorate quickly. Finally, lack of staining can also occur because of
TABLE 1-10 Troubleshooting Variables
Check the Tissue
Check the Pretreatment and Primary Antibody
Check the Detection System and the Controls
Patient tissue
Xylene
Link and label
Fixation
Alcohol
Compatibility
Processing
Buffer/water
Expiration date
Control tissue
Antigen retrieval
Chromogen
Fixation
Preparation
Processing
Incubation time Expiration date
Fixation
Blocking
Results of Test
Optimal fixation
Peroxidase block
Positive staining
Overfixation
Biotin block
Negative staining
Underfixation
Background block
Focal or weak staining
Delayed fixation
Figure 1-25 Pigmented melanophages, not to be confused with chromogen.
Background staining Artifactual staining
Processing
Antibodies
Results of Control
Dehydration
Prediluted, concentrated diluent
Positive staining
Embedding
Expiration date
Negative staining
Sectioning
Storage
Background staining
Mounting
Contamination
Artifactual staining
Slides
Incubation
32
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
TABLE 1-11 Troubleshooting: Weak or Absent Staining Technical Problems and Solutions: Absence of Staining or Weak Staining
Possible Solutions
Inadequate fixation
Avoid delay of fixation (>30 min) or overfixation (>48 h)
Incomplete dehydration during processing
Check protocol for processing; perform regular reagent changes (i.e., alcohol)
Paraffin too hot
Monitor temperature of paraffin (<60° C)
Prolonged or excessive heating
Optimize antigen retrieval time
Staining steps not followed
Review procedure manual
Reagents not working
Check expiration dates, storage parameters, compatibility with other reagents, and pH
Antibody too dilute
Check antibody titration; increase concentration; lengthen incubation time; increase temperature of reaction; check amount of rinsing buffer left on slide
Drying out of tissue during processing
Keep specimen moist as indicated by procedure manual; prevent evaporation with humidity chamber
Insufficient incubation time
Lengthen incubation time to achieve desired intensity of staining; add heat; increase concentration of antibody
Chromogen not working
Add chromogen to labeling solution; monitor for change in color
A
Figure 1-26 Section of liver illustrates endogenous biotin that resulted in nonspecific background staining.
Figure 1-27 Section of lymph node representing “negative control,” omitting the primary antibody and replacing it with an equivalent concentration of immunoglobulin of the same species as the primary (see Table 1-6). Staining that is due to endogenous biotin (shown in Figure 1-26), because it derives from the labeling system, will still be seen in this negative control. Background as a result of the primary antibody, either nonspecificity or excess concentration, would be absent in this control.
B
Figure 1-28 Section of lymph node stained with CD45, showing variable fixation artifact. A, Top left: a narrow rim of intense mahogany stain, probably representing edge artifact that generally should be ignored in interpretation. B, Immediately subjacent: a well-defined zone of cytoplasmic membrane staining in the subcapsular region represents the zone where fixation is optimal. This is the preferred region to interpret. Diminished staining is shown deeper in the block, where the tissue is not adequately fixed, and protein degradation has occurred.
Techniques, Protocols, and Troubleshooting
33
improper treatment in counterstaining, or cover slipping. For example, AEC should not be used with counterstains or mounting media that contain alcohol, xylene, or toluene, because these chemicals can dissolve the soluble colored precipitates formed by the reaction of AEC chromogen and substrate. Following this type of review, the IHC procedure should be repeated with a positive control known to have performed well previously; if this fails, it is best to begin with entirely new reagents throughout. Last but not least, the work of NordiQC and UK NEQAS, discussed previously, reveals that one of the most common causes of poor staining is faulty or inappropriate AR. Figure 1-29 A lymph node section stained with Bcl-2 shows similar variable fixation artifact to that in Figure 1-28 with gradual loss of staining appreciated toward the center of the node. The pathologist must exercise judgment in interpreting such cases. No edge artifact appears in this section.
A
SPECIMEN WITH APPROPRIATE POSITIVE STAINING OF POSITIVE CONTROL
If the positive control slide shows appropriate positive staining of the expected cells, the clinician can assume
B
Figure 1-30 Sections of lymph node stained with CD20. Shown are weak or absent staining (A) and intense membrane staining plus inappropriate cytoplasmic staining (B) of the B cells in the germinal center. Such effects often result from improper concentrations of primary antibody; in A, it is overly dilute, and in B, it is excessively concentrated. The antibody system should be retitrated (see Tables 1-1 and 1-2).
Figure 1-31 Section of normal lung tissue stained with thyroid transcription factor 1 (TTF-1). Specific nuclear positivity of the alveolar lining cells (pneumocytes) is apparent. This is an example of a positive control for TTF-1; normal thyroid gland also could be used. The positive control should be a tissue known to contain the antigen in question, ideally at moderate concentrations (see Table 1-6). If the positive control does not perform as expected, the run is not valid.
Figure 1-32 Section of lymph node stained with CD43. A processing problem with tearing and folding of the tissue is shown here, which subsequently resulted in variable staining intensity and precipitation of the chromogen. All these artifacts can adversely affect interpretation.
34
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Figure 1-33 Section of lymph node stained with CD20 shows low- and high-power views with “bubble artifact.” This is the result of microscopic hydrophobic bubbles formed during the staining process that interfere with uniform distribution of reagent.
A
B
Figure 1-34 Sections of malignant melanoma stained with S-100 protein. A, These spindle-shaped neoplastic cells are overstained to the extent that it is difficult to appreciate true nuclear staining; this effect may result from excess concentration of the primary antibody, or label, or long incubation or drying. B, The variable staining seen here is more difficult to interpret and may result from variable fixation or, in some instances, true biologic heterogeneity of tumor cells. Review of the entire section to determine whether the effect is zonal, as in Figures 1-28 and 1-29, may help resolve the problem.
A
B
Figure 1-35 Cell block section stained with CD20. A, High background staining of the amorphous proteinaceous material is shown. B, This high-power view highlights positive B-lymphoid cells and aberrant nucleolar positivity of scattered T-lymphoid cells, which should not react with CD20. These appearances may result from poor fixation, excess concentration of reagents, inadequate washing, or drying of the section during staining, which in effect produces very high concentrations of reagents.
Techniques, Protocols, and Troubleshooting
35
net effect of antigen recovery; the favored explanation is that whereas fixed tissue seems to be protected from degradation by heating, in comparison to nonfixed tissue, this protection may exist only in aqueous medium, not in paraffin. WEAK STAINING OF SPECIMEN WITH APPROPRIATE STAINING OF POSITIVE CONTROL
Figure 1-36 Section of adenocarcinoma stained with carcinoembryonic antigen. Positive polymorphonuclear neutrophils show strong endogenous peroxidase staining. Misinterpretation is possible if users do not read the detailed interpretative information in the package insert as a guide to determine which “normal” tissue components may be expected to give a positive stain with the antibody in question.
that the procedure was performed correctly and that the reagents were working properly. In such instances, the problem is likely to be preanalytic rather than an aspect of the staining procedure itself. It may, therefore, be the result of improper tissue fixation, processing, or pretreatment, including AR, or a combination of these. Keep in mind, particularly in manual methods, that a single improper step may have occurred in protocol that was limited to the test section. Problems with formalin fixation have been discussed previously (see Tables 1-5 through 1-7) and include delay of fixation, overfixation, underfixation, and variable fixation.60-62,91 For antigens susceptible to autolysis, a delay in fixation may result in absence of staining. For this reason, fixation should begin as soon as possible, preferably within 30 minutes of removing the specimen. Overfixation can also result in absence of staining. Generally, formalin fixation should not exceed 48 hours. If tissue or tissue blocks are overly large, only the periphery of the tissue will be penetrated by the fixative; toward the center of the specimen, the tissue will remain unfixed and raw. In these center areas, the specimen may undergo coagulative fixation by alcohol during tissue dehydration, resulting in variable staining with more intense staining at either the center or the periphery of the specimen, depending on which has occurred, what antibody was used, and whether AR was used (see Figs. 1-28 and 1-29). Less often absence of staining can also result from processing problems Potential processing factors include inadequate dehydration that results from the use of old alcohol reagents. Moreover, heat-sensitive epitopes can be lost by embedding in paraffin that is too hot or by subjecting the tissue to prolonged heating. Therefore the temperature of the paraffin should be monitored so as not to exceed 56° C (see Tables 1-10 and 1-11). This observation is somewhat paradoxical in that the AR subjects the tissue to still higher temperatures with a
Improper fixation or processing of the test tissue, or both, is the most likely cause of weak staining of the specimen when the control stains positively. All the causes discussed previously for absence of staining apply to the situation of weak staining. However, the test tissues and the control slides must have been fixed and processed in an identical manner, otherwise discrepant staining can be expected. Other factors that may cause weak staining with appropriate positive control staining include a lower concentration of antigen in the test tissue compared with the control, meaning that the selection of the control may not be appropriate (see discussion of controls and Table 1-6). In manual methods, inappropriate dilution of antibody can also be the result of leaving too much buffer rinse on the slide before the antibody is applied; this is less of a problem with automated platforms, with which reagent volumes are typically small. Excessively large section size may also give weak or patchy staining if antibody does not diffuse evenly or is insufficient for the large amount of antigen present. BACKGROUND STAINING
Any positive staining that is not the result of antibodyantigen reaction is termed nonspecific background staining. Such staining appears as inappropriate positive staining on the test tissue or the positive control. If also present in the negative control, background is likely due to the labeling steps; if confined to the test specimen and positive control, it may be attributable to the primary antibody and may be ameliorated by assessing again the optimal dilution as described earlier. A number of other conditions can also cause background staining, including the presence of endogenous peroxidase and/ or endogenous biotin (see Fig. 1-26). For details see the section “Blocking Nonspecific Background Staining.” Automated platforms routinely include blocking steps and recommended reagents to reduce this problem. Poorly fixed tissues and areas of necrosis may show background staining in these areas, as will tissue sections that are cut too thick or are fragmented. Such effects may be seen at tissue edges, attributable to reagents dispersing under the tissue margins, where washing is ineffective (see Figs. 1-28 through 1-32). Some other causes of background positivity pertain to the antibody solution itself. Examples include solutions that contain bacteria or particulates that may occur with improper storage or repeated freezing and thawing of the antibodies. Other less common causes of background staining are related to tissue processing, such as the incomplete removal of paraffin. Background staining can also result from the incomplete rinsing of slides between steps or
36
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
TABLE 1-12 Technical Problems and Solutions: Background Staining Problem
Solution
Nonspecific protein binding
Use nonimmune serum from the same animal species as the secondary antibody; add salt to buffer
Incomplete removal of paraffin
Use only completely deparaffinized slides
Poorly fixed or necrotic tissue
Make sure tissue is properly fixed; avoid sampling of necrotic areas
Thick preparation
Cut sections at 3 to 5 mm; prepare cytospins that are monolayer in thickness
Inappropriately concentrated antibody
Check titration; decrease concentration; decrease incubation time; decrease temperature of reaction
Endogenous biotin
Block with avidin
Incomplete rinsing of slides
Follow protocol for proper slide rinsing
Chromogen staining too intense
Monitor timing of chromogensubstrate reaction; filter chromogen; decrease chromogen concentration
by the use of contaminated buffers. Finally, one other consideration that can cause nonspecific positivity is overdevelopment of the chromogen-substrate reaction that can result from incomplete solution or excess concentration of chromogen. Appropriate troubleshooting measures for these problems include filtering of the chromogen solution or decreasing its concentration (Tables 1-12 and 1-13). ARTIFACTUAL STAINING
Artifacts include undissolved precipitates of chromogen or counterstain (see Fig. 1-24). The presence of these TABLE 1-13 Technical Problems and Solutions: Artifactual Staining Problem
Solution
Presence of chromogen or counterstain deposits
Filter the chromogen or counterstain
Black deposits in B5-fixed tissue
Remove the mercury before staining
Endogenous pigments confused with specific positive staining
Check the negative control for the presence of these pigments; use a chromogen of contrasting color
Microbiologic contamination
Change reagents often, filter, use fresh reagents, check expiration dates
artifacts can be corrected by filtering. B5-fixed tissues may show black precipitates spread out across the specimen. This results from incomplete dezenkerization of the tissue and can be corrected by removing the mercury from the tissue before staining. Not uncommonly, endogenous pigments, such as hemosiderin or melanin (see Fig. 1-25), are confused with true histochemical positivity. However, the presence of these pigments will also be seen on the negative control. If interpretation still is difficult, the use of a chromogen of contrasting color—such as AEC, which stains red—can be helpful in providing this distinction. Finally, other artifacts worth considering are the presence of microbial contaminants, such as yeast or bacteria (see Table 1-13). Because of the multistep nature of the ICH procedure, numerous technical problems can arise. Fortunately, many of these can be managed logically and easily with adherence to QC guidelines and strict application of positive and negative controls.62,91 Examples of the staining problems discussed are illustrated in Figures 1-24 through 1-36.
Summary of Amplification Methods Where the IHC stain (signal) intensity is weak, methods have been sought to increase the sensitivity of the method overall. In this context, increased sensitivity means an increase in the intensity of the final stain product. Today, the high level of detection sensitivity achieved by current IHC protocols that include quality antibodies and polymer-based labeling methods generally has made additional amplification unnecessary. A brief review of available approaches follows (Table 1-14). PREDETECTION AMPLIFICATION
In effect, the AR technique is an effective and simple technique of predetection–phase amplification.4,39,40,55-58,143 It is generally accepted, although not well understood, that AR-induced amplification of signal is the result of “restored” antigenicity contingent on recovery of certain epitopes in the formalin-modified protein structure.60,61,143 To the extent that the AR technique serves to restore the natural antigen-antibody reaction, it favors specific binding and does not aggravate nonspecific background staining, thereby providing the potential for an enhanced signal-tonoise ratio. Enzyme digestion methods have also been applied to increase the ability to detect certain antigens,29,123 but generally speaking, these are much less reliable, as indicated by the UK NEQAS surveys (see Table 1-7). DETECTION-PHASE AMPLIFICATION
As described earlier, development of staining methods has continued apace, and highly sensitive and reliable methods are widely available. Further development even offers the promise of detection at the level of single molecules, as with microparticle labeling approaches. Computer-assisted automated stainers have
Techniques, Protocols, and Troubleshooting
37
TABLE 1-14 Classification of Three Basic Signal Amplification Approaches Classification
Basic Principles and Mode of Action
Advantages and Problems
Restoration of formalin-induced modification of protein structure, resulting in dramatic amplification of signal while simultaneously reducing the background noise
Simplest and cheapest procedure (heating) among all methods of amplification; not effective for some antibodies/antigens
Multistep detection systems; PAP, ABC
Increase the accumulation of labeling signal (enzyme or others) 2-fold to 100-fold
The polylabeling technique and polymer-based amplification systems are simpler, cheaper, and faster than other multistep detection systems; a biotin-free detection system avoids the problem of the endogenous biotin reaction
Stepwise amplification
Repeating cycles of detection
Polymeric and tyraminebased amplification
Currently available kits provide for further dilution of primary antibody: more than a hundredfold for some antibodies
Predetection Amplification Antigen retrieval (AR)
Detection Amplification
Postdetection Amplification Procedures are complicated and involve repeating cycles of reactions; labor and costs may be a drawback to widespread application; background staining increases with amplification of signal Enhanced DAB by metal, imidazole
Enhance the color reaction
Anti-EP
Anti-EP + biotinylated link + HRP label, potential for further amplification
Gold/silver enhancement method
Silver enhancement
Modified from Shi S-R, Guo J, Cote RJ, et al: Sensitivity and detection efficiency of a novel two-step detection system (PowerVision) for immunohistochemistry. Appl Immunohistochem Mol Morphol. 1999;7:201-208. ABC, Avidin-biotin conjugate; DAB, diaminobenzidine; EP, end product; HRP, horseradish peroxidase; PAP, peroxidase-antiperoxidase.
led to remarkable improvements in reproducibility and have facilitated the widespread use of highly sensitive detection methods that mostly use polymer-based secondary reagents, as described previously. The quality of reagents and kits available from the major and reputable manufacturers has allowed rapid adoption of these sensitive labeling methods. Also, the manufacturers’ instructions and protocols are more comprehensive and are of increased value in setting up the stain and interpreting the result. Tyramine or catalyzed signal amplification99-101 is the most sensitive method generally available, with newer, even better methods in the pipeline (see “Tyramine Signal Amplification”). In addition, for maximum effect, amplification methods are combined with maximum retrieval approaches,178-180 sometimes with extraordinary effect in terms of the dilution of primary antibody that may be used (well in excess of 1 : 1000 for commercially available reagents) or in the ability to detect minute amounts of antigen that approaches that of single molecules. Highly sensitive detection methods require rigorous control and validation. A major failing is that technologists and pathologists often neglect to read the package
insert, or if they do read it, they ignore the contents or adjust the protocol or reagents. Adjusting a protocol to achieve maximum sensitivity affects the utility and interpretation of the IHC stain and requires careful and thorough validation. For FDA-approved tests, such as those for HER2 or ER, it is important to note that any variation from protocol or reagents requires complete revalidation of the assay that includes proof of performance by the performing labs. POSTDETECTION AMPLIFICATION
Methods of postdetection phase amplification seek to intensify the chromogen reaction, as we have described with reference to chromogens. The two principal drawbacks are an increase in the complexity of the immunostaining procedure, with additional steps that are difficult to control, and often a general increase in nonspecific background staining. This latter effect often means that although the intensity of the stain is increased, the signal-to-noise ratio is not improved, and interpretation may even be more difficult. In general, we do not recommend this approach for routine use.
38
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
Conclusion The widespread application of IHC has entirely transformed diagnostic surgical pathology “from something resembling an art into something more closely resembling a science.”3 Every major surgical pathology textbook now includes extensive treatment of the use of IHC methods in diagnosis and often also includes chapters on methodology. However, as we have described here, all is not entirely well. Reproducibility is a problem that is ameliorated by automation, when used properly, and is at the same time aggravated by the proliferation of automated devices that are not always used in controlled environments. Standardization has thus become the major focus, and it has been emphasized throughout this chapter with reference to published practice guidelines. An ongoing major drawback of IHC is the lack of objective quantitative measurement for target antigens under investigation, especially for proper use of the prognostic markers described elsewhere in this book. In
this respect, the use of proper controls, including QIRS, has been described for calibration of the IHC method, analogous to ELISA tests in the clinical laboratory. Finally, although the focus has been on IHC, much of what has been written regarding standardization of IHC is equally applicable to the use of ISH methods in surgical pathology. In essence pathologists have been practicing “molecular morphology”—the microscopic localization of protein by IHC, and later DNA, and RNA by ISH2,26,91,121—since the late 1970s. To take the next leap forward, we need to “do it right.” Reliable and true quantification is a goal that requires a higher demonstrable level of standardization and reproducibility, stemming from the general use of reference standards coupled with computer-assisted analysis of the reaction product. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Coons AH, Creech HJ, Jones RN: Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med. 47:200, 1941. 2. Taylor CR: The current role of immunohistochemistry in diagnostic pathology. Adv Pathol Lab Med. 7:59, 1994. 3. Taylor CR: An exaltation of experts: Concerted efforts in the standardization of immunohistochemistry. Hum Pathol. 25:2, 1994. 4. Taylor CR, Cote RJ, editors: Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist, ed 2, Philadelphia, 1994, WB Saunders. 5. Taylor CR, Burns J: The demonstration of plasma cells and other immunoglobulin containing cells in formalin-fixed, paraffinembedded tissues using peroxidase labeled antibody. J Clin Pathol. 27:14, 1974. 6. Avrameas S: Enzyme markers: Their linkage with proteins and use in immunohistochemistry. Histochem J. 4:321, 1972. 7. Nakane PK, Pierce GBJ: Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol. 33:307, 1967. 8. Adams JC: Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem. 40:1457, 1992. 9. Bobrow MN, Harris TD, Shaughnessy KJ, et al: Catalyzed reporter deposition: A novel method of signal amplification: Application to immunoassays. J Immunol Methods. 125:279, 1989. 10. Colvin RB, Bhan AK, McCluskey RT, editors: Diagnostic Immunopathology, ed 2, New York, 1995, Raven Press. 11. DeLellis RA, Sternberger LA, Mann RB, et al: Immunoperoxidase techniques in diagnostic pathology: Report of a workshop sponsored by the National Cancer Institute. Am J Clin Pathol. 71:483, 1979. 12. Elias JM, editor: Immunohistopathology: A Practical Approach to Diagnosis, Chicago, 1990, American Society of Clinical Pathologists Press. 13. Heras A, Roach CM, Key ME: Enhanced polymer detection system for immunohistochemistry. Mod Pathol. 8:165A, 1995. 14. Hsu SM, Raine L, Fanger H: A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 75:734, 1981. 15. Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 29:577, 1981. 16. Polak JM, van Noorden S, editors: Immunocytochemistry: Modern Methods and Applications, ed 2, Bristol, UK, 1986, Wright Publishing. 17. Sabattini E, Bisgaard K, Ascani S, et al: The EnVision system: A new immunohistochemical method for diagnostics and research: Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol. 51:506, 1998. 18. Shi S-R, Cote RJ, Taylor CR: Standardization and further development of antigen retrieval immunohistochemistry: Strategies and future goals. J Histotechnol. 22:177, 1999. 19. Sternberger LA, Joseph SA: The unlabeled antibody method: Contrasting color staining of paired pituitary hormones without antibody removal. J Histochem Cytochem. 27:1424, 1979. 20. Vyberg M, Nielsen S: Dextran polymer conjugate two-step visualization system for immunohistochemistry. Appl Immunohistochem. 6:3, 1998. 21. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256:495, 1975. 22. Leong ASY: Commentary: Diagnostic immunohistochemistry— problems and solutions. Pathology. 24:1, 1992. 23. Burns J, Hambridge M, Taylor CR: Intracellular immunoglobulins. A comparative study of three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J Clin Pathol. 27:548–557, 1974. 24. Taylor CR: The nature of Reed-Sternberg cells and other malignant “reticulum” cells. Lancet. 2:802, 1974. 25. Taylor CR: A history of the Reed-Sternberg cell. Biomedicine. 28:196, 1978.
38.e1
26. Taylor CR, Kledzik G: Immunohistologic techniques in surgical pathology: A spectrum of “new” special stains. Hum Pathol. 12:590, 1981. 27. Pinkus GS: Diagnostic immunocytochemistry of paraffinembedded tissues. Hum Pathol. 13:411, 1982. 28. Swanson PE: Editorial: Methodologic standardization in immunohistochemistry: A doorway opens. Appl Immunohistochem. 1:229, 1993. 29. Larsson LI, editor: Immunocytochemistry: Theory and Practice, Boca Raton, FL, 1988, CRC Press. 30. Huang SN: Immunohistochemical demonstration of hepatitis B core and surface antigens in paraffin sections. Lab Invest. 33:88, 1975. 31. Leong AS-Y, Milios J, Duncis CG: Antigen preservation in microwave-irradiated tissues: A comparison with formaldehyde fixation. J Pathol. 156:275, 1988. 32. Fraenkel-Conrat H, Brandon BA, Olcott HS: The reaction of formaldehyde with proteins. IV: Participation of indole groups. J Biol Chem. 168:99, 1947. 33. Fraenkel-Conrat H, Olcott HS: The reaction of formaldehyde with proteins. V: Cross-linking between amino and primary amide or guanidyl groups. J Am Chem Soc. 70:2673, 1948. 34. Fraenkel-Conrat H, Olcott HS: Reaction of formaldehyde with proteins. VI: Cross-linking of amino groups with phenol, imidazole, or indole groups. J Biol Chem. 174:827, 1948. 35. Shi S-R, Key ME, Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 39:741, 1991. 36. Shi S-R, Tandon AK, Cote C, et al: S-100 protein in human inner ear: Use of a novel immunohistochemical technique on routinely processed, celloidin-embedded human temporal bone sections. Laryngoscope. 102:734, 1992. 37. Shi S-R, Tandon AK, Haussmann RR, et al: Immunohistochemical study of intermediate filament proteins on routinely processed, celloidin-embedded human temporal bone sections by using a new technique for antigen retrieval. Acta Otolaryngol (Stockh). 113:48, 1993. 38. Shi S-R, Cote C, Kalra KL, et al: A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidinembedded human temporal bone sections for immunohistochemistry. J Histochem Cytochem. 40:787, 1992. 39. Taylor CR, Shi S-R, Chen C, et al: Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem. 71:263, 1996. 40. Taylor CR, Shi S-R, Cote RJ: Antigen retrieval for immunohistochemistry: Status and need for greater standardization. Appl Immunohistochem. 4:144, 1996. 41. Boon ME, Kok LP: Breakthrough in pathology due to antigen retrieval. Mal J Med Lab Sci. 12:1, 1995. 42. Evers P, Uylings HB, Suurmeijer AJ: Antigen retrieval in formaldehyde-fixed human brain tissue. Methods. 15:133, 1998. 43. Gown AM, de Wever N, Battifora H: Microwave-based antigenic unmasking: A revolutionary new technique for routine immunohistochemistry. Appl Immunohistochem. 1:256, 1993. 44. Cattoretti G, Pileri S, Parravicini C, et al: Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol. 171:83, 1993. 45. Leong ASY, Milios J: An assessment of the efficacy of the microwave antigen-retrieval procedure on a range of tissue antigens. Appl Immunohistochem. 1:267, 1993. 46. Merz H, Rickers O, Schrimel S, et al: Constant detection of surface and cytoplasmic immunoglobulin heavy and light chain expression in formalin-fixed and paraffin-embedded material. J Pathol. 170:257, 1993. 47. Shi S-R, Chaiwun B, Young L, et al: Antigen retrieval using pH 3.5 glycine-HCl buffer or urea solution for immunohistochemical localization of Ki-67. Biotech Histochem. 69:213, 1994. 48. Shi S-R, Cote RJ, Yang C, et al: Development of an optimal protocol for antigen retrieval: A “test battery” approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections. J Pathol. 179:347, 1996.
38.e2
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
49. Shi S-R, Cote RJ, Young L, et al: Use of pH 9.5 TRIS-HCl buffer containing 5% urea for antigen retrieval immunohistochemistry. Biotech Histochem. 71:190, 1996. 50. Shi S-R, Imam SA, Young L, et al: Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem. 43:193, 1995. 51. Shi S-R, Gu J, Kalra KL, et al: Antigen retrieval technique: A novel approach to immunohistochemistry on routinely processed tissue sections. Cell Vision. 2:6, 1995. 52. Suurmeijer AJ, Boon ME: Notes on the application of microwaves for antigen retrieval in paraffin and plastic tissue sections. Eur J Morphol. 31:144, 1993. 53. Taylor CR, Shi S-R, Chaiwun B, et al: Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: Androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques [see comments]. Hum Pathol. 25:263, 1994. 54. von Wasielewski R, Werner M, Nolte M, et al: Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies. Histochemistry. 102:165, 1994. 55. Shi S-R, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry: Past, present, and future. J Histochem Cytochem. 45:327, 1997. 56. Shi S-R, Cote RJ, Young LL, et al: Antigen retrieval immunohistochemistry and molecular morphology in the year 2001. Appl Immunohistochem Mol Morphol. 9:107, 2001. 57. Shi S-R, Cote RJ, Taylor CR: Antigen retrieval techniques: current perspectives. J Histochem Cytochem. 49:931, 2001. 58. Shi S-R, Gu J, Kalra KL, et al: Antigenretrievaltechnique: A novel approach to immunohistochemistry on routinely processed tissue sections. In Gu J, editor: Analytical Morphology: Theory, Applications & Protocols, Natick, MA, 1997, Eaton, p 1. 59. Shi Y, Li G-D, Liu W-P: Recent advances of the antigen retrieval technique. Linchuang yu Shiyan Binglixue Zazhi (J Clin Exp Pathol). 13:265, 1997. 60. Shi S-R, Gu J, Taylor CR, editors: Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology, Natick, MA, 2000, Eaton. 61. Shi S-R, Gu J, Turrens F, et al: Development of the antigen retrieval technique: Philosophy and theoretical base. In Shi S-R, Gu J, Taylor CR, editors: Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology, Natick, MA, 2000, Eaton, pp 17–39. 62. Hewitt SM, Robinowitz M, Bogen SA, et al. Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guidelines—Second Edition, Vol. 31, Wayne, Pennsylvania, USA, 2011, Clinical and Laboratory Standards Institute, No. 4, pp 1–139. 63. UKNEQAS: United Kingdom National External Quality Assessment Service. www.ukneqas.org.uk. Accessed July 24th. 2012. 64. Nordiqc: Nordic immunohistochemical QualityControl. www. nordiqc.org. Accessed July 24th. 2012. 65. Wolff AC, Hammond ME, Schwartz JN, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal; growth factor receptor 2 testing in breast cancer. J Clin Oncopl. 25:118–145, 2007. 66. Goldstein NS, Hewitt SM, Taylor CR, et al and Members of Ad-Hoc Committee on Immunohiostochemistry Standardization: Recommendations for Improved Standardization of Immunohistochemistry. Appl Immunohistochem Mol Morphol,. 15:124–133, 2007. 67. Yaziji H, Taylor CR: Begin at the beginning, with the tissue! The key message underlying the ASCO/CAP Task-Force Guideline Recommendations for HER2 Testing. App Immunohistochem & Mol Morphol. 15(3):239–241, 2007. 68. Yaziji, H, Taylor C, Goldstein N, et al and members of the Standardization Ad-Hoc Consensus Committee: Consensus Recommendations on Estrogen Receptor Testing in Breast Cancer By Immunohistochemistry. Applied Immunohistochem Mol Morph. 16:513–520, 2008. 69. Hanna WM, Hammond E, Taylor CR, et al: Re: High concordance between immunohistochemistry and fluorescence in situ
hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system. Modern Pathology. 21:1278– 1280, 2008. 70. Hammond, ME, Hayes DH, Dowsett M, et al: American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer. Arch Pathol Lab Med. 134:E1–E16, 2010. 71. Mann G, editor: Physiologic histology, Oxford, 1902, Oxford University Press. 72. van Regenmortel MHV: The recognition of proteins and peptides by antibodies. In van Oss CJ, van Regenmortel MHV, editors: Immunochemistry, New York, 1994, Marcel Dekker, pp 277–300. 73. Barlow DJ, Edwards MS, Thornton JM: Continuous and discontinuous protein antigenic determinants. Nature. 322:747, 1986. 74. Johnson CW: Issues in immunohistochemistry. Toxicol Pathol. 27:246, 1999. 75. Nilsson P, Paavilainen L, Larsson K, et al: Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling. Proteomics. 5:4327, 2005. 76. Streefkerk JG: Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J Histochem Cytochem. 29:829, 1972. 77. Burns J: Background staining and sensitivity of the unlabeled antibody-enzyme (PAP) method: Comparison with the peroxidase-labeled antibody sandwich method using formalinfixed paraffin embedded material. Histochemistry. 43:291, 1975. 78. Weir EE, Pretlow TG, Pitts A: Destruction of endogenous peroxidase activity in order to locate cellular antigens by peroxidase-labeled antibodies. J Histochem Cytochem. 22:51, 1974. 79. McMillan EM, Martin D, Wasik R, et al: Demonstration in situ of “T” cells and “T” cell subsets in lichen planus using monoclonal antibodies. J Cutan Pathol. 8:228, 1981. 80. Straus W: Phenylhydrazine as inhibitor of horseradish peroxidase for use in immunoperoxidase procedures. J Histochem Cytochem. 20:949, 1972. 81. Straus W: Use of peroxidase inhibitors for immunoperoxidase procedures. In Feldman R, editor: Proceedings of the first international symposium on immunoenzymatic techniques, Amsterdam, 1976, Elsevier, p 117. 82. Andrew SM, Jasani B: An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers: Application to immunoperoxidase studies on eosinophil-rich tissue preparations. Histochem J. 35:426, 1987. 83. Robinson G, Dawson I: Immunochemical studies of the endocrine cells of the gastrointestinal tract. I: The use and value of peroxidase-conjugated antibody techniques for the localization of gastrin-containing cells in human pyloric antrum. Histochem J. 7:321, 1975. 84. Mason TE, Phifer RF, Spicer SS, et al: An immunoglobulin enzyme bridge method for localizing tissue antigens. J Histochem Cytochem. 17:573, 1969. 85. Sternberger LA, editor: Immunocytochemistry, Englewood Cliffs, NJ, 1974, Prentice-Hall. 86. Cordell JL, Falini B, Erber WN, et al: Immunoenzymatic label of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J Histochem Cytochem. 32:219, 1984. 87. Erber WN, McLachlan J: Use of APAAP technique on paraffin wax embedded bone marrow trephines. J Clin Pathol. 42:1201, 1989. 88. Wagner L, Worman CP: Color-contrast staining of two different lymphocyte subpopulations: A two-color modification of alkaline phosphatase monoclonal anti-alkaline phosphatase complex technique. Stain Technol. 63:129, 1988. 89. Sano T, Smith CL, Cantor CR: Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates. Science. 258:120, 1992. 90. Chen B-X, Szabolcs MJ, Matsushima AY, et al: A strategy for immunohistochemical signal enhancement by end-product amplification. J Histochem Cytochem. 44:819, 1996. 91. Taylor CR, Cote RJ, editors: Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist, ed 3, Philadelphia, 2006, WB Saunders.
References 92. Shi S-R, Guo J, Cote RJ, et al: Sensitivity and detection efficiency of a novel two-step detection system (PowerVision) for immunohistochemistry. Appl Immunohistochem Mol Morphol. 7:201, 1999. 93. Van der Loos CM, Naruko T, Becker AE: The use of enhanced polymer one-step staining reagents for immunoenzyme doublelabeling. Histochemical J. 28:709, 1996. 94. Kammerer U, Kapp M, Gassel AM, et al: A new rapid immunohistochemical staining technique using the EnVision Antibody Complex. J Histochem Cytochem. 49:623, 2001. 95. Bisgaard K, Pluzek K: Use of polymer conjugated in immunohistochemistry; a comparative study of a traditional staining method to a staining method utilizing polymer conjugates [abstract]. Path Int. 46:577, 1996. 96. Tsutsumi Y, Serizawa A, Kawai K: Enhanced polymer one-step staining (EPOS) for proliferation cell nuclear antigen (PCNA) and Ki-67 antigen: Application to intra-operative frozen diagnosis. Path Int. 45:108, 1995. 97. Bricca GM, Brodland DG, Zitelli JA: Immunostaining melanoma frozen sections: the 1-hour protocol. Dermatol Surg. 30:403, 2004. 98. Wiedorn KH, Goldmann T, Henne C, et al: EnVision+, a new dextran polymer-based signal enhancement technique for in situ hybridization(ISH). J Histochem Cytochem. 49:1067, 2001. 99. Bobrow MN, Shaughnessy KJ, Litt GJ: Catalyzed reporter deposition, a novel method of signal amplification. II: Application to membrane immunoassays. J Immunol Methods. 137:103, 1991. 100. Toda Y, Kono K, Abiru H, et al: Application of tyramide signal amplification system to immunohistochemistry: A potent method to localize antigens that are not detectable by ordinary method. Pathol Int. 49:479, 1999. 101. Mengel M, Werner M, von Wasielewski R: Concentration dependent and adverse effects in immunohistochemistry using the tyramine amplification technique. Histochem J. 31:195, 1999. 102. Shi S-R, Gu J, Cote RJ, et al: Standardization of routine immunohistochemistry: Where to begin? In Shi S-R, Gu J, Taylor CR, editors: Antigen retrieval technique: immunohistochemistry and molecular morphology, Natick, MA, 2000, Eaton, pp 255–272. 103. College of American Pathologists: Standard for laboratory accreditation, Skokie, IL, 1987, College of American Pathologists. 104. Taylor CR: The total test approach to standardization of immunohistochemistry. Arch Pathol Lab Med. 124:945, 2000. 105. Wolff AC, Hammond EH, Schwartz JN, et al: ASCO/CAP Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 testing in Breast Cancer. Arch Pathol Lab Med. 131:18, 2007. 106. Rhodes A, Jasani B, Balaton AJ, et al: Frequency of oestrogen and progesterone receptor positivity by immunohistochemical analysis in 7016 breast carcinomas: Correlation with patient age, assay sensitivity, threshold value, and mammographic screening. J Clin Pathol. 53:688, 2000. 107. Rhodes A, Jasani B, Balaton AJ, et al: Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays. Am J Clin Pathol. 115:44, 2001. 108. Vani K, Sompuram SR, Fitzgibbons P, et al: National HER2 proficiency test results using standardized quantitative controls: characterization of laboratory failures. Arch Pathol Lab Med. 132:211, 2008. 109. Bogen SA, Sompuram SR: Recent trends and advances in immunodiagnostics of solid tumors [review]. Biodrugs. 18:387, 2004. 110. Sompuram SR, Vani K, Hafer LJ, et al: Antibodies immunoreactive with formalin-fixed tissue antigens recognize linear protein epitopes. Am J Clin Pathol. 125:82, 2006. 111. Cote RJ, Taylor CR: Immunohistochemistry and Related Marking Techniques. In Damjanov I, Linder J, editors: Anderson’s Pathology, ed 10, St. Louis, 1996, CV Mosby, pp 136–175. 112. Taylor CR: Report of the Immunohistochemistry Steering Committee of the Biological Stain Commission. Proposed format: Package insert for immunohistochemistry products. Biotech Histochem. 67:323, 1992.
38.e3
113. Burry RW: Specificity controls for immunocytochemical methods. J Histochem Cytochem. 48:163, 2000. 114. Swaab DF, Pool CW, VanLeenwen FW: Can specificity ever be proven in immunocytochemical staining? J Histochem Cytochem. 25:388, 1977. 115. Willingham MC: Conditional epitopes: Is your antibody always specific? J Histochem Cytochem. 47:1233, 1999. 116. Skacel M, Skilton B, Peltay JD, et al: Tissue microarrays: A powerful tool for high-throughput analysis of clinical specimens: A review of the method with validation data. Appl Immunohistochem Mol Morphol. 10:1, 2002. 117. Mengel M, Kreipel M, von Wasielewski R: Rapid and large-scale transition of new tumor biomarker to clinical biopsy material by innovative tissue microarray systems. Appl Immunohistochem Mol Morphol. 11:261, 2003. 118. Battifora H: Assessment of antigen damage in immunohistochemistry: The vimentin internal control. Am J Clin Pathol. 96:669, 1991. 119. Taylor CR, Levenson RM: Quantification of Immunohistochemistry—issues concerning methods, utility and semiquantitative assessment. Histopathology. 49:411, 2006. 120. Taylor CR: Quantifiable Internal Reference Standards for Immunohistochemistry; the measurement of quantity by weight. Applied Immunohistochem Mol Morphol. 14:253, 2006. 121. Taylor CR, Becker KF: Liquid Morphology: Immunochemical Analysis of Proteins extracted from Formalin Fixed Paraffin Embedded Tissues: combining Proteomics with Immunohistochemistry. Appl Immunohistochem Mol Morphol. 19:1–9, 2011. 122. Conan Doyle A: The Adventures of Sherlock Holmes, The Adventure of the Beryl Coronet. Strand Mag. 1892. 123. Battifora H, Kopinski M: The influence of protease digestion and duration of fixation on the immunostaining of keratins: A comparison of formalin and ethanol fixation. J Histochem Cytochem. 34:1095, 1986. 124. Cuevas EC, Bateman AC, Wilkins BS, et al: Microwave antigen retrieval in immunocytochemistry: A study of 80 antibodies. J Clin Pathol. 47:448, 1994. 125. Leong AS-Y, Gilham PN: The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology. 21:266, 1989. 126. Shi S-R, Cote RJ, Chaiwun B, et al: Standardization of immunohistochemistry based on antigen retrieval technique for routine formalin-fixed tissue sections. Appl Immunohistochem. 6:89, 1998. 127. Yokoo H, Nakazato Y: A monoclonal antibody that recognizes a carbohydrate epitope of human protoplasmic astrocytes. Acta Neuropathol. 91:30, 1996. 128. Prento P, Lyon H: Commercial formalin substitutes for histopathology. Biotech Histochem. 72:273, 1997. 129. Williams JH, Mepham BL, Wright DH: Tissue preparation for immunocytochemistry. J Clin Pathol. 50:801, 1997. 130. Allison RT, Best T: p53, PCNA and Ki-67 expression in oral squamous cell carcinomas: The vagaries of fixation and microwave enhancement of immunocytochemistry. J Oral Pathol Med. 27:434, 1998. 131. Miller RT, Kubier P, Reynolds B, et al: Blocking of endogenous avidin-binding activity in immunohistochemistry. Appl Immunohistochem Mol Morphol. 7:63, 1999. 132. Zhang PJ, Wang H, Wrona EL, et al: Effects of tissue fixatives on antigen preservation for immunohistochemistry: A comparative study of microwave antigen retrieval on Lillie fixative and neutral buffered formalin. J Histotechnol. 21:101, 1998. 133. Cote RJ, Shi Y, Groshen S, et al: Association of p27kip1 levels with recurrence and survival in patients with stage C prostate carcinoma. J Natl Cancer Inst. 90:916, 1998. 134. Grabau KA, Nielsen O, Hansen S: Influence of storage temperature and high-temperature antigen retrieval buffers on results of immunohistochemical staining in sections stored for long periods. Appl Immunohistochem. 6:209, 1998. 135. Jacobs TW, Prioleau JE, Stillman IE, et al: Loss of tumor markerimmunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst. 88:1054, 1996. 136. Kato J, Sakamaki S, Niitsu Y: More on p53 antigen loss in stored paraffin slides. N Engl J Med. 333:1507, 1995.
38.e4
Techniques of Immunohistochemistry: Principles, Pitfalls, and Standardization
137. Prioleau J, Schnitt SI: p53 antigen loss in stored paraffin slides. N Engl J Med. 332:1521, 1995. 138. Malmstrom PU, Wester K, Vasko J, et al: Expression of proliferative cell nuclear antigen (PCNA) in urinary bladder carcinoma: Evaluation of antigen retrieval methods. APMIS. 100:988, 1992. 139. Wester K, Wahlund E, Sundstrom C, et al: Paraffin section storage and immunohistochemistry. Appl Immunohistochem Mol Morphol. 8:61, 2000. 140. Shi S-R, Liu C, Taylor CR: Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis. J Histochem Cytochem. 55:105, 2007. 141. Shi S-R, Liu C, Pootrakul L, et al: Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry. Am J Clin Pathol. 129:358, 2008. 142. Yamashita S, Okada Y: Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections. J Histochem Cytochem. 53:1421, 2005. 143. Shi SR, Taylor CR, editors: Immunohistochemistry and Antigen retrieval Based Research and Diagnosis, New Jersey, 2010, Wiley. 144. Mason JT, O’Leary TJ: Effects of formaldehyde fixation on protein secondary structure: A calorimetric and infrared spectroscopic investigation. J Histochem Cytochem. 39:225, 1991. 145. Igarashi H, Sugimura H, Maruyama K: Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin-embedded tissue sections. APMIS. 102:295, 1994. 146. Kawai K, Serizawa A, Hamana T, et al: Heat-induced antigen retrieval of proliferating cell nuclear antigen and p53 protein in formalin-fixed, paraffin-embedded sections. Pathol Int. 44:759, 1994. 147. Rait VK, O’Leary TJ, Mason JT: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I— Structural and functional alterations. Lab Invest. 84:292, 2004. 148. Rait VK, Xu L, O’Leary TJ, et al: Modeling formalin fixation and antigen retrieval with bovine pancreatic RBase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. Lab Invest. 84:300, 2004. 149. Sompuram AR, Vani K, Messana E, et al: A molecular mechanism of formalin fixation and antigen retrieval. Am J Clin Pathol. 121:190, 2004. 150. Sompuram SR, Vani K, Bogen SA: A molecular model of antigen retrieval using a peptide array. Am J Clin Pathol. 125:91, 2006. 151. Yamashita S, Okada Y: Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry. J Histochem Cytochem. 53:13, 2005. 152. Evers P, Uylings HB: Microwave-stimulated antigen retrieval is pH and temperature dependent. J Histochem Cytochem. 42:1555, 1994. 153. Biddolph SC, Jones M: Low-temperature, heat-mediated antigen retrieval (LTHMAR) on archival lymphoid sections. Appl Immunohistochem Mol Morphol. 7:289, 1999. 154. Miller RT, Swanson PE, Wick MR: Fixation and epitope retrieval in diagnostic immunohistochemistry: A concise review with practical considerations. Appl Immunohistochem Mol Morphol. 8:228, 2000. 155. Namimatsu S, Ghazizadeh M, Sugisaki Y: Reversing the effects of formalin fixation with citraconic anhydride and heat: A universal antigen retrieval method. J Histochem Cytochem. 53:3, 2005. 156. Shi SR, Liu C, Young L, Taylor CR: Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods. Biotech Histochem. 82:301, 2007. 157. Boon ME, Kok LP, Moorlag HE, et al: Accelerated immunogoldsilver and immunoperoxidase staining of paraffin sections with the use of microwave irradiation: Factors influencing results. Am J Clin Pathol. 91:137, 1989. 158. Chiu KY: Use of microwaves for rapid immunoperoxidase staining of paraffin sections. Med Lab Sci. 44:3, 1987.
159. Leong AS-Y, Milios J: Rapid immunoperoxidase staining of lymphocyte antigens using microwave irradiation. J Pathol. 148:183, 1986. 160. Adams JC: Heavy metal intensification of DAB-based HRP reaction product. J Histochem Cytochem. 29:775, 1981. 161. Hsu SM, Soban E: Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J Histochem Cytochem. 30:1079, 1982. 162. Lan HY, Mu W, Nikolic-Paterson DJ, et al: A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: Use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J Histochem Cytochem. 43:97, 1995. 163. Van Rooijen N: Six methods for separate detection of two different antigens in the same tissue section. J Histochem Cytochem. 28:716, 1980. 164. Krenacs T, Laszik Z, Dobo E: Application of immunogold-silver staining and immunoenzymatic methods in multiple labeling of human pancreatic Langerhans islet cells. Acta Histochem. 85:79, 1989. 165. Lehr HA, van der Loos CM, Teeling P, et al: Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem. 47:119, 1999. 166. Herman GE, Elfont EA, Floyd AD: Overview of automated immunostainers. Methods Mol Biol. 34:383, 1994. 167. Le Neel T, Moreau A, Laboisse C, et al: Comparative evaluation of automated systems in immunohistochemistry. Clin Chim Acta. 278:185, 1998. 168. Fetsch PA, Abati A: Overview of the clinical immunohistochemistry laboratory: Regulations and troubleshooting guidelines. Methods Mol Biol. 115:405, 1999. 169. Moreau A, Le Neel T, Joubert M, et al: Approach to automation in immunohistochemistry. Clin Chim Acta. 278:177, 1998. 170. Bauer KD, Hawes D, de la Torre-Bueno J, et al: Analysis of occult bone marrow metastases using automated cellular imaging. Mod Pathol. 13:220A, 2000. 171. Makarewicz K, McDuffe L, Shi S-R, et al: Immunohistochemical detection of occult metastases using an automated intelligent microscopy system. Presented at the 88th annual meeting of the American Association of Cancer Research, San Diego, CA, 1997, p 269. 172. van der Ploeg M, Duijndam WAL: Matrix models: Essential tools for microscopic cytochemical research. Histochemistry. 84:283, 1986. 173. Riera J, Simpson JF, Tamayo R, et al: Use of cultured cells as a control for quantitative immunocytochemical analysis of estrogen receptor in breast cancer: The Quicgel method. Am J Clin Pathol. 111:329, 1999. 174. Ingram M, Tachy GB, Ward BR, et al: Tissue engineered tumor models. Biotechnic and Histochemistry. 85:213–219, 2010. 175. Kaur P, Ward B, Saha, B, et al: Human Breast Cancer Histioid: An in vitro 3D co-culture model that mimics breast tumor tissue. J Histochem Cytochem. 59:1087–1100, 2011. 176. Martin W, Chon A, Fabiono A, et al: Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol. 24:1016, 2000. 177. Rickers RR, Malinisk RM: Intralaboratory quality assurance of immunohistochemical procedures: Recommended practices for daily application. Arch Pathol Lab Med. 113:673, 1989. 178. Werner M, Von Wasielewski R, Komminoth P: Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol. 105:253, 1996. 179. Kawai K, Osamura RY: Antigen retrieval versus amplification techniques in diagnostic immunohistochemistry. In Shi S-R, Gu J, Taylor CR, editors: Antigen retrieval techniques: Immunohistochemistry and molecular morphology, Natick, MA, 2000, Eaton, pp 249–253. 180. Merz H, Ottesen K, Meyer W, et al: Combination of antigen retrieval techniques and signal amplification of immunohistochemistry in situ hybridization and FISH techniques. In Shi S-R, Gu J, Taylor CR, editors: Antigen retrieval techniques: Immunohistochemistry and molecular morphology, Natick, MA, 2000, Eaton, pp 219–248.
C H A P T E R 2
MOLECULAR ANATOMIC PATHOLOGY: PRINCIPLES, TECHNIQUES, AND APPLICATION TO IMMUNOHISTOLOGIC DIAGNOSIS MARINA N. NIKIFOROVA, YURI E. NIKIFOROV
General Principles of Molecular Biology 39 Genetic Polymorphism and Mutations 41 Specimen Requirements for Molecular Testing 42 Common Techniques for Molecular Analysis 42 Detection of Small-Scale Mutations 49 Detection of Chromosomal Rearrangements 50 Detection of Chromosomal Deletions/Loss of Heterozygosity Analysis 51 Detection of Microsatellite Instability 52 DNA-Based Tissue Identity Testing 54 Summary 55
General Principles of Molecular Biology Immunohistochemistry (IHC) is a common technique used for the detection of protein expression in various tissue samples. In the modern pathology practice, this methodology is expanded and complemented by molecular techniques that test for changes in nucleic acids—in effect, DNA and RNA—to assist the immunohistologic diagnosis. Many of the chapters in this book refer to theranostic and genomic principles that can be investigated with immunohistology and used directly for patient care. The underpinning of these immunohistologic tests requires an understanding of the molecular abnormalities of these disease states and how molecular methods apply
to their study. In addition, the molecular methods discussed here may be valuable in diagnosis when immunohistologic results are nonspecific.
Deoxyribonucleic Acid Genetic information in human cells is encoded in deoxyribonucleic acid (DNA), which is primarily located in the nucleus of each cell. DNA is a doublestranded molecule that consists of two complementary strands of linearly arranged nucleotides, each composed of a phosphorylated sugar and one of four nitrogencontaining bases: adenine (A), guanine (G), thymine (T), or cytosine (C). The order of these four bases encodes genetic information. Two strands of DNA run in opposite directions and are held together through pairing between specific bases—in effect, between adenine and thymine (A : T pairing) and guanine and cytosine (G : C pairing)—that forms a double-stranded helix. As a result, the nucleotide sequence of one DNA strand is complementary to the nucleotide sequence of the other DNA strand.1 The human genome contains approximately 3 billion base pairs (bp) of DNA. The DNA is folded to fit within the nucleus. It is divided among chromosomes and is efficiently packed into chromatin by histones and other accessory proteins. Each normal somatic cell contains two copies of 22 different somatic chromosomes and two sex chromosomes, either XX or XY. Less than 5% of DNA actually encodes protein and other functional products, such as transfer RNA (tRNA), ribosomal RNA (rRNA), micro-RNA (miRNA), and other small nuclear RNAs.2 The majority of human DNA (>95%) consists of noncoding sequences, typically repetitive sequences such as minisatellites, microsatellites, short interspersed elements, and long interspersed elements. Microsatellites are short tandem repeats, and each repeat is from 1 to 13 bp long. Minisatellites are tandemly repeated DNA sequences with a repeat unit of 14 to 500 bp. Microsatellite and minisatellite repeats 39
40
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
are also known as short tandem repeats (STRs). Highly repetitive sequences that contain thousands of repeated units are also found at the telomeric ends of the chromosomes and in the area of the centromere; they play a role in establishing and maintaining chromosome structure and stability. To decode genetic information, the DNA is copied, or transcribed, into mRNA, which is then translocated into the cytoplasm, where it governs translation into protein (Fig. 2-1). Genes are segments of genomic DNA that encode proteins and other functional products. Each gene is typically present in a cell in two copies, one on a maternal and another on a paternal chromosome. Current estimations suggest that about 25,000 distinct genes are present in the human genome. Each gene typically consists of exons, which are proteincoding sequences, and introns, which are noncoding sequences located between the coding regions (see Fig. 2-1).3 Transcription initiation and termination codons flank the portion of a gene that codes for a protein. Gene transcription and silencing are facilitated by promoters and enhancers, which are DNA regions typically located nearby and “upstream” from the gene they regulate, although they may also be located at a great distance.
Ribonucleic Acid Ribonucleic acid (RNA) is a single-stranded molecule that consists of a chain of nucleotides on a sugarphosphate backbone. However, the sugar in RNA is ribose, rather than deoxyribose, and thymine is replaced by uracil. RNA is more susceptible to chemical and enzymatic hydrolysis and is less stable than DNA.4 Several types of RNA exist, and each is different in its structure, function, and location. The most abundant types of RNA are rRNA and tRNA, which comprise up to 90% of the total cellular RNA. They are predominantly located in the cytoplasm and have important functions in protein synthesis: rRNA in a complex with specific proteins forms ribosomes on which proteins are synthesized, and tRNA is responsible for the carrying and adding of the amino acid to the growing polypeptide chain during protein synthesis. Messenger RNA (mRNA) comprises 1% to 5% of total RNA, and each mRNA molecule is a copy of a specific gene and functions to transfer genetic information from the nucleus to the cytoplasm, where it serves as a “blueprint” for protein synthesis. The gene sequence is first transcribed into the primary RNA transcript by RNA polymerase. This transcript is an exact complementary copy of the
Gene Promoter region 5
Intron
Intron Exon 2
Exon 1
UTR Exon (n)
3
Transcription
mRNA 5
Exon 1
Exon 2
Exon (n)
AAAAA 3
Translation
Protein Figure 2-1 Gene structure on the DNA level and the process of transcription and translation. Genes are segments of DNA that contain protein-coding regions (exons), noncoding regions (introns), and regulatory regions that include promoter and enhancer sequences. In the mature mRNA, only protein-coding parts—that is, exons—are preserved, and they contain the genetic information needed for the protein. UTR, Untranslated region.
Genetic Polymorphism and Mutations
gene and includes all exons and introns. Next, intron portions are spliced out from the primary RNA transcript, while it is processed into mature mRNA (see Fig. 2-1).5 Other types of RNA include heterogeneous RNA (hnRNA) and small nuclear RNA (snRNA). Recently, several classes of short RNAs have been discovered, one of which is miRNAs, which are short (19 to 22 nt) single-stranded molecules that function as negative regulators of the coding gene expression.6,7
Protein The abundance of a particular protein within each cell depends on the expression levels of the gene (i.e., how many mRNA copies are transcribed from DNA) and the stability of the protein. Proteins are synthesized on ribosomes in the cell cytoplasm, and mRNA carries genetic information to the ribosomes, which then direct the assembly of polypeptide chains by reading a threeletter genetic code on the mRNA and pairing it with a complementary tRNA linked to an amino acid. The three-bases code, called the codon, defines which specific amino acid is added by the tRNA to the growing polypeptide chain. After synthesis, the protein undergoes posttranslational modification, such as chain cleavage, chain joining, addition of nonprotein groups, and folding into a complex, tridimensional structure.
Genetic Polymorphism and Mutations Variations in DNA sequence are common among individuals. Genetic polymorphism is an alteration in DNA sequence found in the general population at a frequency greater than 1%. Polymorphism may be associated with a single nucleotide change, known as a single-nucleotide polymorphism (SNP), or with variation in a number of repetitive DNA sequences, such as minisatellites or microsatellites, called length polymorphism. Usually, genetic polymorphism does not directly cause a disease, but, rather, it may serve as a predisposing factor. Mutation is a permanent alteration of the DNA sequence of a gene that is found in less than 1% of the population and most likely causes disease. Mutations can be either germline, present in all cells of the body, or somatic, found in tumor cells only. Somatic mutations may provide a selective advantage for cell growth and may initiate cancer development, but they are not transmitted to offspring. In contrast, germline mutations will be passed on to the next generation. Mutations located in a coding sequence, in the regulatory elements, or at the intron-exon boundaries of a gene may affect transcription and/or translation and may result in alteration of the protein structure and function. The sequencing of cancer genomes has revealed that most mutations occur in genes in which the products affect signaling pathways that control important cell functions. It is estimated that most mutations (90%) result in activation of a gene, typically forming an oncogene such as RAS or BRAF; smaller proportions of
41
mutations (10%) lead to loss of function of a tumor suppressor gene, such as TP53. A current list of somatic mutations in cancer can be viewed at the Catalogue of Somatic Mutations in Cancer (COSMIC) database, which documents somatic cancer mutations reported in the literature and identified during the Cancer Genome Project (www.cancer. sanger.ac.uk/cancergenome/projects/cosmic). Not all somatic mutations have a clear biologic effect. Mutations that increase cell growth and survival and are positively selected for tumor development are called driver mutations. Conversely, genetic alterations that do not confer a selective growth advantage to the cell and do not have functional consequences are known as passenger mutations. They may be coincidently present in a cell that acquires a driver mutation and are carried along during clonal expansion, or they occur during clonal expansion of a tumor. It is generally believed that only a small fraction of mutations in a given tumor are represented by driver mutations; thus it has been estimated that a typical human tumor carries approximately 80 mutations that change the amino acid sequences of proteins, of which less than 15 are driver mutations.8 Mutations can be classified based on size and structure into small-scale mutations (sequence mutations) and large-scale mutations (chromosomal alterations). Small-scale mutations include point mutations, which are single-nucleotide substitutions, and small deletions and insertions. Point mutations can be further classified as missense mutations, which lead to amino acid change and result in production of abnormal protein; silent mutations that do not lead to a change in amino acids; and nonsense mutations, when substitution of a single nucleotide results in formation of a stop codon and a truncated protein. Deletion and insertion mutations can result either in deletion or insertion of a number of nucleotides divisible by 3, leading to a change in the number of amino acids and a shorter or longer protein, or it leads to insertion or deletion of a number of nucleotides not divisable by 3, which will cause a shift in the open reading frame of the gene; this affects multiple amino acids and typically produces a stop codon and protein truncation. Large-scale mutations can be due to (1) numerical chromosomal change, that is, loss or duplication of the entire chromosome; (2) chromosomal rearrangement, translocations or inversions that result in an exchange of chromosomal segments between two nonhomologous chromosomes, or within the same chromosome, and typically lead to activation of specific genes located at the fusion point; (3) amplification, when a particular chromosomal region is repeated multiple times on the same chromosome or different chromosomes, resulting in the increased copy number of the gene located within this region; and (4) chromosomal deletion or loss of heterozygosity (LOH), when deletion of a discrete chromosomal region leads to loss of a tumor suppressor gene residing in this area. Functional consequences of each mutation type vary. Generally, mutations result in either activation of the gene— typically forming an oncogene, such as KRAS or RET— or loss of function of a tumor suppressor gene (TP53, PTEN, CDKN1A).
42
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
Specimen Requirements for Molecular Testing Molecular testing in surgical pathology can be performed on a variety of clinical samples, including freshor snap-frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cytology specimens (fresh and fixed fine needle aspiration [FNA] samples), blood, bone marrow, and buccal swabs. Specimen requirement depends on the type of disease and on molecular techniques used for the analysis. Peripheral blood lymphocytes or cells from buccal swabs are typically used for detection of germline mutations responsible for a given inherited disease, such as RET mutations in familiar medullary thyroid carcinoma. Blood and bone marrow biopsy materials are frequently used for detection of chromosomal rearrangements in hematologic malignancies (BCR/ABL1 in acute lymphocytic leukemia). Tumor tissue samples are required to detect somatic mutations such as KRAS point mutation in colorectal cancer, SS18/SSX1 rearrangement in synovial sarcomas, or EGFR mutation in lung adenocarcinomas. Fresh- or snap-frozen tissue is the best sample for testing because freezing minimizes the degradation and provides excellent quality of DNA, RNA, and protein. Such specimens can be successfully used for any type of molecular analysis, including detection of somatic mutations, chromosomal rearrangements, geneexpression arrays, miRNA profiling, and so on. FFPE tissue samples or fixed cytology specimens do not provide such highly preserved nucleic acids; however, these specimens can be successfully used for molecular testing in many situations, particularly for tests that require DNA. Usually, 10% neutral-buffered formalin (NBF) is most commonly used for tissue fixation. However, it leads to fragmentation of DNA; therefore molecular assays need to be optimized when FFPE tissue samples are used by amplification of shorter DNA fragments (250 to 300 bp in length). Prolonged (>24 to 48 h) fixation in 10% NBF adversely affects the quality of nucleic acids; therefore specimens should preferably not be fixed for prolonged times. Tissue specimens that were processed by using bone decalcifying solution cannot be used for molecular analysis because of extensive DNA fragmentation.9 Similarly, it is not recommended to perform molecular testing on specimens exposed to fixatives that contain heavy metals (e.g. Zenker’s, B5, acetic acid-zinc-formalin) because of inhibition of DNA polymerases and other enzymes that are essential for molecular assays. RNA molecules are less stable than DNA and are easily degraded by a variety of ribonuclease enzymes present in abundance in the cell and environment. Therefore only freshly collected or frozen samples are considered to be universally acceptable for RNA-based testing. RNA isolated from FFPE tissue is of poor quality and can be used for some but not all applications, particularly in a setting of clinical diagnostic testing. The amount of tissue required for molecular testing depends on the sensitivity of a particular technique and on the purity of the tumor sample. When selecting a
sample for molecular testing, a representative hematoxylin and eosin (H&E) slide of the tissue must be reviewed by a pathologist to identify a target and determine the purity of the tumor; that is, the proportion of tumor cells and benign stromal and inflammatory cells in the area selected for testing must be evaluated. Manual or laser-capture microdissection can be performed by using unstained tissue sections under the guidance of an H&E slide to enrich the tumor cell population. The minimum percentage of tumor cells required for molecular testing depends on the methodology being used for analysis. In general, a minimum tumor cellularity of 50% and at least 300 to 500 tumor cells are required for Sanger sequencing. For molecular testing of hematologic specimens, blood and bone marrow should be collected in the presence of the anticoagulants ethylendiaminetetraacetic acid (EDTA) or acid-citrate-dextrose (ACD), but not heparin, because even a small residual concentration of heparin will inhibit polymerase chain reaction (PCR) amplification. Conventional cytogenetic analysis requires fresh tissue. Fluorescence in situ hybridization (FISH) can be performed on a variety of specimens including frozen tissue sections, touch preps, paraffin-embedded tissue sections, and cytology slides.
Common Techniques for Molecular Analysis Polymerase Chain Reaction PCR is an amplification technique most frequently used in molecular laboratories. The introduction of PCR has dramatically increased the speed and accuracy of DNA and RNA analysis, and the technique is based on exponential and bidirectional amplification of DNA sequences by using a set of oligonucleotide primers.10 Every PCR run must include the DNA template, two primers complementary to the target sequence, four deoxynucleotide triphosphates—dATP, dCTP, dGTP, and dTTP—DNA polymerase, and magnesium chloride (MgCl2) mixed in the reaction buffer. Three steps take place in the PCR cycle (Fig. 2-2). First, the reaction mixture is heated to a high temperature (95° C), which leads to DNA denaturing, or separation of the double stranded DNA into two single strands. The second step involves annealing of primers, in which the reaction is cooled to 55° C to 65° C to allow primers to attach to their complementary sequences. The third step is DNA extension, in which the reaction is heated to 72° C to allow the enzyme DNA polymerase to build a new DNA strand by adding specific nucleotides to the attached primers. These three steps are repeated 35 to 40 times, and during each cycle, the newly synthesized DNA strands serve as a template for further DNA synthesis. This approach results in the exponential increase in the amount of a targeted DNA sequence and production of 107 to 1011 copies from a single DNA molecule. The efficiency of PCR amplification depends on many factors, which include the quality of the isolated
Common Techniques for Molecular Analysis
5
43
3 DNA
3
5
5
3 Step 1: denaturation
Repeat 3-step cycle 35−40 times
(94° C) 3
5
5
3 Step 2: annealing (55° C−65° C)
3
5
5
3 Step 3: extension (72° C)
3
5
5
3
3
5
>107 of the PCR products Figure 2-2 Schematic representation of the polymerase chain reaction (PCR). A three-step cycle—denaturation, annealing, and extension— is repeated 35 to 40 times to generate more than 107 copies of the targeted DNA fragment.
DNA template, size of the PCR product, optimal primer design, and optimal conditions of the reaction. Quality DNA allows amplification of long products (as high as 3 to 5 kb). However, when dealing with DNA of suboptimal quality—that is, when DNA is isolated from fixed tissue or cytology preparation—the reliable amplification can be achieved on only relatively short DNA sequences (400 to 500 bp or shorter). Once the PCR procedure is complete, the products of amplification should be visualized for analysis and interpretation. A simple way to achieve this is to use agarose gel electrophoresis and ethidium bromide staining. However, this method cannot separate amplification products that differ in size by only few nucleotides; finer separation can be achieved by using polyacrylamide gel or capillary gel electrophoresis (Fig. 2-3). PCR amplification followed by gel electrophoresis is frequently used for detection of small deletions or insertions, microsatellite instability, and LOH. For detection of point mutations, the PCR products should be interrogated by other molecular techniques.
Reverse Transcription Polymerase Chain Reaction Reverse transcription PCR (RT-PCR) is a modification of the standard PCR technique that can be used to amplify mRNA. As a first step, isolated mRNA is converted to a complementary DNA (cDNA) molecule using an RNA-dependent DNA polymerase, also known as reverse transcriptase, during a process called reverse transcription. The complementary DNA can be used as any other DNA molecule for PCR amplification. The primers used for cDNA synthesis can be either non– sequence specific, a mixture of random hexamers or oligo-dT primers, or sequence specific (Fig. 2-4). Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool. The oligo-dT primers are complementary to the poly-A tails of the mRNA molecules and allow synthesis of cDNA only from mRNA molecules. Sequence-specific primers are the most restricted
44
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
AGAROSE GEL ELECTROPHORESIS
L
T1
T2
NC
PCR products
RT-PCR analysis is used in molecular laboratories for the detection of gene rearrangements and gene expression. RT-PCR may also be used to amplify several exonic sequences in one reaction, because it can take advantage of the fact that all introns are spliced out in mRNA, leaving the coding sequences intact and significantly shortening the potential product of amplification. However, it is important to recognize that RNA is easily degradable and has to be handled with great care during the entire process of reverse transcription to avoid degradation.12 Amplification of a housekeeping gene has to accompany each RT-PCR reaction as an internal control to monitor the quality and quantity of RNA in a given sample.
Real-Time Polymerase Chain Reaction A
CAPILLARY GEL ELECTROPHORESIS D19S.559 210
B
240
PCR products
Figure 2-3 Postpolymerase chain reaction (PCR) detection of amplification products. A, Agarose gel electrophoresis shows two PCR products obtained by using DNA from two tumor samples, T1 and T2, which are of similar size. L, DNA ladder (size marker); NC, negative control. B, Capillary gel electrophoresis shows two PCR products of different sizes.
because they are designed to bind selectively to the mRNA molecules of interest, making the entire process of reverse transcription target specific. Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or as two separate reactions.11 RT-PCR performed on freshfrozen tissue provides quality amplification and reliable results. However, when FFPE tissue is used for RT-PCR analysis, the results vary and depend on the level of RNA degradation and length of PCR amplicon. To achieve more stable RT-PCR amplification from FFPE tissues, the target is typically chosen to be less than 150 to 200 nt long.
Real-time PCR uses the main principles of conventional PCR but detects and quantifies the PCR product in real time as the reaction progresses. In addition to all components of conventional PCR, real-time PCR uses fluorescently labeled molecules for the visualization of PCR amplicons. It can be performed in two main formats, with incorporation of DNA dyes such as SYBR Green 1, SYTO9, EvaGreen, or LC Green into the PCR product or taking advantage of fluorescently labeled probes (fluorescence resonance energy transfer [FRET] hybridization probes, TaqMan probes, etc.) annealing to the PCR product.13,14 During PCR, the increasing amounts of fluorescence that result from exponential increase in the amount of amplified DNA sequence are detected by a PCR instrument. The instrument software allows construction of an amplification plot of fluorescence intensity versus cycle number. During the early cycles, the amount of PCR product is low, and fluorescence is not sufficient to exceed the baseline. As the PCR product accumulates, the fluorescence signal will cross the baseline and increase exponentially (Fig. 2-5). At the end of the reaction, the fluorescence reaches a plateau because most of the reagents have been consumed. The real-time detection of amplification allows the detection of PCR product in real time and obviates the need for subsequent gel electrophoresis. Another advantage is that it can use post-PCR melting curve analysis to detect sequence variations at the specific locus. For example, in the LightCycler probe format, binding of hybridization probes to the PCR product in a head-totail fashion initiates the FRET from one probe to another, and detected fluorescence is proportional to the amount of amplified product.15 During postPCR melting curve analysis, the PCR product is gradually heated, and fluorescence is measured at each temperature point. During this process, even a single mismatch between the labeled probe and the amplified sequence will significantly reduce the melting temperature (Tm), defined as the temperature at which 50% of the double-stranded DNA becomes single-stranded. Therefore the presence of a point mutation or SNP in the region covered by a fluorescent probe will be detected as an additional Tm peak on melting curve analysis (see Fig. 2-5).
Common Techniques for Molecular Analysis
45
AAAAA(A)n
mRNA
Reverse transcription
Gene-specific priming
Oligo(dT) priming AAAA(A)n TTTTTT
AAAA(A)n
Random hexamer priming
N6
N6
AAAA(A)n N6
cDNA
PCR amplification Figure 2-4 Principles of reverse-transcriptase PCR (RT-PCR). During reverse transcription, mRNA is used to build a complementary DNA molecule (cDNA) by using either gene-specific, oligo (dT), or random hexamer (N6) priming. The cDNA then can be used as a template for PCR amplification.
Quantitative PCR (qPCR) is a variation of real-time PCR that can be used for evaluation of gene expression levels or gene copy numbers. The quantitative assessment of the initial template used for PCR amplification can be done by comparison of the amount of PCR product of the target sequence with the PCR products generated by amplification of the known quantities of DNA or cDNA. Real-time PCR is frequently used in molecular laboratories, because it is a rapid, less laborious technique compared with other techniques, and it does not require processing of samples after PCR amplification; this minimizes the time of the procedure and risk of contamination by previous PCR products.
PCR–Restriction Fragment Length Polymorphism Analysis Restriction enzymes, or restriction endonucleases, are enzymes that cut DNA at specific nucleotide sequences known as restriction sites. The restriction sites are usually 4 to 8 nt long and are frequently palindromic (the DNA has the same sequences in both directions). The restriction fragment length polymorphism (RFLP) analysis exploits the ability of restriction enzymes to cut DNA at these specific sites. If a given DNA sequence variation, such as a point mutation, alters the restriction site for a specific enzyme, either creating or destroying it, this will change the size of the PCR product, which can be detected by gel electrophoresis. This method is frequently used for detection of known point mutations or sequence polymorphisms (SNPs).16 In addition, it can be used for separation between two amplified sequences that have high similarity in their nucleotide composition. Figure 2-6 illustrates the use of PCR-RFLP to differentiate between SS18/SSX1 and SS18/SSX2 rearrangements, which are common in synovial sarcomas.
Single-Strand Conformation Polymorphism Single-strand conformation polymorphism (SSCP) analysis is a post-PCR technique that can be used to screen for mutations that are not limited to a single hotspot but are randomly distributed throughout the exons. Following the PCR amplification of the region of interest, the PCR products are denatured by heat and exposure to denaturing buffer and are subjected to polyacrylamide gel electrophoresis. If mutation is present within the amplified sequence, it will change the folding conformation of the sequence and alter its electrophoretic mobility. As a result, the wild-type and mutant sequences will migrate differently in the gel. The PCR-SSCP analysis can be used as a screening tool for point mutations and small deletions and insertions.17 However, it does not allow detection of the precise nucleotide change, and this requires usage of an additional technique, such as DNA sequencing.
Allele-Specific Polymerase Chain Reaction and Allele-Specific Hybridization Allele-specific PCR (AS-PCR) and allele-specific hybridization (ASH), also known as dot-blot analysis, are frequently used for detection of point mutations and sequence polymorphisms and are based on the fact that PCR amplification and detection of PCR product by annealing of a probe are critically dependent on the perfect base pairing of primers or probes with template DNA. Under strict PCR conditions, even one nucleotide mismatch at the 3′ end of the primer will prevent PCR amplification. Similarly, strict hybridization conditions and a certain design of oligonucleotide probe will prevent annealing to the PCR product that differs by a single nucleotide. For AS-PCR, the amplification of target DNA is performed in two reactions: one reaction
46
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
of BRAF18 or several of the most common types of point mutations at codons 12 and 13 of KRAS.19 The methods are very sensitive and must be used with caution because they can detect small (<1%) subclones with mutation within a heterogeneous tumor sample.
AMPLIFICATION CURVE 0.578 Plateau
Fluorescence signal (Rn)
0.528 0.478 Exponential phase
0.428
DNA Sequencing Analysis: Conventional Sequencing
0.378 0.328 0.278
Baseline
0.226 0.178 1
5
10
15
A
20
25
30
35
40
Number of cycles MELTING PEAKS (Tm) Wild-type peak
0.123 0.103 0.083
55 bp
0.063
33 bp
SYT
T CGA
SYT
T CGT
SSX1
0.043 0.023
SSX2
0.003 40
B
Taq I
Mutant peak
X
Fluorescence signal (–d/dT)
0.143
Sanger sequencing is the most common technique used in molecular laboratories to detect the exact nucleotide composition of PCR-amplified DNA fragments. It uses chain terminating dideoxynucleoside triphosphates (ddNTPs), which, upon incorporation into the growing DNA strand, terminate its elongation at a particular nucleotide. This results in a mixture of DNA fragments of various lengths, each corresponding to one of four nucleotides positioned at a specific distance from the beginning of the sequence.20 The mixture of labeled
45
50
55
60
65
70
75
80
Temperature (˚C)
Figure 2-5 Real-time polymerase chain reaction (PCR). A, Amplification plot shows a low amount of fluorescence during the initial cycles of amplification (baseline), followed by an exponential increase in fluorescence during the exponential phase, and finally by a plateau at the end of the PCR reaction. B, Post-PCR melting curve analysis demonstrates a single melting peak in the normal DNA sample (wild-type peak) and two melting peaks in the tumor sample, one at the same melting temperature (Tm) as the wild-type peak and another lower Tm peak as the result of a one-nucleotide mismatch with the probe (mutant peak).
has primers complementary to a normal (wild-type) sequence, and another reaction has a forward primer complementary to a mutant sequence and a reverse primer complementary to a normal DNA sequence. Amplification is detected by gel electrophoresis or by real-time PCR. Normal DNA will show amplification with primers complementary to the normal sequence and no amplification with mutant-specific primer, whereas the mutant allele will show a reverse pattern of amplification. For ASH, the PCR products are directly spotted on the nylon membrane. After denaturing, the membrane is hybridized with the labeled oligonucleotide probe complementary to a mutant sequence. Both AS-PCR and ASH can be used for the detection of a specific nucleotide substitution located at a specific position, such as T to A transversion at nucleotide 1799
98 bp
A L
B
(+)
c1
c2
c3
c4
c5
98 bp
98 bp
C
65 bp 33 bp
Figure 2-6 PCR–restriction fragment length polymorphism detection of SYT/SSX1 and SYT/SSX2 rearrangements in synovial sarcoma. A, Taq I restriction enzyme cuts the SYT/SSX1 fusion DNA into two fragments 55 bp and 33 bp long, whereas the SYT/SSX2 fragment remains uncut at 98 bp in length. B, Five synovial sarcoma DNA samples (c1 through c5) are PCR amplified with primers complementary to both SYT/SSX1 and SYT/SSX2 rearrangement types and reveal a 98-bp amplification band in the agarose gel. C, After digestion with Taq I, the PCR products from tumors c1 and c3 and from the SYT/SSX1 positive control (+) are cut into two fragments, indicating the presence of SYT/SSX1 rearrangement, whereas the rest of the tumor samples remain uncut, consistent with SYT/SSX2.
Common Techniques for Molecular Analysis
47
BRAF T1799A (V600E) mutation G AT T T T G GT C TA G C T ACA G N G A A ATC T C GA T G G AG TG G GT 25 33 41 49 57
Figure 2-7 Sequence electropherogram of the polymerase chain reaction (PCR) product of BRAF exon 15. Two overlapping peaks are shown at position 1799, which is diagnostic of a T→A mutation at this position.
DNA fragments is separated by gel electrophoresis. This method has been adopted for automated platforms, and it uses dideoxynucleotides labeled with different fluorescent dyes. An automated sequence analyzer (ABI 3730; Applied Biosystems [Life Technologies, Carlsbad CA]) detects the order of nucleotides and depicts them as a sequence electropherogram (Fig. 2-7). The automated sequencing analysis is easy to perform and is routinely used in molecular laboratories for detecting various mutations. Pyrosequencing is another and more recently developed sequencing technology.21 It is based on the detection of the light emitted during synthesis of a complementary DNA strand for each added nucleotide. The incorporation of deoxynucleotide triphosphate nucleotide into the DNA strand results in the release of pyrophosphate (PPi) molecule in a quantity equimolar to the amount of incorporated nucleotide. The pyrophosphate molecule is then converted to adenosine triphosphate (ATP), and light is produced; this is detected by the instrument and converted to a peak in a pyrogram trace. Each light signal is proportional to the number of incorporated nucleotides. Pyrosequencing can be used for the detection of point mutations, such as KRAS codon 12/13, and of methylated CpG islands.22,23 It is a fast and accurate technique, although it can only be used to analyze short DNA sequences.
Next-Generation Sequencing Introduction of next-generation sequencing (NGS) technology has enabled high-throughput detection of multiple genetic alterations in both constitutional and cancer genomes. NGS offers simultaneous sequencing of thousands to millions of short nucleic acid sequences in a massive, parallel fashion. It provides clear advantages over the conventional sequencing technique, such as Sanger sequencing, by sequencing large regions of the genome at a lower cost and with higher sensitivity. NGS can be performed at different levels of complexity to include whole genome sequencing, whole exome sequencing, whole transcriptome sequencing (mRNA sequencing), and targeted sequencing of multigene
panels. Whereas large-scale analyses are essential for the discovery projects, it is more than likely that targeted panels will offer further advances in routine molecular diagnostics of various diseases, including cancer. Several commercial NGS platforms, such as Life Technologies Ion Torrent and the MiSeq from Illumina (San Diego, CA) are currently available for targeted sequencing, and the technology will continue to develop. They use a variety of chemistries but share similar processing steps. These platforms rely on principles of clonal amplification of single DNA molecules, spatially separated on a solid surface, and on application of sequencing chemistries.24 First, DNA is either fragmented or preamplified with gene-specific primers. Next, universal adapter sequences are added to the ends of the DNA fragment. These oligonucleotide adapters are complementary to PCR primers used for clonal amplification on a solid surface. The Ion Torrent uses emulsion PCR (emPCR) to generate clonal DNA fragments on 3-µm diameter beads, known as ion sphere particles.25 The MiSeq clonally amplifies DNA directly on the surface of a glass flow cell by bridge PCR.26 Each platform uses different sequencing chemistries for signal detection. The Ion Torrent detects signal by the release of hydrogen ions during sequential nucleotide incorporation, and in a way, the sequencing is performed on a chip that works as a very sensitive pH meter.27 The MiSeq uses reversible dye terminator sequencing by synthesis (SBS) chemistry that enables detection of single bases as they are incorporated into growing DNA strands. Both platforms allow sequencing of library fragments from both ends, referred to as paired-end sequencing. Both platforms provide similar performance characteristics, including sequencing of DNA fragments as high as 200 bp read length, 98% to 99.5% of sequencing accuracy, adequate sequence depth or “coverage” (>500× for multigene panels), and total sequence output as high as 1 gigabase (Gb).28 Importantly, they provide highspeed sequencing (5 to 24 hours), and the sequencing is less expensive compared with larger sequencing platforms. The cost can be even further reduced by molecular bar coding of samples that allows multiplexing
48
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
q11.22
q11.23
q21.11
q21.12
q21.2
q21.1
q21.2
q21.3
92 bp 140,453,130 bp
q22.1
q22.2
q23.1
39 bp 140,453,140 bp
43,517,420 bp
No variants found
No variants found
BRAF p.V600E
A
q22.3
RET p.M918T
T
C
T T
B
T T
C
T
C C C C C C C
T
C C
T T V T A BRAF C G A G A T T T C A C T G T A G C T R
A
S
B
T
K
G W
G
A
T M RET
G
G
C A
A
A
B
Figure 2-8 Targeted next-generation sequencing analysis for a panel of 739 mutations in 46 cancer-related genes (AmpliSeq 1.0 panel, Ion Torrent PGM, Life Technologies). A, BRAF V600E mutation in melanoma tissue specimen. B, RET p.M918T mutation in a thyroid fine needle aspiration sample from a patient with medullary thyroid carcinoma. Mutations are visualized by using the Integrative Genomics Viewer (Broad Institute).
patient samples in one run for high throughput. Also, these instruments provide simplified and user-friendly informatics analysis. Targeted NGS is beginning to be implemented into clinical laboratory practice, and at some point, it can replace existing Sanger sequencing or PCR-based assays. It can be used for detection of germline mutations linked to familial cancer syndromes (APC, RET, BRCA1 and BRCA2, etc.), for detection of therapeutically important mutations (KRAS, BRAF, EGFR, etc.), and for cancer diagnosis and prognostication. Targeted NGS testing provides significant cost savings by simultaneously sequencing multiple genetic targets in multiple patient samples and promises to become an important personalized cancer management tool. For example, the AmpliSeq 1.0 cancer panel on the Ion Torrent PGM allows sequencing for 739 mutations in 46
cancer-related genes in eight tumor samples at once. It requires minimal amounts of DNA (10 ng), which can be obtained even from a small biopsy specimen, and it works well on FFPE tissue samples.29 Examples of BRAF V600E mutation in melanoma and RET M918T mutation in medullary thyroid carcinoma detected by the AmpliSeq cancer panel are shown in Figure 2-8.
Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) is a common technique for the detection of gene rearrangements, regions of chromosome deletion and amplification, and numerical chromosomal abnormalities. It uses fluorescently labeled DNA probes that bind to homologous chromosomal regions and can assay interphase nuclei. FISH can be performed on a variety of tissue specimens
Detection of Small-Scale Mutations
that include frozen tissue or FFPE tissue sections, touch preparations from fresh or frozen tissue samples, cultured cells, and cytologic smears. Optimal probe design is an essential part of the FISH technique. Most commercially available and homemade probes are prepared by selecting clones that contain the inserted fragments of the DNA of interest. The most frequently used clones are cosmids, P1 artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Some DNA probes target the centromeric region of chromosomes; they are ideal for detection of numerical chromosomal abnormalities, such as losses or gains of the entire chromosome. Other probes target a specific region of a chromosome and are used for detection of gene rearrangement, deletion, or amplification. Finally, some probes are designed to hybridize to the full length of a chromosome; these probes can be used for identification of multiple regions of loss or gain on a particular chromosome.30 During the FISH procedure, DNA within cells is placed on a slide, and the fluorescently labeled probes of interest are denatured by incubation at high temperature. The probe is then allowed to hybridize to the target DNA. A series of posthybridization washes follows, aimed at removing the probe excess. Finally, after counterstaining of the nuclei, the probe signal is visualized under the fluorescent microscope. The detection of numerical chromosome changes and regions of gene amplification or deletion typically uses a single probe and one-color FISH. The FISH assay for chromosomal translocations may use two strategies. One is a break-apart probe design, in which a single probe that spans a gene of interest is used and will demonstrate a split signal in the presence of translocation. Another strategy is a fusion probe design, labeled with two different fluorochromes. Chromosomal rearrangement will manifest as a pair of fused signals.31,32 The advantage of the break-apart design is in its ability to detect all possible translocations involving a particular gene, such as all types of translocation of the EWSR1 gene in Ewing sarcoma; however, it cannot identify the fusion partner. In contrast, the fusion probe design detects a specific type of rearrangement.
Comparative Genomic Hybridization Comparative genomic hybridization (CGH) is used for identifying gains and losses of a specific chromosomal region within the whole genome.33-35 The procedure is based on simultaneous hybridization of fluorescently labeled tumor DNA and uniquely labeled normal reference DNA to the preparation of normal human metaphase chromosomes (standard CGH) or to the BAC library (array CGH). The ratio of fluorescent staining between the tumor and normal samples along the chromosomes is scored. The equal ratio will indicate the normal amount of tumor DNA in a given chromosomal region. The decrease in intensity of the tumor-specific fluorochrome will indicate the region of chromosomal loss, and the increase in intensity will label the regions of gain.
49
CGH analysis is used as a supplemental technique to conventional cytogenetics. It appears to be more sensitive than conventional cytogenetics because it is capable of detecting chromosomal alterations down to 1 Mb in size. In addition, it can be performed by using DNA isolated from FFPE specimens.36
DNA Microarrays DNA microarrays can be used to determine expression of multiple genes in a single reaction.37 They use a multiplex spotted microarray technology in which thousands of oligonucleotide probes that correspond to human genes are spotted on a solid surface (gene chip) or on microscopic beads. During the analysis, RNA isolated from a tumor sample is converted into cDNA, labeled, and hybridized to the chip or beads. After hybridization, the microarray is washed to eliminate nonspecific binding and is scanned to measure the amount of fluorescence from each spot. The intensity of signals is proportional to the abundance of specific cDNA sequences in a given specimen. DNA microarrays may also be used for whole-genome analysis of SNPs and for chromosome copy number changes.38
Detection of Small-Scale Mutations A number of different techniques are available for the detection of point mutations and small deletions and insertions. In practice, the choice of the method depends on mutation type, location (known hot spot vs. randomly distributed mutations), required sensitivity, specimen type, and test volume. Detection of a point mutation at a specific hot spot, such as KRAS mutation at codons 12 and 13 or a BRAF mutation at codon 600, can be achieved by a variety of molecular techniques that include real-time PCR amplification and post-PCR melting curve analysis, AS-PCR, direct DNA sequencing, pyrosequencing, PCR-RFLP, and others.18,22,39-45 Virtually all of these methods demonstrate reliable detection of point mutations, such as KRAS mutations in colorectal cancer or BRAF mutations in thyroid cancer. DNA sequencing analysis of PCR products is considered the gold standard for detection of point mutations. The automated sequencing analysis is typically used in molecular laboratories. For example, a heterozygous KRAS mutation in colon cancer will present as two overlapping peaks in the sequencing electropherogram (Fig. 2-9). The sensitivity of this method is in the range of 10% to 30%. Real-time PCR–based detection methods are also frequently used in clinical molecular laboratories. They may be even more preferable because they are fast and are run in a closed PCR system that reduces the risk of contamination. For the detection of mutations, two probes complementary to wild-type sequences are designed so that one of the probes spans the known mutation site, such as codons 12 and 13 in the KRAS gene.44 If no mutation is present, the probe will bind perfectly to the DNA sequence, showing a single peak
50
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
KRAS codon 12 mutation
SSCP analysis of TP53 exon 6
C T TG TG GTA G T TGG A G C TGG TGG CGT A GGC A A G A 94
86
78
G
on post-PCR melting curve analysis (Fig. 2-10). In contrast, if a heterozygous mutation is present, the probe will bind to the mutant DNA with one nucleotide mismatch and will melt (dissociate) earlier, producing two melting peaks, a lower Tm peak for the mutant allele, and a higher Tm peak for the wild-type allele. Rarely, the mutation is homozygous and presents as a single melting peak at a lower Tm. The sensitivity of this method for the detection of point mutations, such as KRAS mutations, is generally similar to those of DNA sequencing.44 For detection of point mutations with multiple hot spots, such as mutations in the TP53 gene, the most commonly used techniques are DNA sequencing and SSCP analysis.17 All exons known to harbor mutations
KRAS CODON 12 MUTATIONS Melting peaks
−(d/dT) Fluorescence (640/530)
0.096
Heterozygous Wild type (GGT) Homozygous mutation (GAT) mutation (GTT)
0.076 0.056 0.036 0.016 45
50
55
60
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17
65
70
75
Figure 2-11 Detection of TP53 exon 6 mutations by using singlestrand conformation polymorphism (SSCP) analysis. Polymerase chain reaction products of 17 tumor samples were amplified with a pair of primers flanking exon 6; one primer was radioactively labeled and electrophoresed in polyacrylamide gel. Sample 2 revealed an additional abnormally migrating band (arrow) indicative of a mutation.
are first amplified in several PCR reactions. Next they are subjected to DNA sequencing and analyzed for the presence of a mutation. Alternatively, the SSCP analysis can be used, and only those PCR products that show abnormal migration patterns in the SSCP gel (Fig. 2-11) are selected for DNA sequencing.46,47 Detection of small deletions and insertions can frequently be achieved by DNA sequencing or by gel electrophoresis of the amplified PCR products.48 For example, small (9 to 18 nt) deletions in exon 19 of the EGFR gene common in lung adenocarcinoma can be detected by using PCR amplification followed by polyacrylamide gel electrophoresis. In the presence of a heterozygous deletion, PCR will amplify an affected shorter allele and an intact wild-type allele. Because shorter DNA sequences migrate at higher speed, the gel will show two PCR bands; however, tumor samples negative for the deletion and normal DNA samples will demonstrate a single band of normal size.
Detection of Chromosomal Rearrangements
0.136 0.116
2
T
Figure 2-9 Detection of KRAS mutation in a colon cancer sample by using DNA sequencing. The electropherogram demonstrates a G→T mutation in codon 12.
0.156
1
70
80
Temperature (˚C) Figure 2-10 Detection of KRAS mutation by using real-time polymerase chain reaction (PCR) and post-PCR melting curve analysis. The graph illustrates a single melting peak of the normal DNA sample (wild type, GGT), two melting peaks in the DNA sample that contain a heterozygous mutation (GAT), and a single, lower Tm peak in DNA from a cell line that contains a homozygous mutation (GTT).
Chromosomal rearrangements are typically detected by either FISH or RT-PCR. FISH is the method of choice for detection of gene rearrangements in FFPE samples, whereas RT-PCR is a reliable and sensitive technique for the detection of fusion transcripts in snap-frozen samples, and it can be performed as conventional or real-time RT-PCR.32,49-51 Real-time RT-PCR has several significant advantages: it is fast, it is performed in a closed system with minimal risk of contamination, it is more specific because of the addition of probes, and it allows quantitation of the fusion transcript. For example, SYT/SSX fusion (translocation t[X;18]) in synovial sarcoma can be reliably detected by FISH (Fig. 2-12) with a break-apart probe design that uses a commercially available mixture of two DNA probes located close to each other and complementary to the SYT gene region. The probes are labeled with specific fluorophores (red and green). Although normal interphase cells will show two fused signals (red/green or
Detection of Chromosomal Deletions/Loss of Heterozygosity Analysis
SYT/18q11.2
Figure 2-12 Detection of the SYT gene rearrangement in synovial sarcoma with fluorescence in situ hybridization. In the break-apart probe design, two probes adjacent to each other and spanning the SYT gene are labeled in red and green. In many cells in this tumor, the nuclei contain one yellow or green/red signal, corresponding to the intact gene, and one green and one red signal at a distance, corresponding to the rearranged gene.
by FISH or by PCR amplification of microsatellite loci followed by capillary gel electrophoresis. As an example, the detection of a 1p deletion in oligodendroglioma by FISH is illustrated in Figure 2-14. A set of commercially available probes consists of two probes, one complementary to the frequently deleted chromosomal region on 1p36 and another, differentially labeled control probe that targets the 1q25 region on the long arm of chromosome 1. When 1p deletion is present, the interphase nuclei will demonstrate two signals that correspond to the control probes and only one 1p36 signal. An alternative PCR-based approach uses the amplification of microsatellite repeats located in the area of 1p deletion. Loci of microsatellite repeats are highly
Primer SYT-F SYT
ACGAA
SSX2
SYT
AA G G A
SSX1
Detection of Chromosomal Deletions/Loss of Heterozygosity Analysis Loss of heterozygosity (LOH) results from a deletion of small or large chromosomal regions and often correlates with the loss of important tumor suppressor genes located in these areas. LOH is typically detected
Fluorescence −d(F2/F1) dT
A
Primer SSX-R
Donor probe Acceptor probe 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0
B
4
8
12
16
20
24
28
32
36
40
44
Cycle number
0.03 Fluorescence (F2/F1)
yellow), cells that carry the SYT rearrangement will demonstrate one fused signal and two split signals (one red and one green). Such probe design allows the detection of all types of rearrangements that involve the SYT gene, but it cannot identify the fusion partner; that is, it cannot establish whether the translocation is of the SYT/SSX1, SYT/SSX2, or some other type. SYT/SSX translocation can also be detected by RT-PCR followed by either a post-PCR detection technique or by real-time RT-PCR.52 For example, one possible real-time RT-PCR design exploits the high similarity between the SSX1 and SSX2 gene sequences to develop a pair of primers that flank the two most common fusion types, SYT/SSX1 and SYT/SSX2, and a probe that is complementary to the SSX1 sequence but has two nucleotides mismatched with the SSX2 sequence (Fig. 2-13, A). Such design allows simultaneous amplification of the two rearrangement types in the same reaction (see Fig. 2-13, B) but distinguishes them based on different melting peaks on post-PCR melting curve analysis (see Fig. 2-13, C). This method can be used for the detection of specific types of SYT/SSX translocation in synovial sarcomas, but its best performance requires the input of quality RNA isolated from snap-frozen tissue.
51
SYT −SSX2
SYT −SSX1
0.02 0.01 0.00 −0.01 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70
C
Temperature (°C)
Figure 2-13 Detection of SYT/SSX rearrangement by real-time polymerase chain reaction (PCR) and post-PCR melting curve analysis followed by real-time reverse transcription PCR (RT-PCR). A, The primers are designed to amplify both SYT/SSX1 and SYT/SSX2 fusion points; one of the probes is designed to be complementary to the SSX1 sequence, but it has a two-nucleotide mismatch with the SSX2 sequence. B, All tumor samples that carry SYT/SSX rearrangement are PCR amplified. C, On post-PCR melting curve analysis, the difference in melting peaks distinguishes the SYT/SSX1 and SYT/SSX2 fusion types.
52
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
Detection of Microsatellite Instability
1p36 1q25
Figure 2-14 Detection of 1p deletion in oligodendroglioma by fluorescence in situ hybridization using a probe that corresponds to the commonly deleted region on 1p36, labeled in red, and a control probe for the 1q25 region, labeled in green. Many of the tumor cells show loss of one red signal and preservation of both green signals.
Microsatellite instability (MSI) is caused by defects in the DNA mismatch repair proteins—MSH2, MLH1, PMS1, PMS2, MSH6, or MSH3—and manifests as an abnormal (increased or decreased) length of microsatellite repeats. The presence of microsatellite instability is a sign of DNA mismatch repair deficiency that can either be inherited, caused by a germline mutation in the MSH2 gene such as in hereditary nonpolyposis colon cancer syndrome, or sporadic, a result of hypermethylation of the MLH1 promoter, such as in sporadic colorectal cancer.55,56 Molecular testing for MSI is usually performed by using PCR amplification of DNA regions that contain microsatellite repeats followed by Microsatellite loci Primer 1 Maternal allele Primer 2
polymorphic and therefore frequently have different numbers of repeats in the population. As a result, the probability is high that in a given individual, the maternal and paternal alleles are different sizes. For LOH detection, two primers are designed to flank the microsatellite region (Fig. 2-15, A). PCR amplification of tumor DNA and normal tissue DNA is performed, and PCR products are subjected to capillary gel electrophoresis (Fig. 2-15, B). The PCR products from normal tissue are used to determine whether a given patient is heterozygous for this locus—that is, the patient has two alleles of different sizes—and therefore is informative for LOH analysis. If the locus is informative, the LOH can be determined as either complete absence or a significant decrease in amplification of one of the two alleles. A complete loss of one the alleles is rarely seen because some nonneoplastic cells are almost always present within the tumor. Therefore, LOH calculation is based on the difference in ratios of two allelic peaks in normal tissue compared with the same peaks in tumor tissue. In most cases, an allele ratio that is less than 0.5 or greater than 2.0 is considered evidence of LOH.53,54 Both FISH and PCR-based detection of LOH can be achieved successfully in FFPE samples, and each method has its own limitations. FISH will show a false-negative result in the event of uniparental disomy, when cancer cells have lost one chromosome in the presence of duplication of another chromosomal allele. The PCR-based LOH analysis is able to detect 1p loss in this situation. However, for optimal performance, the PCR-LOH analysis requires normal cells (isolated from normal tissue or blood) and has to rely on amplification of several markers located in the same region, because some of the microsatellite loci will be noninformative.
Paternal allele
Primer 1
Primer 2
A Sample Name 05 576A4 (NBL) 30
Panel OS D1S. 199 D1S.199 60 90 120
4800 3200 1600 0
sz 99.02 ht 5125
Normal
SO
150
180
150
180
sz 105.63 ht 3524
D1S. 199
06 57781 T
D1S.199 30 6300 4200 2100 0 Tumor
60
90
sz 99.02 ht 6988
120
sz 105.62 ht 295
B Figure 2-15 Detection of loss of heterozygosity in a tumor sample. A, The polymerase chain reaction (PCR) primers are designed to flank a region of microsatellite repeats that has a different number of repetitive units on the maternal and paternal alleles. The allele that contains a smaller number of microsatellite repeats will yield a shorter PCR product, and one with a larger number of repeats will yield a longer PCR product. B, Capillary gel electropherograms demonstrate amplification of two alleles in the normal tissue sample and show virtually complete loss of the larger allele in the tumor sample.
Detection of Microsatellite Instability
capillary gel electrophoresis. Typically, DNA isolated from normal and tumor tissue is separately amplified by PCR with fluorescent-labeled primers. The electrophoretic patterns of PCR products from the normal and tumor tissue are compared to identify insertions or deletions of repetitive units in the tumor sample. The National Cancer Institute guidelines for MSI testing recommend a panel of five microsatellite loci that
include three dinucleotide repeat markers (D2S123, D5S346, D17S250) and two mononucleotide repeat markers (BAT 25 and BAT 26). This panel is knows as the Bethesda panel.55,56 High-frequency MSI (MSI-H) is defined as instability found in two or more of the five markers (Fig. 2-16), and low-frequency MSI (MSI-L) is defined as one unstable marker. The microsatellite stable (MSS) status is established when none of the markers
MSI ANALYSIS Dinucleotide Microsatellite Markers Panel OS Normal D2S. 123 150 180 210 240 270 900 600 300 0 sz 210.43 ht 930 Tumor 150 900 600 300 0
OS 240
300
270
45 9000 6000 3000 0
Panel D5S.346 76 105
OS 135
300
45
Panel D5S.346 76 105
165
OS 135
80 600 400 200 0
195
97.5 2400 1500 800 0
165
97.5
195
sz 112.71 ht 5210
1200 800 400 0
100 4500 3000 1500 0
128
SO 144
160
Panel BAT25 112.5 127.5
OS 142.5
SO 157.5
172.5
Panel BAT25 112.5 127.5
OS 142.5
SO 157.5
172.5
Panel D17S.250 140
OS 180
SO 220
260
sz 151.43 ht 4123 Tumor 100
1200 800 400 0
OS
160
sz 120.99 ht 543
Normal
Figure 2-16 Detection of microsatellite instability (MSI) using the National Cancer Institute (NCI) recommended panel of five microsatellite markers. In this colon cancer sample, all five markers show microsatellite instability, which manifests as a change in the size of microsatellite repeats seen on capillary gel electropherograms of tumor polymerase chain reaction (PCR) products. Electropherograms of normal tissue PCR products are used as a negative control.
144
sz 120.98 ht 2513 Tumor
SO
Panel BAT26 96 112
SO
Added sz 116.88 ht 108 Normal
SO
sz 112.76 ht 7537 Tumor
Mononucleotide Microsatellite Markers Normal Panel OS BAT26 80 96 112 128 900 600 300 0 sz 116.91 ht 867 Tumor
SO
sz 208.52 ht 786
Normal
6300 4200 2100 0
Panel D26.123 180 210
SO
53
Panel D17S.250 140
OS 180
sz 151.29 ht 1014
SO 220
260
54
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
shows instability. The test can be performed by using DNA isolated from either snap-frozen or FFPE tissue, and it provides reliable and reproducible detection of MSI.
DNA-Based Tissue Identity Testing In pathology practice, DNA-based tissue identity testing is typically used for detection of tissue contaminants or “floaters” and for mislabeled specimens. The detection of tissue contaminants is particularly important when distinction between the actual tissue sample and the
tissue fragment in question is not obvious based on microscopic or IHC characteristics. Tissue identity testing is performed by using PCR amplification of DNA isolated from the obviously “correct” tissue fragment, or from the patient’s blood, and from the fragment in question. It is based on comparison of the length of multiple hypervariable DNA regions, such as microsatellite repeats, between the two specimens.57-59 Typically, multiplex PCR is performed, and as many as 16 different microsatellite loci are amplified in the same reaction. The most frequently used microsatellite loci are tetranucleotide repeats, which offer larger differences in the allele size for ease of interpretation. Markers
Renal biopsy Tumor fragment in question
D3S1358
TH01 150
7
D21S812
150
28 29
12 13
14 16
D8S1179
D21S812 210
D8S1179 150
210
32
13 14 D13S317 210
8
9
D3S1358
TH01 150
11 14
6
9
33.2
D13S317 210
10 11
Figure 2-17 DNA-based tissue identity testing. In this case, hematoxylin and eosin–stained sections of a renal biopsy revealed, in addition to the renal tissue, a discrete fragment of tumor tissue, which was suspected to be a contaminant. Unstained sections were used to separately microdissect the renal biopsy tissue and the tumor fragment in question for DNA extraction and polymerase chain reaction amplification for 16 polymorphic microsatellite loci. The capillary gel electropherograms show different sized alleles for all of the tested markers, indicating that these two tissue fragments belong to different individuals, and the tissue fragment in question is indeed a contaminant.
Summary
on chromosome X and Y are included for gender determination. Multiple PCR products are amplified with primers labeled with different fluorophores and separated by capillary gel electrophoresis. The size of each PCR product is identified and compared between the two samples (Fig. 2-17). Because microsatellites are highly polymorphic in the population, the test is typically informative and allows the clinician to determine whether the two DNA profiles are identical or different (i.e., belonging to different patients). The PCR-based tissue identity test requires a very small amount of DNA and can be successfully performed by using FFPE tissue samples or hematoxylin and eosin–stained tissue fragments removed from glass slides.
55
Summary The intent of this chapter is for the reader to appreciate the supplemental information that can be obtained from these molecular methods in the diagnostic arena. A basic knowledge of these molecular techniques enhances the utilization skills of the anatomic pathologist when confronted with nonspecific or ambiguous immunohistologic results for tumor diagnosis. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Watson JD, Crick FH: Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 171:737–738, 1953. 2. Lander ES, Linton LM, Birren B, et al: Initial sequencing and analysis of the human genome. Nature. 409:860–921, 2001. 3. Chargaff E: Structure and function of nucleic acids as cell constituents. Fed Proc. 10:654–659, 1951. 4. Shen LX, Cai Z, Tinoco I, Jr: RNA structure at high resolution. Faseb J. 9:1023–1033, 1995. 5. Sharp PA: Splicing of messenger RNA precursors. Science. 235:766–771, 1987. 6. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281–297, 2004. 7. Ambros V: The functions of animal microRNAs. Nature. 431:350– 355, 2004. 8. Wood LD, Parsons DW, Jones S, et al: The genomic landscapes of human breast and colorectal cancers. Science. 318:1108–1113, 2007. 9. Moore JL, Aros M, Steudel KG, et al: Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis. Biotechniques. 32:296–298, 2002. 10. Mullis KB, Faloona FA: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155:335– 350, 1987. 11. Freeman WM, Walker SJ, Vrana KE: Quantitative RT-PCR: pitfalls and potential. BioTechniques. 26:112–122, 124–115, 1999. 12. Micke P, Ohshima M, Tahmasebpoor S, et al: Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens. Lab Invest. 86:202–211, 2006. 13. Ginzinger DG: Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol. 30:503–512, 2002. 14. Krypuy M, Newnham GM, Thomas DM, et al: High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in nonsmall cell lung cancer. BMC Cancer. 6:295, 2006. 15. Pryor RJ, Wittwer CT: Real-time polymerase chain reaction and melting curve analysis. Methods Mol Biol. 336:19–32, 2006. 16. Dang GT, Cote GJ, Schultz PN, et al: A codon 891 exon 15 RET proto-oncogene mutation in familial medullary thyroid carcinoma: a detection strategy. Mol Cell Probes. 13:77–79, 1999. 17. Nikiforov YE, Nikiforova MN, Gnepp DR, et al: Prevalence of mutations of ras and p53 in benign and malignant thyroid tumors from children exposed to radiation after the Chernobyl nuclear accident. Oncogene. 13:687–693, 1996. 18. Jin L, Sebo TJ, Nakamura N, et al: BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid. Diagn Mol Pathol. 15:136–143, 2006. 19. Bjorheim J, Lystad S, Lindblom A, et al: Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillary electrophoresis and allele specific polymerase chain reaction. Mutat Res. 403:103–112, 1998. 20. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci U S A. 74:5463–5467, 1977. 21. Ronaghi M: Pyrosequencing sheds light on DNA sequencing. Genome Res. 11:3–11, 2001. 22. Ogino S, Kawasaki T, Brahmandam M, et al: Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn. 7:413–421, 2005. 23. Ogino S, Kawasaki T, Nosho K, et al: LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 122:2767–2773, 2008. 24. Glenn TC: Field guide to next-generation DNA sequencers. Mol Ecol Resour. 11:759–769, 2011. 25. Dressman D, Yan H, Traverso G, et al: Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci U S A. 100:8817–8822, 2003. 26. Bentley DR, Balasubramanian S, Swerdlow HP, et al: Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 456:53–59, 2008.
55.e1
27. Rothberg JM, Hinz W, Rearick TM, et al: An integrated semiconductor device enabling non-optical genome sequencing. Nature. 475:348–352, 2011. 28. Meldrum C, Doyle MA, Tothill RW: Next-generation sequencing for cancer diagnostics: a practical perspective. Clin Biochem Rev/ Aust Assoc Clin Biochem. 32:177–195, 2011. 29. Nikiforova M, Durso MB, Kelly LM, et al: Testing for 740 Mutations in Thyroid Samples Using Targeted Next Generation Sequencing Approach. Edited by Quebec, CA, 2012, p A-79. 30. Tonnies H: Modern molecular cytogenetic techniques in genetic diagnostics. Trends Mol Med. 8:246–250, 2002. 31. Nikiforova MN, Stringer JR, Blough R, et al: Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science. 290:138–141, 2000. 32. Zhu Z, Ciampi R, Nikiforova MN, et al: Prevalence of RET/PTC rearrangements in thyroid papillary carcinomas: effects of the detection methods and genetic heterogeneity. J Clin Endocrinol Metab. 91:3603–3610, 2006. 33. Cowell JK, Wang YD, Head K, et al: Identification and characterisation of constitutional chromosome abnormalities using arrays of bacterial artificial chromosomes. Br J Cancer. 90:860–865, 2004. 34. Forozan F, Karhu R, Kononen J, et al: Genome screening by comparative genomic hybridization. Trends Genet. 13:405–409, 1997. 35. Kallioniemi A, Kallioniemi OP, Sudar D, et al: Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science. 258:818–821, 1992. 36. Johnson NA, Hamoudi RA, Ichimura K, et al: Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells. Lab Invest. 86:968–978, 2006. 37. Schulze A, Downward J: Navigating gene expression using microarrays–a technology review. Nat Cell Biol. 3:E190–E195, 2001. 38. Bier FF, von Nickisch-Rosenegk M, Ehrentreich-Forster E, et al: DNA microarrays. Adv Biochem Eng/Biotechnol. 109:433–453, 2008. 39. Sapio MR, Posca D, Troncone G, et al: Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA). Eur J Endocrinol/Eur Fed Endocr Soc. 154:341–348, 2006. 40. Rowe LR, Bentz BG, Bentz JS: Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis. J Clin Pathol. 60:1211–1215, 2007. 41. Hayashida N, Namba H, Kumagai A, et al: A rapid and simple detection method for the BRAF(T1796A) mutation in fine-needle aspirated thyroid carcinoma cells. Thyroid. 14:910–915, 2004. 42. Nikiforova MN, Kimura ET, Gandhi M, et al: BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab. 88:5399–5404, 2003. 43. Kimura ET, Nikiforova MN, Zhu Z, et al: High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 63:1454–1457, 2003. 44. Nikiforova MN, Lynch RA, Biddinger PW, et al: RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma. J Clin Endocrinol Metab. 88:2318–2326, 2003. 45. van Krieken JH, Jung A, Kirchner T, et al: KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch. 453:417–431, 2008. 46. Kambouris M, Jackson CE, Feldman GL: Diagnosis of multiple endocrine neoplasia [MEN] 2A, 2B and familial medullary thyroid cancer [FMTC] by multiplex PCR and heteroduplex analyses of RET proto-oncogene mutations. Hum Mutat. 8:64–70, 1996. 47. Ceccherini I, Hofstra RM, Luo Y, et al: DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret protooncogene. Oncogene. 9:3025–3029, 1994. 48. Pan Q, Pao W, Ladanyi M: Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn. 7:396–403, 2005.
55.e2
Molecular Anatomic Pathology: Principles, Techniques, and Application to Immunohistologic Diagnosis
49. Nikiforova MN, Biddinger PW, Caudill CM, et al: PAX8-PPARgamma rearrangement in thyroid tumors: RT-PCR and immunohistochemical analyses. Am J Surg Pathol. 26:1016–1023, 2002. 50. Nikiforova MN, Caudill CM, Biddinger P, et al: Prevalence of RET/PTC rearrangements in Hashimoto’s thyroiditis and papillary thyroid carcinomas. Int J Surg Pathol. 10:15–22, 2002. 51. Qian X, Jin L, Shearer BM, et al: Molecular diagnosis of Ewing’s sarcoma/primitive neuroectodermal tumor in formalin-fixed paraffin-embedded tissues by RT-PCR and fluorescence in situ hybridization. Diagn Mol Pathol. 14:23–28, 2005. 52. Nikiforova MN, Groen P, Mutema G, et al: Detection of SYT-SSX rearrangements in synovial sarcomas by real-time one-step RT-PCR. Pediatr Dev Pathol. 8:162–167, 2005. 53. Johnson MD, Vnencak-Jones CL, Toms SA, et al: Allelic losses in oligodendroglial and oligodendroglioma-like neoplasms: analysis using microsatellite repeats and polymerase chain reaction. Arch Pathol Lab Med. 127:1573–1579, 2003. 54. Marsh JW, Finkelstein SD, Demetris AJ, et al: Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival. Liver Transpl. 9:664–671, 2003.
55. Boland CR, Thibodeau SN, Hamilton SR, et al: A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 58:5248–5257, 1998. 56. Umar A, Boland CR, Terdiman JP, et al: Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst. 96:261–268, 2004. 57. O’Briain DS, Sheils O, McElwaine S, et al: Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers. Am J Clin Pathol. 106:758–764, 1996. 58. Kessis TD, Silberman MA, Sherman M, et al: Rapid identification of patient specimens with microsatellite DNA markers. Mod Pathol. 9:183–188, 1996. 59. Hunt JL, Swalsky P, Sasatomi E, et al: A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks. Arch Pathol Lab Med. 127:213–217, 2003.
C H A P T E R 3
IMMUNOHISTOLOGY OF INFECTIOUS DISEASES EDUARDO J. EYZAGUIRRE, DAVID H. WALKER, SHERIF R. ZAKI
Overview 56 Viral Infections 56 Bacterial Infections 62 Fungal Infections 65 Protozoal Infections 66 Emerging Infectious Diseases 67 Pathologists, Immunohistochemistry, and Bioterrorism 68 Beyond Immunohistology: Molecular Diagnostic Applications 70 Summary 72
Overview During the last two decades, the approach to histopathologic diagnosis has been dramatically transformed by immunohistochemistry (IHC), specifically in the diagnosis and classification of tumors and, more recently, in the diagnosis of infectious diseases in tissue samples.1 Pathologists play an important role in recognizing infectious agents in tissue samples from patients; in many cases, when fresh tissue is not available for culture, pathologists can provide a rapid morphologic diagnosis from frozen samples that can facilitate clinical decisions regarding patient treatment.2 In addition, pathologists have played a central role in the identification of emerging and reemerging infectious agents; in the description of pathogenetic processes of diseases such as hantavirus pulmonary syndrome (HPS) and other viral hemorrhagic fevers, leptospirosis, and rickettsial and ehrlichial infections; and in the diagnosis of anthrax during the bioterrorist attacks of 2001.3-7 Traditionally, microbial identification in infectious diseases has been made primarily by using cultures and serologic assays. However, fresh tissue is not always available for culture, and culture of fastidious pathogens can be difficult and may take weeks or months to yield results. Moreover, culture alone cannot distinguish 56
colonization or contamination from tissue invasion. In addition, serologic results can be difficult to interpret in the setting of immunosuppression, or when only a single sample is available for evaluation. Some microorganisms have distinctive morphologic characteristics that allow their identification in formalin-fixed tissues by using routine and special stains. Nevertheless, in many instances it is difficult or even impossible to identify an infectious agent specifically by conventional morphologic methods. IHC is one of the most powerful techniques in surgical pathology. Interest has been increasing in the use of specific antibodies to viral, bacterial, fungal, and parasitic antigens for the detection and identification of the causative agents in many infectious diseases. The use of a specific antibody to detect a microbial antigen was first performed by Coons and associates8 to detect pneumococcal antigen in tissues. The advantages of IHC over conventional staining methods (Box 3-1) and the contributions of IHC in the management of infectious diseases (Box 3-2) are substantial. In many instances, IHC has shown high specificity, allowing the differentiation of morphologically similar microorganisms.9 IHC is especially useful when microorganisms are difficult to identify by routine or special stains, are fastidious to grow, or exhibit atypical morphology (Box 3-3).10-14 It is important to understand that there may be widespread occurrence of common antigens among bacteria and pathogenic fungi, and both monoclonal and polyclonal antibodies must be tested for possible crossreactivity with other organisms.15 Finally, it is important to emphasize that IHC has several steps, and all of them can affect the final result; however, in general, the only limitations are the availability of specific antibodies and the preservation of epitopes.16 Table 3-1 lists some commercially available antibodies for diagnostic use in surgical pathology.
Viral Infections IHC has played an important role not only in the diagnosis of a large number of viral infections but also in the study of their pathogenesis and epidemiology. Conventionally, the diagnosis of viral infections has relied
Viral Infections
Box 3-1 ADVANTAGES OF IMMUNOHISTOCHEMISTRY FOR THE DIAGNOSIS OF INFECTIOUS DISEASES • Opportunity for rapid results • Reduced risk of exposure to serious infectious diseases by performance on formalin-fixed, paraffin-embedded tissue • High sensitivity allows identification of infectious agents even before morphologic changes occur • Opportunities for retrospective diagnosis of individual patients and for in-depth study of the disease • Specific identification of infectious agents with many monoclonal antibodies and some polyclonal antibodies
on cytopathic changes observed by routine histopathologic examination. Several viral pathogens produce characteristic intracellular inclusions that allow pathologists to make a presumptive diagnosis of viral infection. However, for some viral infections, the characteristic cytopathic changes are subtle and sparse, requiring a meticulous search.17 Moreover, only 50% of known viral diseases are associated with characteristic intracellular inclusions.18 In addition, the most commonly used fixative in histopathology, formalin, is a poor fixative for demonstrating the morphologic and tinctorial features of viral inclusions.19 When viral inclusions are not detected in hematoxylin and eosin (H&E) stained sections, or when the viral inclusions present cannot be differentiated from those of other viral diseases, IHC techniques offer a more reliable approach to reach a specific diagnosis.
Hepatitis B Hepatitis B virus (HBV) infection constitutes an important cause of chronic hepatitis in a significant proportion of patients. In many instances, the morphologic changes induced by HBV in hepatocytes are not typical enough to render a presumptive diagnosis of HBV infection. In other instances, there may be so little hepatitis B surface antigen (HBsAg) that it cannot be demonstrated by techniques such as orcein staining. In these cases, IHC techniques to detect HBsAg are more sensitive than histochemical methods and are helpful in reaching a diagnosis.20 Immunostaining for HBsAg has Box 3-2 CONTRIBUTIONS OF IMMUNOHISTOCHEMISTRY TO THE DIAGNOSIS OF INFECTIOUS DISEASES • Allows identification of new human pathogens • Allows microbiologic-morphologic correlation that establishes the pathogenic significance of microbiologic results • Provides a rapid morphologic diagnosis that allows early treatment of serious infectious diseases • Contributes to understanding of the pathogenesis of infectious diseases • Provides a diagnosis when fresh tissue is not available or when culture methods do not exist
57
Box 3-3 APPLICATIONS OF IMMUNOHISTOCHEMISTRY IN THE DIAGNOSIS OF INFECTIOUS DISEASES • Identification of microorganisms that are difficult to detect by routine or special stains • Detection of microorganisms that are present in low numbers • Detection of microorganisms that stain poorly • Identification of microorganisms that exhibit atypical morphology
been used in the diagnosis of HBV and in the study of carrier states.21,22 Of cases with positive serologic results for HBsAg, IHC demonstrates cytoplasmic HBsAg in 80% or more.23 By immunoperoxidase localization, hepatitis B core antigen (HBcAg) can be demonstrated within the nuclei or the cytoplasm of hepatocytes, or both. Cytoplasmic expression of HBcAg usually is associated with a higher grade of hepatitis activity,23 and diffuse immunostaining of nuclei for HBcAg generally suggests uncontrolled viral replication in the setting of immunosuppression.24 Immunostaining for HBsAg and HBcAg is useful in the diagnosis of recurrent hepatitis B infection in liver allografts, particularly when present with atypical histopathologic features.25
Herpesviruses Histologically, the diagnosis of herpes simplex virus (HSV) infection involves the detection of multinucleated giant cells that contain characteristic molded, ground glass–appearing nuclei and Cowdry type A intranuclear inclusions with angulated edges. When abundant viral inclusions exist within infected cells, the diagnosis is usually straightforward. However, diagnosis can be difficult when the characteristic intranuclear inclusions or multinucleated cells, or both, are absent, or when the amount of tissue in a biopsy specimen is small.26 In these cases, IHC with either polyclonal or monoclonal antibodies against HSV antigens has proved to be a sensitive and specific technique used to diagnose HSV infections (Fig. 3-1).27-30 Although polyclonal antibodies against major HSV glycoprotein antigens are sensitive, they do not allow distinction between HSV-1 and HSV-2; this is because the two viruses are antigenically similar.31 In addition, the histologic features of HSV infection are not specific and can also occur in patients with varicella-zoster virus (VZV) infection. Monoclonal antibodies against the VZV envelope glycoprotein GP1 are sufficiently sensitive and specific to allow a clear-cut distinction between HSV and VZV infections.27,32,33 IHC has also been useful in demonstrating the association of human herpes virus 8 (HHV-8) with Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman disease.34-38 Diagnosis of Kaposi sarcoma may be problematic because of its broad morphologic spectrum and similar appearance to other benign and
58
Immunohistology of Infectious Diseases
TABLE 3-1 Commercially Available Antibodies for Immunohistochemical Diagnosis of Infectious and Prion Diseases Microorganism
Antibody/Clone
Dilution
Pretreatment
Source
Adenovirus
Mab/20/11 and 2/6
1 : 2000
Proteinase K
Chemicon
Bartonella henselae
Mab
1 : 100
HIAR
Biocare Medical
BK virus
Mab/BK T.1
1 : 8000
Trypsin
Chemicon
Candida albicans
Mab/1B12
1 : 400
HIAR
Chemicon
Chlamydia pneumoniae
Mab/RR402
1 : 200
HIAR
Accurate
Cryptosporidium spp.
Mab/Mabc1
1 : 100
HIAR
Novocastra
Cytomegalovirus
Mab/DDG9/CCH2
1 : 50
HIAR
Novocastra
Clostridium spp.
Rabbit polyclonal
1 : 1000
None
Biodesign
Giardia intestinalis
Mab/9D5.3.1
1 : 50
HIAR
Novocastra
Hepatitis B core antigen
Rabbit polyclonal
1 : 2000
HIAR
Dako
Hepatitis B surface antigen
Mab/3E7
1 : 100
HIAR
Dako
Herpes simplex 1 and 2 viruses
Rabbit polyclonal
1 : 3200
HIAR
Dako
Helicobacter pylori
Rabbit polyclonal
1 : 40
Proteinase K
Dako
Human herpesvirus 8
Mab/LNA-1
1 : 500
HIAR
Novocastra
Klebsiella pneumoniae
Rabbit polyclonal
1 : 200
Proteinase K
Biogenex
Listeria monocytogenes
Rabbit polyclonal
1 : 5000
Proteinase K
Difco
Mycoplasma pneumoniae
Mab/1.B.432
1 : 25
HIAR
US Biological
Parvovirus B19
Mab/R92F6
1 : 500
HIAR
Novocastra
Pneumocystis carinii
Mab/3F6
1 : 20
HIAR
Novocastra
Plasmodium falciparum
Mab/BDI400
1 : 1000
Proteinase K
Biodesign
Prion
Mab/3F4 Mab/12F10 Mab/KG9
1 : 200 1 : 1000 1 : 1000
Antigen retrieval Proteinase K Proteinase K
Dako Cayman Chemical TSE Resource Center
Respiratory syncytial virus
Mab/5H5N
1 : 200
HIAR
Novocastra
Staphylococcus aureus
Rabbit polyclonal
1 : 500
Proteinase K
Biodesign
Treponema pallidum
Rabbit polyclonal
HIAR
Biodesign
Toxoplasma gondii
Rabbit polyclonal
1 : 320
HIAR
Biogenex
West Nile virus
Mab/5H10
1 : 400
Proteinase K
Bioreliance
HIAR, Heat-induced antigen retrieval.
malignant neoplastic vascular lesions. Immunostaining of latent associated nuclear antigen 1 (LANA-1) is useful to confirm the diagnosis of Kaposi sarcoma, particularly when difficult early lesions closely resemble the appearance of interstitial granuloma annulare, and when the neoplasm is found in an unusual location. Immunostaining also allows distinction of Kaposi sarcoma from several morphologically similar vasoproliferative lesions.39-41 Immunostaining is restricted to the nuclei of spindle cells and endothelial cells of the slitlike vascular spaces (Fig. 3-2). IHC has also demonstrated expression of HHV-8 LANA-1 in mesothelial cells of human immunodeficiency virus (HIV)–associated recurrent pleural effusions.42 Cytomegalovirus (CMV) continues to be an important opportunistic pathogen in immunocompromised
patients, and it is estimated that 30% of transplant recipients experience CMV disease.43 The range of organ involvement in posttransplant CMV disease is wide; hepatitis occurs in 40% of liver transplant recipients,44 and pneumonitis is more frequently seen in heart and heart-lung transplant patients.45 Other organs commonly affected are the gastrointestinal tract and the peripheral and central nervous systems. Histologic diagnosis of CMV in fixed tissues usually rests on identifying characteristic cytopathic effects that include intranuclear inclusions, cytoplasmic inclusions, or both. However, histologic examination lacks sensitivity, and in some cases, atypical cytopathic features can be confused with reactive or degenerative changes.46 Additionally, as many as 38% of patients with gastrointestinal CMV disease fail to demonstrate any inclusions.47 In these
Viral Infections
59
Figure 3-1 Photomicrograph of cervical biopsy from a patient with herpes simplex virus infection shows abundant nuclear and cytoplasmic antigen (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Figure 3-3 Colon biopsy of a patient with steroid-refractory ulcerative colitis. Rare epithelial cells show intranuclear cytomegalovirus antigen (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
cases, IHC using monoclonal antibodies against early and late CMV antigens allows the detection of CMV antigens in the nucleus and cytoplasm of infected cells (Fig. 3-3). The sensitivity of IHC for detecting CMV infection ranges from 78% to 93%.47,48 In addition, IHC may allow detection of CMV antigens early in the course of the disease, when cytopathic changes have not yet developed.49-54 For example, CMV early nuclear antigen is expressed 9 to 96 hours after cellular infection and indicates early active viral replication. IHC has been used to detect CMV infection in patients with steroid refractory ulcerative colitis, and the routine use of IHC for the detection of CMV in the evaluation of these patients is now recommended.55,56 CMV immunostaining has been used to detect occult CMV infection of the central nervous system (CNS) in liver transplant patients who develop neurologic
complications.57 It has also been used to demonstrate a high frequency of CMV antigens in tissues from firsttrimester abortions.58 CMV is the most common opportunistic organism found in liver biopsies from transplant patients; nonetheless, the incidence of CMV hepatitis appears to be decreasing owing to better prophylactic treatments.59 Although CMV hepatitis presents with characteristic neutrophilic aggregates within the liver parenchyma, atypical features suggestive of acute rejection or changes indistinguishable from those of any other viral hepatitis are occasionally observed.60 In addition, parenchymal neutrophilic microabscesses have been described in cases with no evidence of CMV infection.61 In these cases, immunostaining for CMV antigens is most useful in determining the diagnosis of CMV infection.62 The sensitivity of IHC is better than light microscopy identification of viral inclusions and compares favorably with culture and in situ hybridization (ISH).49,51,52,54,63 Additionally, IHC assays can be completed faster than the shell vial culture technique, allowing the rapid results that are so important for early anti-CMV therapy.54 Other herpesvirus infections that have been diagnosed with IHC methods include HHV-6 infection64 and Epstein-Barr viral (EBV) infection.65 IHC has been used to identify EBV latent membrane protein 1 (LMP-1) in cases of Hodgkin lymphoma and posttransplant lymphoproliferative disorder (Fig. 3-4).66
Adenoviruses
Figure 3-2 Lymph node biopsy from a patient with Kaposi sarcoma. The spindle cells show strong nuclear staining for human herpesvirus 8 latency-associated nuclear antigen 1. Endothelial cells of wellformed vascular spaces are negative (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Adenovirus has been increasingly recognized as a cause of morbidity and mortality among immunocompromised patients, owing to transplant and congenital immunodeficiency.67,68 Rarely, adenovirus infection has been described in HIV-infected patients.69-71 Characteristic adenovirus inclusions are amphophilic, intranuclear, homogeneous, and glassy. However, in some cases, the infection may contain only rare cells that show the
60
Immunohistology of Infectious Diseases
be overlooked. IHC staining has been of value in differentiating adenovirus colitis from CMV colitis.70,75
Parvovirus B19 Infection
Figure 3-4 Epstein-Barr virus latent membrane protein 1 within cytoplasm of characteristic Reed-Sternberg cells in a case of Hodgkin lymphoma (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
characteristic cytopathic effect.70 In addition, other viral inclusions that include CMV, human papillomavirus (HPV), HSV, and VZV can be mistaken for adenovirus inclusions and vice versa. In these circumstances, IHC assay may be necessary for a definitive diagnosis. A monoclonal antibody that is reactive with all 41 serotypes of adenovirus has been used in an IHC technique to demonstrate intranuclear adenoviral antigen in immunocompromised patients (Fig. 3-5).70-74 Histologic diagnosis of adenovirus colitis is difficult, and it is usually underdiagnosed. Moreover, in immunosuppressed patients, the incidence of coinfection with other viruses is high, and the presence of adenovirus tends to
Figure 3-5 Adenovirus pneumonia in a heart transplant patient who developed acute respiratory distress syndrome and respiratory failure. Infected cells within necrotizing exudate show intranuclear reactivity with antibody to adenovirus antigen. Some cells show inclusions with a clear halo; thus a differential diagnosis from cytomegalovirus is difficult on hematoxylin and eosin stain (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Parvovirus B19 has been associated with asymptomatic infections, erythema infectiosum, acute arthropathy, aplastic crisis, hydrops fetalis, chronic anemia, and red cell aplasia. In addition, parvovirus B19 infection has been recognized as an important cause of severe anemia in immunocompromised leukemic patients receiving chemotherapy.76 The diagnosis of parvovirus infection can be achieved by identifying typical findings in bone marrow specimens, including decreased or absent red cell precursors, giant pronormoblasts, and eosinophilic or amphophilic intranuclear inclusions in erythroid cells.77,78 Because intravenous immunoglobulin (Ig) therapy is effective, a rapid and accurate diagnostic method is important. IHC with a monoclonal antibody against VP1 and VP2 capsid proteins has been used as a rapid and sensitive method to establish the diagnosis of parvovirus B19 infection in formalin-fixed, paraffin-embedded (FFPE) tissues.79-82 IHC is of particular help in detecting parvovirus B19 antigen in cases with sparse inclusions, to study cases not initially identified by examination of routinely stained tissue sections, or in cases of hydrops fetalis with advanced cytolysis (Fig. 3-6).79,83,84 Several studies have found a strong correlation among results obtained from morphologic, IHC, ISH, and polymerase chain reaction (PCR) methods.78,79,82,84
Viral Hemorrhagic Fevers Since the 1960s, numerous emerging and reemerging agents of viral hemorrhagic fevers have attracted the attention of pathologists.3-5 Investigators have played an important role in identifying these agents and in supporting epidemiologic, clinical, and pathogenetic studies
Figure 3-6 Hydrops fetalis caused by parvovirus B19 infection. Normoblasts within the villous capillaries show intranuclear viral antigen (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Viral Infections
Figure 3-7 Yellow fever. Abundant yellow fever viral antigens are seen within hepatocytes and Kupffer cells (immunoperoxidase staining with 3-amino-9-ethylcarbazole and hematoxylin counterstain; ×400).
of emerging viral hemorrhagic fevers.4,5,7 Viral hemorrhagic fevers are often fatal. They are clinically difficult to diagnose, in the absence of bleeding or organ manifestations, and they frequently require handling and testing of potentially dangerous biologic specimens. In addition, histopathologic features are not pathognomonic, and they can resemble other viral, rickettsial, and bacterial infections (e.g., leptospirosis). IHC is essential and has been successfully and safely applied to the diagnosis and study of the pathogenesis of these diseases. Several studies have established the utility of IHC as a sensitive, safe, and rapid diagnostic method for the diagnosis of viral hemorrhagic fevers such as yellow fever (Fig. 3-7),85-87 dengue hemorrhagic fever,87,88 Crimean-Congo hemorrhagic fever,89 Argentine hemorrhagic fever,90 Venezuelan hemorrhagic fever,91 and Marburg disease.92 Additionally, a sensitive, specific, and safe immunostaining method has been developed to diagnose Ebola hemorrhagic fever in formalin-fixed skin biopsies (Fig. 3-8).93 IHC demonstrated that Lassa virus targets primarily endothelial cells, mononuclear inflammatory cells, and hepatocytes (Fig. 3-9).93-95
61
Figure 3-8 Extensive Ebola viral antigens are seen primarily within fibroblasts in the dermis of a skin specimen from a fatal case of Ebola hemorrhagic fever (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×20).
cytopathic changes observed in BK virus infection are not pathognomonic and can be observed in other viral infections. Moreover, in early BK virus infection, histologic changes may be minimal or completely absent, although IHC can identify viral antigen.99,100 In this setting, IHC with an antibody against the large T antigen of SV40 virus has been effective in demonstrating BK virus infection (Fig. 3-10).96,99,101-103 The JC virus is a double-stranded DNA human polyomavirus that causes progressive multifocal leukoencephalopathy (PML). This fatal demyelinating disease is characterized by bizarre giant astrocytes and cytopathic changes in oligodendrocytes. In addition to detection by antibodies to SV40-T antigen, IHC with a polyclonal rabbit antiserum against the protein VP1 is a specific, sensitive, and rapid method used to confirm the diagnosis of PML.104-107 JC virus antigen is usually seen within oligodendrocytes (Fig. 3-11) and occasional astrocytes, and antigen-bearing cells are more commonly seen in early lesions.
Polyomaviruses Polyomavirus (BK virus) infections are frequent during infancy; in immunocompetent individuals, the virus remains latent in the kidneys, CNS, and B lymphocytes. In this population, the infection reactivates and spreads to other organs. BK virus nephropathy is an important cause of graft failure in patients who have undergone renal transplant,96 and prevalence varies from 2% to 4.5% in different transplant centers.96,97 Because specific clinical signs and symptoms are lacking in BK virus nephropathy, the diagnosis can only be made histologically in a graft biopsy.98 In the kidney, the infection is associated with mononuclear interstitial inflammatory infiltrates and tubular atrophy, findings that can be difficult to distinguish from acute rejection.98 The
Figure 3-9 Liver specimen from a patient with Lassa fever. Scattered hepatocytes and reticuloendothelial cells show reactivity with monoclonal antibody to Lassa virus (naphthol-fast red substrate and hematoxylin counterstain; original magnification ×100).
62
Immunohistology of Infectious Diseases
Figure 3-10 Immunohistochemical detection of simian virus (SV)40-T antigen in the nuclei of tubular cells in a renal transplant patient with BK virus–associated nephropathy (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Figure 3-12 Immunostaining of respiratory syncytial virus antigens in desquamated bronchial and alveolar lining cells with a monoclonal antibody (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Other Viral Infections
Other viral agents that can be diagnosed with IHC methods include enteroviruses,117-120 eastern equine encephalitis virus,121-123 and rotaviruses.124-126 IHC staining has been used in the histopathologic diagnosis of viral hepatitis C (HCV); however, IHC for this virus is not as effective as serologic assays and detection of HCV RNA in serum.
IHC has also been used to confirm the diagnosis of respiratory viral diseases such as influenza A virus and respiratory syncytial virus (RSV) infections (Fig. 3-12) when cultures were not available.108-111 The diagnosis of rabies relies heavily on histopathologic examination of tissues to demonstrate its characteristic cytoplasmic inclusions, Negri bodies. In a significant percentage of cases, Negri bodies are inconspicuous and so few that confirming the diagnosis of rabies is extremely difficult.112 Furthermore, in nonendemic areas, the diagnosis of rabies usually is not suspected clinically, or the patient may come to medical attention with ascending paralysis. In these settings, IHC staining is a very sensitive, specific, and safe diagnostic tool for detection of rabies (Fig. 3-13).112-116
Figure 3-11 Progressive multifocal leukoencephalopathy. SV40-T antigen in the nuclei of enlarged oligodendrocytes in a patient with JC virus infection (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Bacterial Infections Among bacterial infections, the greatest number of IHC studies has been performed in the investigation of Helicobacter pylori. A few studies have evaluated the use of IHC for other bacterial, mycobacterial, rickettsial, and spirochetal infections.
Figure 3-13 Immunostaining of rabies viral antigens in neurons of the central nervous system with a rabbit polyclonal antibody. Red precipitate corresponds to Negri inclusions by hematoxylin and eosin staining (immunoalkaline phosphatase with naphtholfast red substrate and hematoxylin counterstain; original magnification ×40).
Bacterial Infections
Figure 3-14 Numerous curved Helicobacter pylori in the superficial gastric mucus are clearly demonstrated by immunoperoxidase staining in this patient with chronic active gastritis (immuno peroxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Antigen retrieval is generally not required for the IHC demonstration of bacteria in fixed tissue. However, interpreting the results can be complicated, because many of these antibodies cross-react with other bacteria. Moreover, antibodies may react with only portions of the bacteria, and they may label remnants of bacteria or spirochetes when viable organisms are no longer present.
Helicobacter pylori Infection Gastric infection by H. pylori results in chronic, active gastritis and is strongly associated with lymphoid hyperplasia, gastric lymphomas, and gastric adenocarcinoma. Heavy infections with numerous organisms are easily detected on routine H&E-stained tissues; however, the detection rate is only 66%, with many false-positive and false-negative results.127,128 Conventional histochemical methods such as silver stains are more sensitive than H&E in detecting H. pylori. Nonetheless, for detecting scant numbers of organisms, it has been proven that IHC has high specificity and sensitivity, is less expensive when all factors are considered, is superior to conventional histochemical methods, and has a low interobserver variation (Fig. 3-14).127 Treatment for chronic active gastritis and H. pylori infection can change the shape of the microorganism. This change can make it difficult to identify and differentiate the organism from extracellular debris or mucin globules. In these cases IHC improves the rate of successful identification of the bacteria, even when histologic examination and cultures are falsely negative.129-132
Whipple Disease Whipple disease affects primarily the small bowel and mesenteric lymph nodes; less commonly, the CNS and other organs such as the heart are affected. Numerous
63
foamy macrophages characterize the disease, and the diagnosis usually relies on the demonstration of periodic acid–Schiff (PAS)-positive intracytoplasmic bacteria. Nevertheless, the presence of PAS-positive macrophages is not pathognomonic; they can be observed in other diseases such as Mycobacterium avium infections, histoplasmosis, Rhodococcus equi infections, and macroglobulinemia. Tropheryma whipplei is a rare cause of endocarditis that shares many histologic features with other culture-negative endocarditides, such as those caused by Coxiella burnetii and Bartonella spp.133 The development of specific antibodies against these microorganisms has significantly enhanced the ability to detect them in the heart valves of patients with culturenegative endocarditis.134 IHC staining with rabbit polyclonal antibody provides a sensitive and specific method for the rapid diagnosis of intestinal and extraintestinal Whipple disease and for follow-up of treatment response.135-137
Rocky Mountain Spotted Fever Confirmation of Rocky Mountain spotted fever (RMSF) usually requires the use of serologic methods to detect antibodies to spotted fever group (SFG) rickettsiae; however, most patients with RMSF lack diagnostic titers during the first week of disease. IHC has been successfully used to detect SFG rickettsiae in formalin-fixed tissue sections, and it is superior to histochemical methods (Fig. 3-15).138,139 Several studies illustrate the value of IHC in diagnosing suspected cases of RMSF and the use of skin biopsies with high specificity and sensitivity to confirm fatal cases of seronegative RMSF.10,140-144 Rickettsia rickettsii cannot be distinguished from other SFG rickettsiae such as R. parkeri or R. conorii, because they cross-react.
Figure 3-15 Immunohistologic demonstration of Rickettsia rickettsii within vascular endothelium in the pons of a patient with fatal Rocky Mountain spotted fever (immunoperoxidase staining with 3-amino-9-ethylcarbazole and hematoxylin counterstain; ×600).
64
Immunohistology of Infectious Diseases
Bartonella Infections Bartonella are slow-growing, fastidious, gram-negative, Warthin-Starry–stained bacteria associated with bacillary angiomatosis, peliosis hepatis, cat-scratch disease, trench fever, relapsing bacteremia, and disseminated granulomatous lesions of liver and spleen.145 Bartonella are important agents of blood culture–negative endocarditis. Traditional techniques such as histology, electron microscopy, and serology have been used to identify the agents of culture-negative endocarditis. However, Bartonella spp., C. burnetii, and T. whipplei endocarditis share many morphologic features that do not allow for a specific histologic diagnosis.146 Besides, serologic tests for Bartonella may show cross-reactivity with C. burnetii and Chlamydia spp.147 Immunostaining has been successfully used to identify B. henselae and B. quintana in the heart valves of patients with blood culture–negative endocarditis, and this has significantly enhanced the ability to establish a specific diagnosis in these cases.148,149 A polyclonal rabbit antibody that does not differentiate between B. henselae and B. quintana has also been used to detect these microorganisms in cat-scratch disease (Fig. 3-16), bacillary angiomatosis, and peliosis hepatis.150,151 A commercially available monoclonal antibody specific for B. henselae is also available and has been used to demonstrate the organism in a case of spontaneous splenic rupture caused by this bacterium.152
Syphilis Syphilis, caused by Treponema pallidum, a fastidious organism that has not been cultivated, continues to be a public health problem.153 The diagnosis of syphilis relies on serology and the identification of T. pallidum by dark-field microscopy. However, these methods have
Figure 3-17 Syphilis. Skin biopsy from a patient with secondary syphilis. Scattered intact Treponema pallidum are easily visible with a rabbit polyclonal antibody against T. pallidum (immuno peroxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
low sensitivity and specificity,154 and serologic methods can be negative in early stages of the disease and in immunosuppressed patients, such as those coinfected with HIV.155 In tissue sections, the usual method for detecting spirochetes is through silver impregnation stains (Warthin-Starry or Steiner). These stains, however, can be technically difficult to perform and interpret, are nonspecific, and frequently show marked background artifacts, because silver stains also highlight melanin granules and reticulin fibers. Detection rates of spirochetes with silver stains vary from 33% to 71%.156 It has been shown that immunostaining of biopsy specimens with anti–T. pallidum polyclonal antibody (Fig. 3-17) is more sensitive and specific than silverstaining methods, with sensitivities that range from 71% to 94%.153,156,157
Mycobacterium tuberculosis Infection
Figure 3-16 Photomicrograph of a lymph node biopsy from a patient with cat-scratch disease shows abundant extracellular, clumped coccobacilli of Bartonella henselae in necrotic foci (immunoalkaline phosphatase with monoclonal antibody against B. henselae, naphthol-fast red substrate, and hematoxylin counterstain; ×200). Courtesy Dr. Suimin Qiu, University of Texas Medical Branch.
Identification of M. tuberculosis is routinely achieved by acid-fast staining, culture of biopsy specimens, or both. Nevertheless, staining for acid-fast bacilli (AFB) has a low sensitivity and is not specific, because it does not allow differentiation of mycobacterial species.158 Furthermore, cultures may take several weeks, and sensitivity is low in paucibacillary lesions.159 Anti– bacillus Calmet-Guerin polyclonal antibody has been used in the histologic diagnosis of mycobacterial infections and shows better sensitivity than AFB staining, although it is not superior to AFB stains in paucibacillary lesions and does not allow for differentiation between M. tuberculosis and other mycobacteria.160 Recently, a polyclonal antibody against the M. tuberculosis–secreted antigen MPT64 was used in cases of mycobacterial lymphadenitis; it showed good sensitivity (90%) and specificity (83%) and performed better than AFB staining in cases of paucibacillary disease and was comparable to nested PCR.161
Fungal Infections
Other Bacterial Infections Other bacterial diseases that can be identified by IHC in formalin-fixed tissue include leptospirosis, a zoonosis that usually presents as an acute febrile syndrome but occasionally can have unusual manifestations, such as pulmonary hemorrhage with respiratory failure or abdominal pain.162-164 Rabbit polyclonal antibodies have been used in IHC to detect leptospiral antigens in the gallbladder and lungs from patients with unusual presentations (Fig. 3-18).162-165 Lyme disease has protean clinical manifestations, and Borrelia burgdorferi is difficult to culture from tissues and fluids. In addition, cultures are rarely positive before 2 to 4 weeks of incubation. B. burgdorferi can be identified in tissues by immunostaining with polyclonal or monoclonal antibodies. Although IHC is more specific than silver impregnation staining, the sensitivity of immunostaining is poor, and the microorganisms are difficult to detect, because of the low numbers present in tissue sections.166,167 Q fever is a zoonosis caused by Coxiella burnetii and is characterized by protean and nonspecific manifestations. Acute Q fever can manifest as atypical pneumonia or granulomatous hepatitis, frequently with characteristic fibrin-ring granulomas. This microorganism is recognized as one agent that causes blood culture– negative chronic endocarditis.168 A monoclonal antibody has been used to specifically identify C. burnetii in cardiac valves of patients with chronic Q fever endocarditis.12,169 Recently, IHC has been successfully used to identify Streptococcus pneumoniae in formalin-fixed organs with an overall sensitivity of 100% and a specificity of 71% when compared with cultures.170 IHC assays are used to identify Clostridium spp., Staphylococcus aureus, and Streptococcus pyogenes171,172; Haemophilus influenzae173-175; Chlamydia spp.176-178; Legionella pneumophila and L. dumoffii179-181; Listeria monocytogenes182-184; Salmonella
Figure 3-18 Leptospira. Immunostaining of intact leptospires and granular forms of leptospiral antigens in a kidney specimen of a patient who died of pulmonary hemorrhage (immunoalkaline phosphatase with rabbit polyclonal antisera with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×63).
65
spp.185,186; and rickettsial infections other than RMSF, such as boutonneuse fever, epidemic typhus, murine typhus,187 rickettsialpox,188,189 African tick bite fever,138 and scrub typhus.190
Fungal Infections The great majority of fungi are readily identified by H&E staining alone or in combination with histochemical stains, such as PAS and Gomori methenamine silver (GMS). However, these stains cannot distinguish morphologically similar fungi with potential differences in susceptibility to antimycotic drugs. In addition, several factors may influence the appearance of fungal elements, which may appear atypical in tissue sections because of steric orientation, age of the fungal lesion, effects of antifungal chemotherapy, type of infected tissue, and host immune response.191 Currently, the final identification of fungi relies on culture techniques; however, culture may take several days or longer to yield a definitive result, and surgical pathologists rarely have access to fresh tissue. In past years, IHC has been used to identify various fungal elements in FFPE tissue.192-194 IHC methods have the advantage of providing rapid and specific identification of several fungi and allowing pathologists to identify unusual, filamentous hyphal and yeast infections and to accurately distinguish them from confounding artifacts.193,195 In addition, IHC allows pathologists to correlate microbiologic and histologic findings of fungal infections and to distinguish them from harmless colonization. IHC can also be helpful when more than one fungus is present; in these cases, dual immunostaining techniques can highlight the different fungal species present in the tissue.196 An important limitation of IHC in the identification of fungi is the well-known, widespread occurrence of common antigens among pathogenic fungi that frequently results in cross-reactivity with polyclonal antibodies and even with some monoclonal antibodies.193,195-197 Therefore assessing crossreactivity by using a panel of fungi is a very important step in the evaluation of IHC methods.193,194 Candida spp. are often stained weakly with H&E, and sometimes the yeast form may be difficult to differentiate from Histoplasma capsulatum, Cryptococcus neoformans, and even Pneumocystis jiroveci. Polyclonal and monoclonal antibodies against Candida genus antigens are sensitive and strongly reactive and do not show cross-reactivity with other fungi tested.193,194,198,199 In particular, two monoclonal antibodies against Candida albicans mannoproteins show high sensitivity and specificity. Monoclonal antibody 3H8 recognizes primarily filamentous forms of C. albicans, whereas monoclonal antibody 1B12 highlights yeast forms.199-203 Identification of Cryptococcus neoformans usually is not a problem when the fungus produces a mucicarminestained capsule. However, infections by capsule-deficient strains are more difficult to diagnose, and the disease can be confused with histoplasmosis, blastomycosis, or Candida glabrata infection. Also, in long-standing infections the yeast often appears atypical and fragmented.
66
Immunohistology of Infectious Diseases
Polyclonal antibodies raised against C. neoformans yeast cells are sensitive and specific.193,194 More recently, monoclonal antibodies have been produced that allow identification and differentiation of varieties of C. neoformans in formalin-fixed tissue. The antibodies are highly sensitive (97%) and specific (100%) and can differentiate C. neoformans var. neoformans from C. neoformans var. gattii.204,205 Sporothrix schenckii may be confused in tissue sections with Blastomyces dermatitidis and fungal agents of phaeohyphomycosis. In addition, yeast cells of S. schenckii may be sparsely present in tissues. Antibodies against yeast cells of S. schenckii are sensitive but demonstrate cross-reactivity with Candida spp.; however, after specific adsorption of the antibody with Candida yeast cells, the cross-reactivity of the antibodies is eliminated.193,194 Invasive aspergillosis is a frequent cause of fungal infection, with high morbidity and mortality rates in immunocompromised patients.206 The diagnosis is often difficult and relies heavily on histologic identification of invasive septate hyphae and culture confirmation. Nevertheless, several filamentous fungi such as Fusarium spp., Pseudallescheria boydii, and Scedosporium spp. share similar morphology with Aspergillus spp. in H&Estained tissues.207 A precise and rapid diagnosis of invasive aspergillosis is important, because early diagnosis is associated with improved clinical response, and it allows planning of the correct duration and choice of antimycotic therapy. Researchers have shown that the yield of cultures in histologically proven cases is low, ranging from 25% to 50%.206,208-211 Several polyclonal and monoclonal antibodies against Aspergillus antigens have been tested in formalin-fixed tissues with variable sensitivities, and most of them cross-react with other fungi.197,212,213 More recently, monoclonal antibodies (WF-AF-1, 164G, and 611F) against Aspergillus galactomannan have shown high sensitivity and specificity in identifying A. fumigatus, A. flavus, and A. niger in formalin-fixed tissues without cross-reactivity with other filamentous fungi.211,214,215 Cysts and trophozoites of Pneumocystis jiroveci can be detected in bronchoalveolar lavage specimens using monoclonal antibodies that yield results that are slightly more sensitive than GMS, Giemsa, or Papanicolaou staining (Fig. 3-19).194,216,217 IHC is most helpful in cases of extrapulmonary pneumocystosis or in the diagnosis of P. jiroveci pneumonia when atypical pathologic features are present (e.g., hyaline membranes or granulomatous pneumocystosis in which microorganisms are usually very sparse). Penicillium marneffei can cause a disseminated infection in immunocompromised patients.218,219 Morphologically the organisms must be differentiated from Histoplasma capsulatum, Cryptococcus neoformans, and Candida albicans. The monoclonal antibody endothelial barrier antigen 1 against the galactomannan of Aspergillus spp. cross-reacts with and detects P. marneffei in tissue sections.209,220 IHC has also been used to detect Blastomyces, Coccidioides, and Histoplasma.193,194,221 However, the antibodies have significant cross-reactivity with several other fungi.
Figure 3-19 Immunodeficient patient with Pneumocystis jiroveci pneumonia. Cohesive aggregates of cyst forms and trophozoites within alveolar spaces are demonstrated by a monoclonal antibody against Pneumocystis with an immunoperoxidase technique (immunoperoxidase staining with diaminobenzidine and hematoxylin counterstain; ×400).
Protozoal Infections Protozoa usually can be identified in tissue sections stained with H&E or Giemsa stain; however, because of the small size of the organisms and the subtle distinguishing features, an unequivocal diagnosis cannot always be made. The role of IHC in the detection of protozoal infections has been particularly valuable when the morphology of the parasite is distorted by tissue necrosis or autolysis. In addition, in immunocompromised patients, toxoplasmosis can have an unusual disseminated presentation with numerous tachyzoites without bradyzoites (Fig. 3-20).222,223 IHC has also been useful in cases with unusual presentation of the disease.224 The diagnosis of leishmaniasis in routine practice usually is not difficult; however, in certain circumstances, pathologic diagnosis may be problematic, as is the case in chronic granulomatous leishmaniasis with small numbers of parasites, when the microorganism occurs in unusual locations, or when necrosis distorts the morphologic appearance of the disease.225 In these cases, IHC staining has been a valuable diagnostic tool.225-228 The highly sensitive and specific monoclonal antibody p19-11 recognizes different species of Leishmania and allows differentiation from morphologically similar microorganisms (Toxoplasma, Trypanosoma cruzi, and P. marneffei).225 IHC assays with polyclonal antibodies specific for Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba spp. are used to demonstrate amebic trophozoites and cysts in areas of necrosis and can allow their differentiation from macrophages in cases of amebic meningoencephalitis.229 IHC has also been used to identify Cryptosporidium,230 Entamoeba histolytica,231 T. cruzi,232-234 Babesia spp.,235 Giardia lamblia,236 Plasmodium falciparum, and P. vivax in fatal cases of malaria237 in FFPE tissue samples.
Emerging Infectious Diseases
Figure 3-20 Human immunodeficiency virus–infected patient with toxoplasmic encephalitis. Immunoperoxidase staining highlights cyst forms and scattered tachyzoites (diaminobenzidine substrate with hematoxylin counterstain; ×400).
Emerging Infectious Diseases In 1992 the Institute of Medicine defined emerging infectious diseases (EIDs) as those caused by new, previously unidentified microorganisms or those for which the incidence in humans has increased within the previous two decades or is likely to increase in the near future.238 The list of pathogens newly recognized since 1973 is long and continues to increase. Recognizing emerging infections is a challenge, and many new infectious agents remain undetected for years before they become an identified public health problem.239 EIDs are a global phenomenon that requires a global response. The Centers for Disease Control and Prevention (CDC) has defined the strategy to detect and prevent EIDs.239 The anatomic pathology laboratory plays a critical role in the initial and rapid detection of EIDs.240,241 IHC, besides assisting in the identification of new infectious agents, has contributed to the understanding of the pathogenesis and epidemiology of EIDs.
67
Figure 3-21 Hantavirus antigen–containing endothelial cells of the pulmonary microvasculature in the lung of a hantavirus pulmonary syndrome patient as detected by immunohistochemistry by using a mouse monoclonal antibody (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×100).
clinical picture is variable and nonspecific: it can range from subclinical infection to flaccid paralysis and encephalitis characterized morphologically by perivascular mononuclear cell inflammatory infiltrates, neuronal necrosis, edema, and microglial nodules, particularly prominent in the brainstem, cerebellum, and spinal cord.247-251 The diagnosis of WNV encephalitis is usually established by identifying virus-specific IgM in cerebrospinal fluid (CSF) and/or serum and by demonstrating viral RNA in serum, CSF, or other tissue.252 Immuno staining with monoclonal or polyclonal antibodies has been successfully used to diagnose WNV infection in immunocompromised patients with an inadequate antibody response (Fig. 3-22).248
Enterovirus 71 Encephalomyelitis Enterovirus 71 (EV71) has been associated with hand, foot, and mouth disease; herpangina; aseptic meningitis;
Hantavirus Pulmonary Syndrome In 1993 several previously healthy individuals died of rapidly progressive pulmonary edema, respiratory insufficiency, and shock in the southwestern United States.242,243 IHC was central in identifying the viral antigens of an unknown hantavirus.244,245 IHC analysis was also important in detecting the occurrence of unrecognized cases of HPS before 1993 and in showing the distribution of viral antigen in endothelial cells of the microcirculation, particularly in the lung (Fig. 3-21).244,246
West Nile Virus Encephalitis West Nile virus (WNV) was originally identified in Africa in 1937, and the first cases of WNV encephalitis in the United States were described in 1999. The
Figure 3-22 West Nile virus (WNV). Immunostaining of flaviviral antigens in neurons and neuronal processes in the central nervous system of an immunosuppressed patient who died of WNV encephalitis (Flavivirus-hyperimmune mouse ascitic fluid naphthol-fast red substrate with hematoxylin counterstain; original magnification ×40).
68
Immunohistology of Infectious Diseases
and poliomyelitis-like flaccid paralysis. More recently, EV71 has been associated with unusual cases of fulminant encephalitis, pulmonary edema and hemorrhage, and heart failure.253,254 Severe and extensive encephalomyelitis of the cerebral cortex, brainstem, and spinal cord has also been described. IHC staining with monoclonal antibody against EV71 has played a pivotal role in the linking of EV71 infection to fulminant encephalitis (Fig. 3-23). Viral antigen is observed within neurons, neuronal processes, and in mononuclear inflammatory cells.255-257
Nipah Virus Infection Nipah virus is a recently described paramyxovirus that causes an acute febrile encephalitic syndrome with a high mortality rate.258-260 Pathology played a key role in identifying the causative agent. Histopathologic findings include vasculitis with thrombosis, microinfarctions, syncytial giant cells, and viral inclusions.258,260 Although characteristic of this disease, syncytial giant endothelial cells are seen only in 25% of cases,258 and viral inclusions of similar morphology can be seen in other paramyxoviral infections. Immunostaining provides a useful tool for unequivocal diagnosis of the disease, demonstrating viral antigen within neurons and endothelial cells of most organs (Fig. 3-24).5,258
Ehrlichioses The tick-transmitted, intracellular, gram-negative bacteria Ehrlichia chaffeensis, E. ewingii, E. muris-like (EML) organism, and Anaplasma phagocytophilum cause human monocytotropic ehrlichiosis, ehrlichiosis ewingii, a newly emerging unnamed disease, and human granulocytotropic anaplasmosis, respectively. The acute febrile illnesses usually present with cytopenias, myalgias, and mild to moderate hepatitis.261-264 Diagnosis of ehrlichiosis depends upon finding the characteristic monocytic and/or granulocytic cytoplasmic inclusions (morulae), performing a PCR analysis of blood, and detecting specific antibodies in blood.
Figure 3-23 Enterovirus 71 (EV 71). Positive staining of EV71 viral antigens in neurons and neuronal processes of a fatal case of enterovirus encephalitis (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×40).
Figure 3-24 Nipah virus. Immunostaining of Nipah virus antigens in neurons and neuronal processes in the central nervous system of a patient with a fatal case of Nipah virus encephalitis (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×63).
However, morulae are rare and are often missed on initial evaluation; H&E-stained sections often fail to show organisms, even when IHC reveals abundant ehrlichial antigen; and antibody titers may take several weeks to rise to diagnostic levels.261,265 Additionally, immunocompromised patients may not develop anti ehrlichial antibodies before death.261,263 In these cases, immunostaining for Ehrlichia or Anaplasma is a sensitive and specific diagnostic method.261,263,264,266 IHC has been a very valuable tool used to identify and study several other EIDs such as Ebola hemorrhagic fever;93-95 Hendra virus encephalitis5,267,268; leptospirosis163-165; emerging tick-borne rickettsioses caused by R. parkeri269 and R. africae270; and, recently, a new coronavirus associated with severe acute respiratory syndrome (SARS).271,272 SARS was first recognized during a global outbreak of severe pneumonia that occurred in late 2002 in Guangdong Province, China, and then erupted in February 2003 with cases in more than two dozen countries in Asia, Europe, North America, and South America. Early in the investigation, clinical, pathologic, and laboratory studies focused on previously known agents of respiratory illness. Subsequently, a virus was isolated from the oropharynx of a SARS patient and was identified by ultrastructural characteristics as belonging to the family Coronaviridae.271,272 Various reports have described diffuse alveolar damage as the main histopathologic finding in SARS patients, and SARS-associated coronavirus (SARS-CoV) has been demonstrated in human and experimental animal tissues by IHC (Fig. 3-25) and ISH assays.273-282
Pathologists, Immunohistochemistry, and Bioterrorism Currently, concern is increasing about the use of infectious agents as potential biologic weapons. Biologic
Pathologists, Immunohistochemistry, and Bioterrorism
69
public health response to a biologic attack.284-286 Two important components of this response plan include the rapid diagnosis and characterization of biologic agents. Pathologists using newer diagnostic techniques such as IHC, ISH, and PCR will have a direct impact on the rapid detection and control of emerging infectious diseases from natural or intentional causes.287 IHC provides a simple, safe, sensitive, and specific method for rapid detection of biologic threats, either at the time of investigation or retrospectively, and it facilitates rapid implementation of effective public health responses.
Anthrax Figure 3-25 Severe acute respiratory syndrome (SARS). Coro navirus antigen–positive pneumocytes and macrophages in the lung of a SARS patient (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×63).
warfare agents vary from rare exotic viruses to common bacterial agents, and the intentional use of biologic agents to cause disease can simulate naturally occurring outbreaks or may have unusual characteristics.283 The CDC has issued recommendations for a complete
A
C
IHC staining of Bacillus anthracis with monoclonal antibodies against cell-wall and capsule antigens has been successfully used in the recognition of bioterrorismrelated anthrax cases and represented an important step in early diagnosis and treatment of these cases (Fig. 3-26, A-C).5,288-292 Gram-staining and culture isolation of B. anthracis are the usual methods to diagnose anthrax; nevertheless, previous antibiotic treatment affects culture yield and gram-staining identification of the bacteria.290 IHC has demonstrated high sensitivity and specificity for the detection of B. anthracis in skin biopsies, pleural biopsies, transbronchial biopsies, and pleural fluids (see Fig. 3-26).289-291,293 In addition, immunostaining has been very useful for determining the
B
Figure 3-26 Anthrax. A, Photomicrograph of a pleural effusion cell block from a patient with bioterrorism-associated inhalation anthrax shows bacillary fragments and granular antigen staining with the anti–Bacillus anthracis capsule antibody (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×63). B, Skin biopsy from a patient with cutaneous anthrax shows abundant granular antigen and bacillary fragments with B. anthracis cell-wall antibody (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×40). C, Photomicrograph of mediastinal lymph node from a patient with inhalational anthrax shows abundant granular antigen and bacillary fragments with anti–B. anthracis cell-wall antibody (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×63).
70
Immunohistology of Infectious Diseases
Figure 3-27 Tularemia. Immunohistochemistry of a lymph node shows a stellate abscess with Francisella tularensis antigen–bearing macrophages in the central necrotic area by using a mouse monoclonal antibody against the lipopolysaccharide of F. tularensis (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×40).
route of entry of the bacteria and identification of the mode of spread of the disease.290,294
Tularemia IHC staining is also valuable in the rapid identification of Francisella tularensis in formalin-fixed tissue sections. Tularemia can have a variable clinical and pathologic presentation that can simulate other infectious diseases such as anthrax, plague, cat-scratch disease, or lymphogranuloma venereum. Moreover, the microorganisms are difficult to demonstrate in tissue sections, even with gram-staining or silver-staining methods. A mouse monoclonal antibody against the lipopolysaccharide of F. tularensis has been used to demonstrate intact bacteria and granular bacterial antigen in the lungs, spleen, lymph nodes, and liver with high sensitivity and specificity (Fig. 3-27).295,296
Plague A mouse monoclonal antibody directed against the fraction 1 antigen of Yersinia pestis has been used to detect intracellular and extracellular bacteria in dermal blood vessels, lungs, lymph nodes, spleen, and liver (Fig. 3-28).297-302 This technique is potentially useful for the rapid diagnosis of plague in formalin-fixed skin biopsies. In addition, IHC may allow distinction of primary (inhalational) and secondary (hematogenous spread to lung) pneumonic plague by identifying Y. pestis in different lung locations, such as in the alveoli versus the interstitium.297 IHC methods that use polyclonal or monoclonal antibodies have been applied to the identification of several other potential biologic terrorism agents, including the causative agents of brucellosis,5 Q fever,5,138,168,169 viral encephalitides (eastern equine encephalitis; Fig. 3-29),5,121-123 rickettsioses (typhus and RMSF),138-141,187 and viral hemorrhagic fevers (Ebola and Marburg).5,89-95
Figure 3-28 Immunohistochemical stain of lung containing abundant bacterial and granular Yersinia pestis antigen in the alveolar spaces with a mouse monoclonal antibody against F1 capsular antigen (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×20).
Beyond Immunohistology: Molecular Diagnostic Applications During the past several decades, an enormous advance has been made in molecular technology that has dramatically influenced the diagnosis and study of infectious diseases: the application of molecular probes to the study and diagnosis of infectious diseases. This technology is a great adjunct to IHC as a diagnostic method and often allows for even more rapid and specific identification of organisms.303-309 With the rapid advances in molecular diagnostic techniques, a corresponding increased interest has been seen in the use of paraffin-embedded specimens for nucleic acid hybridization assays. Two main, basic hybridization formats have been used in the diagnostic pathology laboratory for the diagnosis of infectious diseases: ISH and PCR. ISH is analogous to IHC in that it allows for the cellular identification and localization of microbial pathogens. Instead of microbial antigens, the targets of ISH are specific RNA or DNA sequences. Many viruses (Figs. 3-30 through 3-33), bacteria
Figure 3-29 Immunostaining of viral antigens in neurons and neuronal processes in central nervous system with a mouse anti–eastern equine encephalitis virus antibody (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×10).
Beyond Immunohistology: Molecular Diagnostic Applications
71
Figure 3-30 Crimean-Congo hemorrhagic fever (CCHF). Localization of CCHF viral RNA as seen in a single CCHF-infected hepatocyte (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×250).
Figure 3-32 Parvovirus infection. Confirmation of B19-infected cells in bone marrow of a patient infected with human immunodeficiency virus by using a digoxigenin-labeled B19 riboprobe and in situ hybridization. Staining is mainly nuclear and is seen in multiple cells that contain classic parvovirus inclusions (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×250).
Figure 3-31 Influenza A. In situ hybridization shows localization of viral nucleic acids in bronchial epithelium with an influenza A hemagglutinin digoxigenin–labeled probe (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×158).
Figure 3-33 Severe acute respiratory syndrome (SARS). Lung shows diffuse alveolar damage and SARS-CoV nucleic acids, primarily in pneumocytes, as seen by colorimetric in situ hybridization (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×158).
72
Immunohistology of Infectious Diseases
Figure 3-34 Ehrlichia chaffeensis. Organisms appear as red inclusions within monocytes by in situ hybridization (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×250).
Figure 3-36 Polyomavirus (BK virus) infection. BK virus–infected cells in urothelium as seen by using a colorimetric in situ hybridization and a DNA probe. Staining is both nuclear and cytoplasmic and is seen in multiple cells that contain classic viral inclusions (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×100).
(Fig. 3-34), and other microorganisms can be localized in tissues by ISH, including EBV, HPV (Fig. 3-35), polyomaviruses (Fig. 3-36), Mycobacterium leprae, Legionella, Haemophilus influenzae, Zygomycetes, and Aspergillus.108,114,207,261,282,310-332 PCR has the advantage of increased sensitivity, minimal tissue requirements, and the potential to sequence the amplified product for a more specific identification of the microbial genotype or strain of the agent involved. PCR assays are available for most microorganisms that have been, or potentially can be, adapted for use on formalin-fixed tissues.11,124,172,293,333-345
Summary
Figure 3-35 Human papillomavirus (HPV). In situ hybridization in a patient with a benign cervical lesion. HPV RNA is localized within the nucleus and cytoplasm of koilocytotic cells (immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain; original magnification ×250).
IHC, ISH, and PCR should be regarded as complementary diagnostic methods for use in the diagnostic pathology laboratory. Each laboratory needs to consider the advantages and limitations of each as they apply to individual cases and overall laboratory needs. This everexpanding field behooves all pathologists interested in the field of infectious diseases to keep abreast of the changing technology and its ever-increasing application in the diagnostic arena. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Cartun RW: Use of immunohistochemistry in the surgical pathology laboratory for the diagnosis of infectious diseases. Pathol Case Rev. 4:260–265, 1999. 2. Watts JC: Surgical pathology in the diagnosis of infectious diseases (Editorial). Am J Clin Pathol. 102:711–712, 1994. 3. Schwartz DA, Bryan RT: Infectious disease pathology and emerging infections: Are we prepared? Arch Pathol Lab Med. 120:117–124, 1996. 4. Schwartz DA: Emerging and reemerging infections: Progress and challenges in the subspecialty of infectious disease pathology. Arch Pathol Lab Med. 121:776–784, 1997. 5. Zaki SR, Paddock CD: The emerging role of pathology in infectious diseases. In Scheld WM, Armnstrong D, Hughes JM, editors. Emerging Infections 3, Washington, D.C., 1999, ASM Press, pp 181–200. 6. Medical Examiners, Coroners, and Biologic Terrorism: A Guidebook for Surveillance and Case Management. MMWR Morbidity and Mortality Weekly Report. 53(RR-8):1–53, 2004. 7. Zaki SR, Peters CJ: Viral hemorrhagic fevers. In Connor DH, Chandler FW, Schwartz DA, et al, editors. Pathology of Infectious Diseases, Stamford, CT, 1997, Appleton & Lange, pp 347– 364. 8. Coons AH, Creech HJ, Jone RN, et al: The demonstration of pneumococcal antigen in tissues by use of fluorescent antibodies. J Immunol. 45:159–170, 1942. 9. Cohen PR: Tests for detecting herpes simplex virus and varicellazoster virus infections. Dermatol Clin. 12:51–68, 1994. 10. Procop GW, Burchette JL, Jr, Howell DN, et al: Immunoperoxidase and immunofluorescent staining of Rickettsia rickettsii in skin biopsies. Arch Pathol Lab Med. 121:894–899, 1997. 11. Guarner J, Greer PW, Whitney A, et al: Pathogenesis and diagnosis of human meningococcal disease using immunohistochemical and PCR assay. Am J Clin Pathol. 122:754–764, 2004. 12. Lepidi H, Houpikina P, Liang Z, et al: Cardiac valves in patients with Q fever endocarditis: microbiological, molecular, and histologic studies. J Infect Dis. 187:1097–1106, 2003. 13. Eyzaguirre EJ, Haque AK: Application of immunohistochemistry to infections. Arch Pathol Lab Med. 132:424–431, 2008. 14. Lepidi H, Fenollar F, Dumler JS: Cardiac valves in patients with Whipple endocarditis: microbiological, molecular, quantitative histologic, and immunohistochemical studies of 5 patients. J Infect Dis. 190:935–945, 2004. 15. Jeavons L, Hunt L, Hamilton A: Immunochemical studies of heat-shock protein 80 of Histoplasma capsulatum. J Med Vet Mycol. 32:47–57, 1994. 16. Werner M, Chott A, Fabiano A, et al: Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol. 24:1016–1019, 2000. 17. Woods GL, Walker DH: Detection of infection or infectious agents by use of cytologic and histologic stains. Clin Microbiol Rev. 9:382–404, 1996. 18. Chandler FW: Invasive microorganisms. In Spicer SS, editor. Histochemistry in Pathology Diagnosis, New York, NY, 1987, Marcel Dekker, pp 77–101. 19. Clausen PP, Thomsen P: Demonstration of hepatitis B surface antigen in liver biopsies. A comparative investigation of immunoperoxidase and orcein staining on identical sections on formalin-fixed, paraffin-embedded tissue. Acta Pathol Microbiol Scand [A]. 86A:383, 1978. 20. Thomsen P, Clausen PP: Occurrence of hepatitis B-surface antigen in a consecutive material of 1539 liver biopsies. Acta Pathol Microbiol Immunol Scand [A]. 91:71, 1983. 21. Al Adnani MS, Ali SM: Patterns of chronic liver disease in Kuwait with special reference to localization of hepatitis B surface antigen. J Clin Pathol. 37:549, 1984. 22. Taylor C: Lung, pancreas, colon and rectum, stomach, liver. In Taylor CR, Cote RJ, editors. Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist, ed 2, Philadelphia, 1994, Saunders, pp 292–317. 23. Park YN, Han KH, Kim KS, et al: Cytoplasmic expression of hepatitis B core antigen in chronic hepatitis B virus infection: role of precore stop mutants. Liver. 19:199–205, 1999. 24. McDonald JA, Harris S, Waters JA, et al: Effect of human immunodeficiency virus (HIV) infection on chronic hepatitis B hepatic viral display. J Hepatol. 4:337–342, 1987.
72.e1
25. Hubscher SG, Portmann BC: Transplantation pathology. In Burt AD, Portmann BC, Ferrell LD, editors. MacSween’s Pathology of the Liver, ed 5, Philadelphia, 2007, Elsevier, pp 815–879. 26. Feiden W, Borchard F, Burrig KF, et al: Herpes esophagitis: I. Light microscopical immunohistochemical investigations. Virchows Arch [A]. 404:167–176, 1984. 27. Nikkels AF, Delvenne P, Sadzot-Delvaux C, et al: Distribution of varicella zoster virus and herpes simplex virus in disseminated fatal infections. J Clin Pathol. 49:243–248, 1996. 28. Greenson JK, Beschorner WE, Boitnott JK, et al: Prominent mononuclear cell infiltrate is characteristic of herpes esophagitis. Hum Pathol. 22:541–549, 1991. 29. Wang JY, Montone KT: A rapid simple in situ hybridization method for herpes simplex virus employing a synthetic biotinlabeled oligonucleotide probe: a comparison with immuno histochemical methods for HSV detection. J Clin Lab Anal. 8:105–115, 1994. 30. Kobayashi TK, Ueda M, Nishino T, et al: Brush cytology of herpes simplex virus infection in oral mucosa: use of the ThinPrep processor. Diag Cytopath. 18:71–75, 1998. 31. Nicoll JAR, Love S, Burton PA, et al: Autopsy findings in two cases of neonatal herpes simplex virus infection: detection of virus by immunohistochemistry, in situ hybridization and the polymerase chain reaction. Histopathology. 24:257–264, 1994. 32. Nikkels AF, Debrus S, Sadzot-Delvaux C, et al: Comparative immunohistochemical study of herpes simplex and varicellazoster infections. Virchows Archiv A Pathol Anat. 422, 1993. 33. Cohen PR: Tests for detecting herpes simplex virus and varicellazoster virus infections. Dermat Clin. 12:51–68, 1994. 34. Katano H, Sato Y, Kurata T, et al: Expression and localization of human herpesvirus 8-encoded proteins in primary effusion lymphoma, Kaposi’s sarcoma, and multicentric Castleman’s disease. Virology. 269:335–344, 2000. 35. Katano H, Suda T, Morishita Y, et al: Human herpesvirus 8-associated solid lymphomas that occur in AIDS patients takes anaplastic large cell morphology. Mod Pathol. 13:77–85, 2000. 36. Ely SA, Powers J, Lewis D, et al: Kaposi’s sarcoma-associated herpesvirus-positive primary effusion lymphoma arising in the subarachnoid space. Hum Pathol. 30:981–984, 1999. 37. Katano H, Sato Y, Kurata T, et al: High expression of HHV-8encoded ORF73 protein in spindle-shape cells of Kaposi’s sarcoma. Am J Pathol. 155:47–52, 1999. 38. Said JW, Shintaku IP, Asou H, et al: Herpesvirus 8 inclusions in primary effusion lymphoma: report of a unique case with T-cell phenotype. Archiv Pathol Lab Med. 123:257–260, 1999. 39. Cheuk W, Wong KO, Wong CS, et al: Immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish Kaposi sarcoma from its mimickers. Am J Clin Pathol. 121:335–342, 2004. 40. Robin YM, Guillou L, Michels JJ, et al: Human herpesvirus 8 immunostaining. A sensitive and specific method for diagnosing Kaposi sarcoma in paraffin-embedded sections. Am J Clin Pathol. 121:330–334, 2004. 41. Wada DA, Perkins SL, Tripp S, et al: Human herpesvirus 8 and iron staining are useful in differentiating Kaposi sarcoma from interstitial granuloma annulare. Am J Clin Pathol. 127:263–270, 2007. 42. Bryant-Greenwood P, Sorbara L, Filie AC, et al: Infection of mesothelial cells with human herpesvirus 8 in human immunodeficiency virus-infected patients with Kaposi sarcoma, Castleman disease, and recurrent pleural effusions. Mod Pathol. 16:145–153, 2003. 43. de la Hoz RE, Stephens G, Sherlock C: Diagnosis and treatment approaches to CMV infections in adult patients. J Clin Virol. 25(suppl 2):S1–S12, 2002. 44. Bronsther O, Makowka L, Jaffe R: The occurrence of cytomegalovirus hepatitis in liver transplant patients. J Med Virol. 24:423– 434, 1988. 45. Drummer JS, White LT, Ho M, et al: Morbidity of cytomegalovirus infection in recipients of heart or heart-lung transplant who receive cyclosporine. J Infect Dis. 152:1182–1191, 1985. 46. Anwar F, Erice A, Jessurun J: Are there cytopathic features associated with cytomegalovirus infection predictive of resistance to antiviral therapy? Ann Diag Pathol. 3:19–22, 1999. 47. Cote L, Drouet E, Bissuel F, et al: Diagnostic value of amplification of human cytomegalovirus DNA from gastrointestinal
72.e2
Immunohistology of Infectious Diseases
biopsies from human immunodeficiency virus-infected patients. J Clin Microbiol. 31:2066–2069, 1993. 48. Rawlinson WD: Broadsheet number 50: Diagnosis of human cytomegalovirus infection and disease. Pathology. 31, 1999. 49. Sheehan MM, Coker R, Coleman DV: Detection of cytomegalovirus (CMV) in HIV+ patients: comparison of cytomorphology, immunohistochemistry and in situ hybridization. Cytopath. 9:29–37, 1998. 50. Kutza AS, Muhl E, Hackstein H, et al: High incidence of active cytomegalovirus infection among septic patients. Clin Infect Dis. 26:1076–1082, 1998. 51. Saetta A, Agapitos E, Davaris PS: Determination of CMV placentitis: Diagnostic application of the polymerase chain reaction. Virchows Arch. 432:159–162, 1998. 52. Solans EP, Yong S, Husain AN, et al: Bronchioloalveolar lavage in the diagnosis of CMV pneumonitis in lung transplant recipients: an immunocytochemical study. Diagn Cytopath. 16:350– 352, 1997. 53. Nebuloni M, Pellegrinelli A, Ferri A, et al: Etiology of microglial nodules in brains of patients with acquired immunodeficiency syndrome. J Neurovirol. 6:46–50, 2000. 54. Rimsza LM, Vela EE, Frutiger YM, et al: Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies. Am J Clin Pathol. 106:544–548, 1996. 55. Kandiel A, Lashner B: Cytomegalovirus colitis complicating inflammatory bowel disease. Am J Gastroenterol. 101:2857– 2865, 2006. 56. Kambham N, Vij R, Cartwright CA, et al: Cytomegalovirus infection in steroid-refractory ulcerative colitis. A case-control study. Am J Surg Pathol. 28:365–373, 2004. 57. Ribalta T, Martinez AJ, Jares P, et al: Presence of occult cytomegalovirus infection in the brain after orthotopic liver transplantation. An autopsy study of 83 cases. Virchows Arch. 440:166–171, 2002. 58. Cruz-Spano L, Lima-Pereira FE, Gomes da Silva-Basso N, et al: Human cytomegalovirus infection and abortion: an immunohistochemical study. Med Sci Monit. 8:BR230–BR235, 2002. 59. Seehofer D, Rayes N, Tullius SG, et al: CMV hepatitis after liver transplantation: incidence, clinical course, and long term follow-up. Liver Transp. 8:1138–1146, 2002. 60. Lautenschlager I, Hockerstedt K, Taskinen E: Histologic findings associated with CMV infection in liver transplantation. Transplant Proc. 35:819, 2003. 61. Lamps LW, Pinson CW, Raiford DS, et al: The significance of microabscesses in liver transplant biopsies: a clinicopathological study. Hepatology. 28:1532–1537, 1998. 62. Barkholt LM, Ehrnst A, Veress B: Clinical use of immuno histopathologic methods for the diagnosis of cytomegalovirus hepatitis in human liver allograft biopsy specimens. Scand J Gastroenterol. 29:553–560, 1994. 63. Colina F, Jucá NT, Moreno E, et al: Histological diagnosis of cytomegalovirus hepatitis in liver allografts. J Clin Pathol. 48:351–357, 1995. 64. Lones MA, Shintaku IP, Weiss LM, et al: Posttransplant lymphoproliferative disorder in liver allograft biopsies: a comparison of three methods for the demonstration of Epstein Barr virus. Hum Pathol. 28:533–539, 1997. 65. Challoner PB, Smith KT, Parker JD, et al: Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci U S A. 92:7440–7444, 1995. 66. Anderson J: Epstein-Barr virus and Hodgkin’s lymphoma. HERPES. 13:12–16, 2006. 67. Flomenberg P, Babbitt J, Drobyski WR, et al: Increasing incidence of adenovirus disease in bone marrow transplant recipients. J Infect Dis. 169:775–781, 1994. 68. Strickler JG, Singleton TP, Copenhaver GM, et al: Adenovirus in the gastrointestinal tracts of immunosuppressed patients. Am J Clin Pathol. 97:555–558, 1992. 69. Yi ES, Powell HC: Adenovirus infection of the duodenum in an AIDS patient: an ultrastructural study. Ultrastruct Pathol. 18:549–551, 1994. 70. Yan Z, Nguyen S, Poles M, et al: Adenovirus colitis in human immunodeficiency virus infection: an underdiagnosed entity. Am J Surg Pathol. 22:1101–1106, 1998.
71. Dombrowski F, Eis-Hubinger AM, Ackermann T, et al: Adenovirus-induced liver necrosis in a case of AIDS. Virchows Archiv. 431:469–472, 1997. 72. Simsir A, Greenebaum E, Nuovo G, et al: Late fatal adenovirus pneumonitis in a lung transplant recipient. Transplantation. 65:592–594, 1998. 73. Saad RS, Demetris AJ, Lee RG, et al: Adenovirus hepatitis in the adult allograft liver. Transplantation. 64:1483–1485, 1997. 74. Ohori NP, Michaels MG, Jaffe R, et al: Adenovirus pneumonia in lung transplant recipients. Hum Pathol. 26:1073–1079, 1995. 75. Marc J, Dieterich DT: The histopathology of 103 consecutive colonoscopy biopsies from 82 symptomatic patients with acquired immunodeficiency syndrome. Arch Pathol Lab Med. 125:1042–1046, 2001. 76. El-Mahallawy HA, Mansour T, El-Din SE, et al: Parvovirus B19 infection as a cause of anemia in pediatric acute lymphoblastic leukaemia during maintenance chemotherapy. J Pediatr Hematol Oncol. 26:403–406, 2004. 77. Brown KE, Young NS: Parvovirus B19 infection and hematopoiesis. Blood Rev. 9:176–182, 1995. 78. Jordan JA, Penchansky L: Diagnosis of human parvovirus B19induced anemia: correlation of bone marrow morphology with molecular diagnosis using PCR and immunohistochemistry. Cell Vision. 2:279–282, 1995. 79. Morey AL, O’Neil HJ, Coyle PV, et al: Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffinembedded tissues. J Pathol. 166:105–108, 1992. 80. Puvion-Dutilleul F, Puvion E: Human parvovirus B19 as a causative agent for rheumatoid arthritis. Proc Nat Acad Sci U S A. 95:8227–8232, 1998. 81. Yufu Y, Matsumoto M, Miyamura T, et al: Parvovirus B19associated haemophagocytic syndrome with lymphadenopathy resembling histiocytic necrotizing lymphadenitis (Kikuchi’s disease). Br J Haematol. 96:868–871, 1997. 82. Vadlamudi G, Rezuke N, Ross JW, et al: The use of monoclonal antibody R92F6 and polymerase chain reaction to confirm the presence of parvovirus B19 in bone marrow specimens of patients with acquired immunodeficiency syndrome. Arch Pathol Lab Med. 123:768–773, 1999. 83. Wright C, Hinchliffe SA, Taylor C: Fetal pathology in intrauterine death due to parvovirus B19 infection. Br J Obstet Gynaecol. 103:133–136, 1996. 84. Essary LR, Vnencak-Jones CL, Manning SS, et al: Frequency of parvovirus B19 infection in nonimmune hydrops fetalis and utility of three diagnostic methods. Hum Pathol. 29:696–701, 1998. 85. Monath TP, Ballinger ME, Miller BR, et al: Detection of yellow fever viral RNA by nucleic acid hybridization and viral antigen by immunohistochemistry in fixed human liver. Am J Trop Med Hyg. 40:663–668, 1989. 86. De Brito T, Siqueira SA, Santos RT, et al: Human fatal yellow fever. Immunohistochemical detection of viral antigens in the liver, kidney and heart. Pathol Res Pract. 188:177–181, 1992. 87. Hall WC, Crowell TP, Watts DM, et al: Demonstration of yellow fever and dengue antigens in formalin-fixed paraffin-embedded human liver by immunohistochemical analysis. Am J Trop Med Hyg. 45:408–417, 1991. 88. Ramos C, Sanchez G, Pando RH, et al: Dengue virus in the brain of a fatal case of hemorrhagic dengue fever. J Neurovirol. 4:465– 468, 1998. 89. Burt FJ, Swanepoel R, Shieh W-J, et al: Immunohistochemical and in situ localization of Crimean-Congo hemorrhagic fever (CCHF) virus in human tissues and implications for CCHF pathogenesis. Arch Pathol Lab Med. 121:839–846, 1997. 90. Maiztegui JI, Laguens RP, Cossio PM, et al: Ultrastructural and immunohistochemical studies in five cases of Argentine hemorrhagic fever. J Infect Dis. 132:35–53, 1975. 91. Hall WC, Geisbert TW, Huggins JW, et al: Experimental infection of guinea pigs with Venezuelan hemorrhagic fever virus (Guanarito): a model of human disease. Am J Trop Med Hyg. 55:81–88, 1996. 92. Geisbert TW, Jaax NK: Marburg hemorrhagic fever: report of a case studied by immunohistochemistry and electron microscopy. Ultrastruct Pathol. 22:3–17, 1998.
References 93. Zaki SR, Shieh W-J, Greer PW, et al: A novel immunohistochemical assay for the detection of Ebola virus in skin: implications for diagnosis, spread, and surveillance of Ebola hemorrhagic fever. J Infect Dis. 179(Suppl 1):S36–S37, 1999. 94. Ksiazek TG, Rollin PE, Williams AJ, et al: Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of Congo. J Infect Dis. 179:S177–S187, 1999. 95. Wyers M, Formenty P, Cherel Y, et al: Histopathological and immunohistochemical studies of lesions associated with Ebola virus in a naturally infected chimpanzee. J Infect Dis. 179(Suppl 1):S54–S59, 1999. 96. Nickeleit V, Hirsch HH, Zeiler M, et al: BK-virus nephropathy in renal transplants-tubular necrosis, MHC-class II expression and rejection in a puzzling game. Nephrol Dial Transplant. 15:324–332, 2000. 97. White LH, Casian A, Hilton R, et al: BK virus nephropathy in renal transplant patients in London. Transplantation. 85:1008– 1015, 2008. 98. Drachenberg CB, Papadimitriou JC, Ramos E: Histologic versus molecular diagnosis of BK polyomavirus-associated nephropathy: a shifting paradigm? Clin J Am Soc Nephrol. 1:374–379, 2006. 99. Vogler C, Wang Y, Brink DS, et al: Renal pathology in the pediatric transplant patient. Adv Anat Pathol. 14:202–216, 2007. 100. Dall A, Hariharan S: BK virus nephritis after renal transplantation. Clin J Am Soc Nephrol. 3(suppl 2):S68–S75, 2008. 101. Latif S, Zaman F, Veeramachaneni R, et al: BK polyomavirus in renal transplants: role of electron microscopy and immunostaining in detecting early infection. Ultrastruct Pathol. 31:199–207, 2007. 102. Nebuloni M, Tosoni A, Boldorini R, et al: BK virus renal infection in a patient with the acquired immunodeficiency syndrome. Arch Pathol Lab Med. 123:807–811, 1999. 103. Elli A, Banfi G, Battista-Fogazzi G, et al: BK polyomavirus interstitial nephritis in a renal transplant patient with no previous acute rejection episodes. J Nephrol. 15:313–316, 2002. 104. Jochum W, Weber T, Frye S, et al: Detection of JC virus by antiVP1 immunohistochemistry in brains with progressive multifocal leukoencephalopathy. Acta Neuropathol. 94:226–231, 1997. 105. Chima SC, Agostini HT, Ryschlkeewitsch CF, et al: Progressive multifocal leukoencephalopathy and JC virus genotypes in West African patients with acquired immunodeficiency syndrome. A pathologic and DNA sequence analysis of 4 cases. Arch Pathol Lab Med. 123:395–403, 1999. 106. Aoki N, Mori M, Kato K, et al: Antibody against synthetic multiple antigen peptides (MAP) of JC virus capsid protein (VP1) without cross reaction to BK virus: a diagnostic tool for progressive multifocal leukoencephalopathy. Neurosci Lett. 205:111– 114, 1996. 107. Silver SA, Arthur RR, Rozan YS, et al: Diagnosis of progressive multifocal leukoencephalopathy by stereotactic brain biopsy utilizing immunohistochemistry and the polymerase chain reaction. Acta Cytol. 39:35–44, 1995. 108. Guarner J, Shieh WJ, Dawson J, et al: Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs. Am J Clin Pathol. 114:227–233, 2000. 109. Guarner J, Paddock CD, Shieh WJ, et al: Histopathologic and immunohistochemical features of fatal influenza virus infection in children during the 2003-2004 season. Clin Infect Dis. 43:132–140, 2006. 110. Nielson KA, Yunis EJ: Demonstration of respiratory syncytial virus in an autopsy series. Pediatr Pathol. 10:491–502, 1990. 111. Wright C, Oliver KC, Fenwick FI, et al: A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffinembedded tissue. J Pathol. 182:238–244, 1997. 112. Jogai S, Radotra BD, Banerjee AK: Immunohistochemical study of human rabies. Neuropathology. 20:197–203, 2000. 113. Jogai S, Radotra BD, Banerjee AK: Rabies viral antigen in extracranial organs: a postmortem study. Neuropathol Appl Neurobiol. 28:334, 2002. 114. Warner CK, Zaki SR, Shieh WJ, et al: Laboratory investigation of humans from vampire bat rabies in Peru. Am J Trop Med Hyg. 60:502–507, 1999.
72.e3
115. Sinchaisri TA, Nagata T, Yoshikawa Y, et al: Immunohistochemical and histopathological study of experimental rabies infection in mice. J Vet Med Sci. 54:409–416, 1992. 116. Jackson AC, Ye H, Phelan CC, et al: Extraneural organ involvement in human rabies. Lab Invest. 79:945–951, 1999. 117. Yousef GE, Mann GF, Brown IN, et al: Clinical and research application of an enterovirus group-reactive monoclonal antibody. Intervirol. 28:199–205, 1987. 118. Hohenadl C, Klingel K, Rieger P, et al: Investigation of the coxsackievirus B3 nonstructural proteins 2B, 2C, and 3AB: generation of specific polyclonal antisera and detection of replicating virus in infected tissue. J Virol Methods. 47:279–295, 1994. 119. Zhang H, Li Y, Peng T, et al: Localization of enteroviral antigen in myocardium and other tissues from patients with heart muscle disease by an improved immunohistochemical technique. J Histochem Cytochem. 48:579–584, 2000. 120. Li Y, Bourlet T, Andreoletti L, et al: Enteroviral capsid protein VP1 is present in myocardial tissues from some patients with myocarditis or dilated cardiomyopathy. Circulation. 101:231– 234, 2000. 121. Del Piero F, Wilkins PA, Dubovi EJ, et al: Clinical, pathologic, immunohistochemical, and virologic findings of eastern equine encephalitis in two horses. Vet Pathol. 38:451–456, 2001. 122. Patterson JS, Maes RK, Mullaney TP, et al: Immunohistochemical diagnosis of eastern equine encephalomyelitis. J Vet Diag Invest. 8:156–160, 1996. 123. Garen PD, Tsai TF, Powers JM: Human eastern equine encephalitis: immunohistochemistry and ultrastructure. Mod Pathol. 12:646–652, 1999. 124. Tatti KM, Gentsch J, Shieh WJ, et al: Molecular and immunological methods to detect rotavirus in formalin-fixed tissue. J Virol Methods. 105:305–319, 2002. 125. Morrison C, Gilson T, Nuovo GJ, et al: Histologic distribution of fatal rotaviral infection: an immunohistochemical and reverse transcriptase in situ polymerase chain reaction analysis. Hum Pathol. 32:216–221, 2001. 126. Cioc AM, Nuovo GJ: Histologic and in situ viral findings in the myocardium in cases of sudden, unexpected death. Mod Pathol. 15:914–922, 2001. 127. Toulaymant M, Marconi S, Garb J, et al: Endoscopic biopsy pathology of Helicobacter pylori gastritis. Arch Pathol Lab Med. 123:778–781, 1999. 128. El-Zimaity HMT, Graham DY, Al-Assis MT, et al: Interobserver variation in the histopathological assessment of Helicobacter pylori gastritis. Hum Pathol. 27:35–41, 1996. 129. Marcio L, Angelucci D, Grossi L, et al: Anti-Helicobacter pylori specific antibody immunohistochemistry improves the diagnostic accuracy of Helicobacter pylori in biopsy specimen from patients treated with triple therapy. Am J Gastroenterol. 93:223– 226, 1998. 130. Rotimi O, Cairns A, Gray S, et al: Histological identification of Helicobacter pylori: comparison of staining methods. J Clin Pathol. 53:756–759, 2000. 131. Goldstein NS: Chronic inactive gastritis and coccoid Helicobacter pylori in patients treated for gastroesophageal reflux disease or with H. pylori eradication therapy. Am J Clin Pathol. 118:719–726, 2002. 132. Eshun JK, Black DD, Casteel HB: Comparison of immunohistochemistry and silver stain for the diagnosis of pediatric Helicobacter pylori infection in urease-negative gastric biopsies. Ped Devel Pathol. 4:82–88, 2001. 133. Lepidi H, Fenollar F, Dumler JS, et al: Cardiac valves in patients with Whipple endocarditis: microbiological, molecular, quantitative histologic, and immunohistochemical studies of 5 patients. J Infect Dis. 190:935–945, 2004. 134. Houpikian P, Raoult D: Diagnostic methods: current best practices and guidelines for identification of difficult-to-culture pathogens in infective endocarditis. Infect Dis Clin N Am. 16:377–392, 2007. 135. Baisden BL, Lepidi H, Raoult D, et al: Diagnosis of Whipple disease by immunohistochemical analysis. A sensitive and specific method for the detection of Tropheryma whipplei (the Whipple bacillus) in paraffin-embedded tissue. Am J Clin Pathol. 118:742–748, 2002.
72.e4
Immunohistology of Infectious Diseases
136. Lepidi H, Fenollar F, Gerolami R, et al: Whipple disease: Immunospecific and quantitative immunohistochemical study of intestinal biopsy specimens. Hum Pathol. 34:589–596, 2003. 137. Lepidi H, Costedoat N, Piette JC, et al: Immunohistological detection of Tropheryma whipplei (Whipple bacillus) in lymph nodes. Am J Med. 113:334–336, 2002. 138. Dumler JS, Walker DH: Diagnostic tests for Rocky Mountain spotted fever and other rickettsial diseases. Dermat Clin. 12:25– 36, 1994. 139. Parola P, Paddock CD, Raoult D: Tick-borne rickettsioses around the world: Emerging diseases challenging old concepts. Clin Microbiol Rev. 18:719–756, 2005. 140. Walker DH, Burday MS, Folds JD: Laboratory diagnosis of Rocky Mountain spotted fever. South Med J. 73:1443–1447, 1980. 141. Paddock CD, Greer PW, Ferebee TL, et al: Hidden mortality attributable to Rocky Mountain spotted fever: immunohistochemical detection of fatal, serologically unconfirmed disease. J Infect Dis. 179:1469–1476, 1999. 142. Kaplowitz LG, Lange JV, Fischer JJ, et al: Correlation of rickettsial titers, circulating endotoxin, and clinical features in Rocky Mountain spotted fever. Arch Intern Med. 143:1149–1151, 1983. 143. Dumler JS, Gage WR, Pettis GL, et al: Rapid immunoperoxidase demonstration of Rickettsia rickettsii in fixed cutaneous specimens from patients with Rocky Mountain spotted fever. Am J Clin Pathol. 93:410–414, 1990. 144. Chapman AS, Bakken JS, Folk SM, et al: Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichiosis, and anaplasmosis—United States: A practical guide for physicians and other health-care and public health professionals. MMWR Recomm Rep. 55:1–27, 2006. 145. Spach DH, Koehler JE: Bartonella-associated infections. Infect Dis Clin North Am. 12:137–155, 1998. 146. Lepidi H, Durack DT, Raoult D: Diagnostic methods: Current best practices and guidelines for histologic evaluation in infective endocarditis. Infect Dis Clin North Am. 16:339–361, 2002. 147. Albrich WC, Kraft C, Fisk T, et al: A mechanic with a bad valve: Blood-culture-negative endocarditis. Lancet Infect Dis. 4:777– 784, 2004. 148. Lepidi H, Fournier PE, Raoult D: Quantitative analysis of valvular lesions during Bartonella endocarditis. Am J Clin Pathol. 114:880–889, 2000. 149. Baorto E, Payne RM, Slater LN, et al: Culture-negative endocarditis caused by Bartonella henselae. J Pediatr. 132:1051–1054, 1998. 150. Reed JA, Brigati DJ, Flynn SD, et al: Immunohistochemical identification of Rochalimaea henselae in bacillary (epithelioid) angiomatosis, parenchymal bacillary peliosis, and persistent fever with bacteremia. Am J Surg Pathol. 16:650–657, 1992. 151. Min KW, Reed JA, Welch DF, et al: Morphologically variable bacilli of cat scratch disease are identified by immunohistochemical labeling with antibodies to Rochalimaea henselae. Am J Clin Pathol. 101:607–610, 1994. 152. Daybell D, Paddock CD, Zaki SR, et al: Disseminated infection with Bartonella henselae as a cause of spontaneous splenic rupture. Clin Infect Dis. 39:21–24, 2004. 153. Buffet M, Grange PA, Gerhardt P, et al: Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry. J Invest Dermatol. 127:2345–2350, 2007. 154. Jethwa HS, Schmitz JL, Dallabetta G, et al: Comparison of molecular and microscopic techniques for detection of Treponema pallidum in genital ulcers. J Clin Microbiol. 33:180–183, 1995. 155. Kingston AA, Vujevich J, Shapiro M, et al: Seronegative secondary syphilis in 2 patients coinfected with human immunodeficiency virus. Arch Dermatol. 141:431–433, 2005. 156. Hoang MP, High WA, Molberg KH: Secondary syphilis: A histologic and immunohistochemical evaluation. J Cutan Pathol. 31:595–599, 2004. 157. Guarner J, Greer PW, Bartlett J, et al: Congenital syphilis in a newborn: an immunopathologic study. Mod Pathol. 12:82–87, 1999. 158. Ulrichs T, Lefmann K, Reich M, et al: Modified immunohistological staining allows detection of Ziehl-Neelsen-negative Mycobacterium tuberculosis organisms and their precise localization in human tissues. J Pathol. 205:633–640, 2005.
159. Daniel TM: The rapid diagnosis of tuberculosis: A selective review. J Lab Clin Med. 116:277–282, 1990. 160. Carabias E, Palenque R, Serrano JM, et al: Evaluation of an immunohistochemical test with polyclonal antibodies raised against mycobacteria used in formalin-fixed tissue compared with mycobacterial specific culture. APMIS. 106:385–388, 1998. 161. Mustafa T, Wiker HG, Mfinanga SGM, et al: Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculosis lymphadenitis. Mod Pathol. 19:1606–1614, 2006. 162. Zaki SR, Shieh WJ, and the Epidemic Working Group: Leptospirosis associated with an outbreak of acute febrile illness and pulmonary hemorrhage, Nicaragua 1995. Lancet. 347:535–536, 1996. 163. Trevejo RT, Rigau-Pérez JG, Ashford DA, et al: Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua. J Infect Dis. 1998(178):1457–1463, 1995. 164. Guarner J, Shieh WJ, Morgan J, et al: Leptospirosis mimicking acute cholecystitis among athletes participating in a triathlon. Hum Pathol. 32:750–752, 2001. 165. Nicodemo AC, Duarte MI, Alves VA, et al: Lung lesions in human leptospirosis: microscopic, immunohistochemical, and ultrastructural features related to thrombocytopenia. Am J Trop Med Hyg. 56:181–187, 1997. 166. Lebech AM, Clemmensen O, Hansen K: Comparison of in vitro, immunohistochemical staining, and PCR for detection of Borrelia burgdorferi in tissue from experimentally infected animals. J Clin Microbiol. 33:2328–2333, 1995. 167. Aberer E, Kersten A, Klade H, et al: Heterogeneity of Borrelia burgdorferi in the skin. Am J Dermatopathol. 18:571–579, 1996. 168. Brouqui P, Raoult D: Endocarditis due to rare and fastidious bacteria. Clin Microbiol Rev. 14:177–207, 2001. 169. Brouqui P, Dumler JS, Raoult D: Immunohistologic demonstration of Coxiella burnetii in the valves of patients with Q fever endocarditis. Am J Med. 97:451–458, 1994. 170. Guarner J, Packard MM, Nolte KB, et al: Usefulness of immunohistochemical diagnosis of Streptococcus pneumoniae in formalin-fixed, paraffin-embedded specimens compared with culture and gram stain techniques. Am J Clin Pathol. 127:617– 618, 2007. 171. Guarner J, Bartlett J, Reagan S, et al: Immunohistochemical evidence of Clostridium sp, Staphylococcus aureus, and group A Streptococcus in severe soft tissue infections related to injection drug use. Hum Pathol. 37:1482–1488, 2006. 172. Guarner J, Sumner J, Paddock CD, et al: Diagnosis of invasive group A streptococcal infections by using immunohistochemical and molecular assays. Am J Clin Pathol. 126:148– 155, 2006. 173. Terpstra WJ, Groeneveld K, Eijk PP, et al: Comparison of two nonculture techniques for detection of Hemophilus influenzae in sputum: In situ hybridization and immunoperoxidase staining with monoclonal antibodies. Chest. 94:126S, 1988. 174. Groeneveld K, van Alphen L, van Ketel RJ, et al: Nonculture detection of Haemophilus influenzae in sputum with monoclonal antibodies specific for outer membrane lipoprotein P6. J Clin Microbiol. 27:2263, 1989. 175. Forsgren J, Samuelson A, Borrelli S, et al: Persistence of nontypeable Haemophilus influenzae in adenoid macrophages: a putative colonization mechanism. Act Oto-Laryngol. 116:766–773, 1996. 176. Shurbaji MS, Dumler JS, Gage WR, et al: Immunohistochemical detection of chlamydial antigens in association with cystitis. Am J Pathol. 93:363, 1990. 177. Paukku M, Puolakkainen M, Paavonen T, et al: Plasma cell endometritis is associated with Chlamydia trachomatis infection. Am J Clin Pathol. 112:211–215, 1999. 178. Naas J, Gnarpe JA: Demonstration of Chlamydia pneumoniae in tissue by immunohistochemistry. APMIS. 107:882–886, 1999. 179. Suffin SC, Kaufmann AF, Whitaker B, et al: Legionella pneumophila. Identification in tissue sections by a new immunoenzymatic procedure. Arch Pathol Lab Med. 104:283–286, 1980. 180. Maruta K, Miyamoto H, Hamada T, et al: Entry and intracellular growth of Legionella dumoffii in alveolar epithelial cells. Am J Respir Crit Care Med. 157:1967–1974, 1998.
References 181. Fiore AE, Nuorti JP, Levine OS, et al: Epidemic Legionnaires’ disease two decades later: Old sources, new diagnostic methods. Clin Infect Dis. 26:426–433, 1998. 182. Parkash V, Morotti RA, Joshi V, et al: Immunohistochemical detection of Listeria antigens in the placenta in perinatal listeriosis. Int J Gynecol Pathol. 17:343–350, 1998. 183. Chiba M, Fukushima T, Koganei K, et al: Listeria monocytogenes in the colon in a case of fulminant ulcerative colitis. Scand J Gastroenterol. 33:778–782, 1998. 184. Weinstock D, Horton SB, Rowland PH: Rapid diagnosis of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissue. Vet Pathol. 32:193–195, 1995. 185. Pospischil A, Wood RL, Anderson TD: Peroxidase-antiperoxidase and immunogold labeling of Salmonella typhimurium and Salmonella cholerasuis var kunzendorf in tissues of experimentally infected swine. Am J Vet Res. 51:619–624, 1990. 186. Thygesen P, Martinsen C, Hougen HP, et al: Histologic, cytologic, and bacteriologic examination of experimentally induced Salmonella typhimurium infection in Lewis rats. Comp Med. 50:124–132, 2000. 187. Walker DH, Feng HM, Ladner S, et al: Immunohistochemical diagnosis of typhus rickettsioses using an anti-lipopolysaccharide monoclonal antibody. Mod Pathol. 10:1038–1042, 1997. 188. Koss T, Carter EL, Grossman ME, et al: Increased detection of rickettsialpox in a New York City hospital following the anthrax outbreak of 2001: Use of immunohistochemistry for the rapid confirmation of cases in an era of bioterrorism. Arch Dermatol. 139:1545–1552, 2003. 189. Walker DH, Hudnall SD, Szaniawski WK, et al: Monoclonal antibody-based immunohistochemical diagnosis of rickettsialpox: the macrophage is the principal target. Mod Pathol. 12:529– 533, 1999. 190. Moron CG, Popov VL, Feng HM, et al: Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus. Mod Pathol. 14:752–759, 2001. 191. Schwarz J: The diagnosis of deep mycoses by morphological methods. Hum Pathol. 13:519–533, 1982. 192. Marques MEA, Coelho KIR, Bacchi CE: Comparison between histochemical and immunohistochemical methods for the diagnosis of sporotrichosis. J Clin Pathol. 45:1089–1093, 1992. 193. Reed JA, Hemaan BA, Alexander JL, et al: Immunomycology: Rapid and specific immunocytochemical identification of fungi in formalin-fixed, paraffin-embedded material. J Histochem Cytochem. 41:1217–1221, 1993. 194. Jensen HE, Schonheyder H, Hotchi M, et al: Diagnosis of systemic mycosis by specific immunohistochemical tests. APMIS. 104:241–258, 1996. 195. Fukuzawa M, Inaba H, Hayama M, et al: Improved detection of medically important fungi by immunoperoxidase staining with polyclonal antibodies. Virchows Arch. 427:407–414, 1995. 196. Kauffman L: Immunohistologic diagnosis of systemic mycosis: An update. Eur J Epidemiol. 8:377–382, 1992. 197. Verweij PE, Smedts F, Poot T: Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections. J Clin Pathol. 49:798–801, 1996. 198. Breier F, Oesterreicher C, Brugger S, et al: Immunohistochemistry with monoclonal antibody against Candida albicans mannan antigen demonstrates cutaneous Candida granulomas as evidence of Candida sepsis in an immunosuppressed host. Dermatology. 194:293–296, 1997. 199. Marcilla A, Monteagudo C, Mormeneo S, et al: Monoclonal antibody 3H8: A useful tool in the diagnosis of candidiasis. Microbiol. 145:695–701, 1999. 200. Monteagudo C, Marcilla A, Mormeneo S, et al: Specific immunohistochemical identification of Candida albicans in paraffinembedded tissue with a new monoclonal antibody (1B12). Am J Clin Pathol. 103:130–135, 1995. 201. Jarvensivu A, Hietanen J, Rautemaa R, et al: Candida yeast in chronic periodontitis tissues and subgingival microbial biofilms in vivo. Oral Diseases. 10:106–112, 2004. 202. Williams DW, Jones HS, Allison RT: Immunohistochemical detection of Candida albicans in formalin fixed, paraffin embedded material. J Clin Pathol. 51:857–859, 1998. 203. Jarvensivu A, Rautemaa R, Sorsa T, et al: Specificity of the monoclonal antibody 3H8 in the immunohistochemical identification of Candida species. Oral Diseases. 12:428–433, 2006.
72.e5
204. Kockenberger MB, Canfield PJ, Kozel TR, et al: An immunohistochemical method that differentiates Cryptococcus neoformans varieties and serotypes in formalin-fixed paraffin-embedded tissues. Med Mycol. 39:523–533, 2001. 205. Tsunemi T, Kamata T, Fumimura Y, et al: Immunohistochemical diagnosis of Cryptococcus neoformans var. gatti infection in chronic meningoencephalitis: The first case in Japan. Intern Med. 40:1241–1244, 2001. 206. Chamilos G, Luna M, Lewis RE, et al: Invasive fungal infections in patients with hematologic malignancies in a tertiary care cancer center: An autopsy study over a 15-year period (19892003). Haematologica. 91:986–989, 2006. 207. Hayden RT, Isolato PA, Parrett T, et al: In situ hybridization for the differentiation of Aspergillus, Fusarium, and Pseudallescheria species in tissue sections. Diagn Mol Pathol. 12: 21–26, 2003. 208. Rickerts V, Mousset S, Lambrecht E, et al: Comparison of histopathological analysis, culture, and polymerase chain reaction assays to detect invasive mold infections from biopsy specimens. Clin Infect Dis. 44:1078–1083, 2007. 209. Hope WW, Walsh TJ, Denning DW: Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis. 5:609–622, 2005. 210. Tarrand JJ, Lichterfeld M, Warraich I, et al: Diagnosis of invasive septated mold infections. A correlation of microbiological culture and histologic or cytologic examination. Am J Clin Pathol. 119:854–858, 2003. 211. Choi JK, Mauger J, McGowan KL: Immunohistochemical detection of Aspergillus species in pediatric tissue samples. Am J Clin Pathol. 121:18–25, 2004. 212. Pierard GE, Arrese-Estrada J, Pierard-Franchimont C, et al: Immunohistochemical expression of galactomannan in the cytoplasm of phagocytic cells during invasive aspergillosis. Am J Clin Pathol. 96:373–376, 1991. 213. Phillips P, Weiner MH: Invasive aspergillosis diagnosed by monoclonal and polyclonal reagents. Hum Pathol. 18:1015–1024, 1987. 214. Fenelon LE, Hamilton AJ, Figueroa JI, et al: Production of specific monoclonal antibodies to Aspergillus species and their use in immunohistochemical identification of aspergillosis. J Clin Microbiol. 37:1221–1223, 1999. 215. Jensen HE, Salonen J, Ekfors TO: The use of immunohistochemistry to improve sensitivity and specificity in the diagnosis of systemic mycoses in patients with haematological malignancies. J Pathol. 181:100–105, 1997. 216. Wazir JE, Brown I, Martin-Bates E, et al: EB9, a new antibody for the detection of trophozoites of Pneumocystis carinii in bronchoalveolar lavage specimens in AIDS. J Clin Pathol. 47:1108– 1111, 1994. 217. Wazir JE, Macrorie SG, Coleman DV: Evaluation of the sensitivity, specificity, and predictive value of monoclonal antibody 3F6 for the detection of Pneumocystis carinii pneumonia in bronchoalveolar lavage specimens and induced sputum. Cytopathol. 5:82–89, 1994. 218. Cooper CR, McGinnis MR: Pathology of Penicillium marneffei: An emerging acquired immunodeficiency syndrome-related pathogen. Arch Lab Pathol Med. 121:798–804, 1997. 219. Chaiwun B, Khunamornpong S, Sirivanichai C, et al: Lymphadenopathy due to Penicillium marneffei infection: Diagnosis by fine needle aspiration cytology. Mod Pathol. 15:939–943, 2002. 220. Arrese Estrada J, Stynen D, Van Cutsem J, et al: Immunohistochemical identification of Penicillium marneffei by monoclonal antibody. Int J Dermatol. 31:410–412, 1992. 221. Burke DG, Emancipator SN, Smith MC, et al: Histoplasmosis and kidney disease in patients with AIDS. Clin Infect Dis. 25:281–284, 1997. 222. Arnold SJ, Kinney MC, McCormick MS, et al: Disseminated toxoplasmosis: Unusual presentations in the immunocompromised host. Arch Pathol Lab Med. 121:869–873, 1997. 223. Warnke C, Tuazon CU, Kovacs A, et al: Toxoplasma encephalitis in patients with acquired immunodeficiency syndrome: Diagnosis and response to therapy. Am J Trop Med Hyg. 36:509, 1987. 224. Ganji M, Tan A, Maitar ML, et al: Gastric toxoplasmosis in a patient with acquired immunodeficiency syndrome. A case report and review of the literature. Arch Pathol Lab Med. 127:732–734, 2003. 225. Hofman V, Brousset P, Mougneau E, et al: Immunostaining of visceral leishmaniasis caused by Leishmania infantum using
72.e6
Immunohistology of Infectious Diseases
monoclonal antibody (19-11) to the Leishmania homologue of receptors for activated C-kinase. Am J Clin Pathol. 120:567–574, 2003. 226. Azadeh B, Sells PG, Ejeckman GC, et al: Localized Leishmania lymphadenitis immunohistochemical studies. Am J Clin Pathol. 102:11–15, 1994. 227. Kenner JR, Aronson NE, Bratthauer GL, et al: Immunohistochemistry to identify Leishmania parasites in fixed tissues. J Cutan Pathol. 26:130–136, 1999. 228. Amato VS, Duarte MIS, Nicodemo AC, et al: An evaluation of clinical, serologic, anatomopathologic and immunohistochemical findings for fifteen patients with mucosal leishmaniasis before and after treatment. Rev Inst Med Trop S Paulo. 40:23–30, 1998. 229. Guarner J, Bartlett J, Shieh WJ, et al: Histopathologic spectrum and immunohistochemical diagnosis of amebic meningoencephalitis. Mod Pathol. 20:1230–1237, 2007. 230. Bonnin A, Petrella T, Dubremetz JF, et al: Histopathologic methods for diagnosis of cryptosporidiosis using monoclonal antibodies. Eur J Clin Microbiol Infect Dis. 9:664–665, 1990. 231. Perez de Suarez E, Perez-Schael I, Perozo-Ruggeri G, et al: Immunocytochemical detection of Entamoeba histolytica. Trans R Soc Trop Med Hyg. 81:624–626, 1987. 232. Guarner J, Bartlett J, Zaki SR, et al: Mouse model for Chagas disease: Immunohistochemical distribution of different stages of Trypanosoma cruzi in tissues throughout infection. Am J Trop Med Hyg. 65:152–158, 2001. 233. Anez N, Carrasco H, Parada H, et al: Myocardial parasite persistence in chronic chagasic patients. Am J Trop Med Hyg. 60:726– 732, 1999. 234. Reis MM, Higuchi Mde L, Benvenuti LA, et al: An in situ quantitative immunohistochemical study of cytokines and IL-2R+ in chronic human chagasic myocarditis: correlation with the presence of myocardial Trypanosoma cruzi antigens. Clin Immunol Immunopathol. 83:165–172, 1997. 235. Torres-Velez FJ, Nace EK, Won KY, et al: Development of an immunohistochemical assay for the detection of babesiosis in formalin-fixed, paraffin-embedded tissue samples. Am J Clin Pathol. 120:833–838, 2003. 236. Sanad MM, Darwish RA, Nasr ME, et al: Giardia lamblia and chronic gastritis. J Egypt Soc Parasitol. 26:481–495, 1996. 237. Genrich GL, Guarner J, Paddock CD, et al: Fatal malaria infection in travelers: Novel immunohistochemical assays for the detection of Plasmodium falciparum in tissues and implications for pathogenesis. Am J Trop Med Hyg. 76:251–259, 2007. 238. Institute of Medicine: Emerging infections: microbial threats to health in the United States, Washington, DC, 1992, National Academy Press. 239. CDC: Preventing Emerging Infectious Diseases: A Strategy for the 21st Century. Overview of the Updated CDC Plan. MMWR Recomm Rep. 47:1–14, 1998. 240. Perkins BA, Flood JM, Danila R, et al: Unexplained deaths due to possibly infectious causes in the United States: Defining the problem and designing surveillance and laboratory approaches. Emerg Infect Dis. 2:47–53, 1998. 241. Houpikina P, Raoult D: Traditional and molecular techniques for the study of emerging bacterial diseases: One laboratory’s perspective. Emerg Infect Dis. 8:122–131, 2002. 242. Khan AS, Khabbaz RF, Armstrong LR, et al: Hantavirus pulmonary syndrome: The first 100 US cases. J Infect Dis. 173:1297– 1303, 1996. 243. Moolenaar RL, Dalton C, Lipman HB, et al: Clinical features that differentiate hantavirus pulmonary syndrome from three other acute respiratory illnesses. Clin Infect Dis. 21:643–649, 1995. 244. Nolte KB, Feddersen RM, Foucar K, et al: Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent. Hum Pathol. 26:110–120, 1995. 245. Zaki SR, Greer PW, Coffield LM, et al: Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am J Pathol. 146:552–579, 1995. 246. Zaki SR, Khan AS, Goodman RA, et al: Retrospective diagnosis of hantavirus pulmonary syndrome, 1978-1993. Implications for emerging infectious diseases. Arch Pathol Lab Med. 120:134– 139, 1996.
247. Shieh WJ, Guarner J, Layton M, et al: The role of pathology in an investigation of an outbreak of West Nile encephalitis in New York, 1999. Emerg Infect Dis. 6:370–372, 2000. 248. Cushing MM, Brat DJ, Mosunjac MI, et al: Fatal West Nile virus encephalitis in a renal transplant recipient. Am J Clin Pathol. 121:26–31, 2004. 249. Petersen RL, Roehrig JT, Hughes JM: West Nile virus encephalitis. N Engl J Med. 347:1225–1226, 2002. 250. Sampson BA, Ambrosi C, Charlot A, et al: The pathology of human West Nile virus infection. Hum Pathol. 31:527–532, 2000. 251. Guarner J, Shieh WJ, Hunter S, et al: Clinicopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis. Hum Pathol. 35:983–990, 2004. 252. Lanciotti RS, Kerst AJ, Nasci RS, et al: Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase PCR assay. J Clin Microbiol. 38:4066–4071, 2000. 253. Ho M, Chen ER, Hsu KH, et al: An epidemic of enterovirus 71 infection in Taiwan. Taiwan enterovirus epidemic working group. N Engl J Med. 341:929–935, 1999. 254. Chan LG, Parashar UD, Lye MS: Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: Clinical and pathological characteristics of the disease. Clin Infect Dis. 31:678–683, 2000. 255. Wong KT, Chua KB, Lam SK: Immunohistochemical detection of infected neurons as a rapid diagnosis of enterovirus 71 encephalomyelitis. Ann Neurol. 45:271–272, 1999. 256. Yan JJ, Wang JR, Liu CC, et al: An outbreak of enterovirus 71 infection in Taiwan 1998: A comprehensive pathological, virological, and molecular study on a case of fulminant encephalitis. J Clin Virol. 17:13–22, 2000. 257. Shieh WJ, Jung SM, Hsueh C, et al: Pathologic studies of fatal causes in outbreak of hand, foot, and mouth disease. Taiwan. Emerg Infect Dis. 7:146–148, 2001. 258. Wong KT, Shieh WJ, Kumar S, et al: Nipah virus infection. Pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol. 161:2153–2167, 2002. 259. Goh KJ, Tan CT, Chew NK, et al: Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. N Engl J Med. 342:1229–1235, 2000. 260. Chua KB, Bellini WJ, Rota PA, et al: Nipah virus: A recently emergent deadly paramyxovirus. Science. 288:1432–1435, 2000. 261. Dawson JE, Paddock CD, Warner CK, et al: Tissue diagnosis of Ehrlichia chaffeensis in patients with fatal ehrlichiosis by use of immunohistochemistry, in situ hybridization, and polymerase chain reaction. Am J Trop Med Hyg. 65:603–609, 2001. 262. Walker DH, Dumler JS: Human monocytic and granulocytic ehrlichiosis. Discovery and diagnosis of emerging tick-borne infections and the critical role of the pathologist. Arch Pathol Lab Med. 121:785–791, 1997. 263. Paddock CD, Suchard DP, Grumbach KL, et al: Fatal seronegative ehrlichiosis in a patient with HIV infection. N Engl J Med. 329:1164–1167, 1993. 264. Lepidi H, Bunnell JE, Martin ME, et al: Comparative pathology and immunohistology associated with clinical illness after Ehrlichia phagocytophila-group infections. Am J Trop Med Hyg. 62:29–37, 2000. 265. Childs JE, Sumner JW, Nicholson WL, et al: Outcome of diagnostic tests using samples from patients with culture-proven human monocytic ehrlichiosis: Implications for surveillance. J Clin Microbiol. 37:2997–3000, 1999. 266. Sehdev AE, Dumler JS: Hepatic pathology in human monocytic ehrlichiosis. Ehrlichia chaffeensis infection. Am J Clin Pathol. 119:859–865, 2003. 267. Hooper PT, Russell GM, Selleck PW, et al: Immunohistochemistry in the identification of a number of new diseases in Australia. Vet Microbiol. 68:89–93, 1999. 268. Williamson MM, Hooper PT, Selleck PW, et al: Experimental Hendra virus infection in pregnant guinea-pigs and fruit bats (Pteropus poliocephalus). J Comp Pathol. 122: 201–207, 2000. 269. Paddock CD, Summer JW, Comer JA, et al: Rickettsia parkeri: A newly recognized cause of spotted fever rickettsiosis in the United States. Clin Infect Dis. 38:805–811, 2004.
References 270. Lepidi H, Fournier PE, Raoult D: Histologic features and immunodetection of African tick-bite fever eschar. Emerg Infect Dis. 12:1332–1337, 2006. 271. Ksiazek TG, Erdman D, Goldsmith CS, et al: A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 348:1953–1966, 2003. 272. Peiris JS, Lai ST, Poon LL, et al: Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet. 361:1319–1325, 2003. 273. Nakajima N, Asahi-Ozaki Y, Nagata N, et al: SARS coronavirusinfected cells in lung detected by new in situ hybridization technique. Jpn J Infect Dis. 56:139–141, 2003. 274. Kuiken T, Fouchier RA, Schutten M, et al: Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet. 362:263–270, 2003. 275. Chong PY, Chui P, Ling AE, et al: Analysis of deaths during the severe acute respiratory syndrome (SARS) epidemic in Singapore: Challenges in determining a SARS diagnosis. Arch Pathol Lab Med. 128:195–204, 2004. 276. McAuliffe J, Vogel L, Roberts A, et al: Replication of SARS coronavirus administered into the respiratory tract of African green, rhesus and cynomolgus monkeys. Virology. 5:8– 15, 2004. 277. Roberts A, Vogel L, Guarner J, et al: SARS coronavirus infection of golden Syrian hamsters. J Virol. 79:503–511, 2005. 278. Chen PC, Hsiao CH, Re: To KF, et al: Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: An in-situ hybridization study of fatal cases. J Pathol. 203:729–731, 2004. 279. Re: To KF, Tong JH, Chan PK, et al: Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: An in-situ hybridization study of fatal cases. J Pathol. 202:157–163, 2004. 280. Chow KC, Hsiao CH, Lin TY, et al: Detection of severe acute respiratory syndrome-associated coronavirus in pneumocytes of the lung. Am J Clin Pathol. 121:574–580, 2004. 281. Ding Y, He L, Zhang Q, et al: Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-Co-V) in SARS patients: Implications for pathogenesis and virus transmission pathways. J Pathol. 203:622–630, 2004. 282. Sheih WJ, Cheng-Hsiang H, Paddock CD, et al: Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan. Hum Pathol. 36:303–309, 2005. 283. Ashford DA, Kaiser RB, Bales ME, et al: Planning against biological terrorism: Lessons from outbreak investigations. Emerg Infect Dis. 9:515–519, 2003. 284. Inglesby TV, O’Toole T, Henderson DA: Preventing the use of biological weapons: Improving response should prevention fail. Clin Infect Dis. 30:926–929, 2000. 285. Lillibridge SR, Bell AJ, Roman RS: Thoughts for the new millennium: Bioterrorism. Centers for Disease Control and Prevention bioterrorism preparedness and response. Am J Infect Control. 27:463–464, 1999. 286. Franz DR, Zajtchuk R: Biological terrorism: Understanding the threat, preparation, and medical response. Dis Month. 46:125– 190, 2000. 287. Guarner J, Zaki SR: Histopathology and immunohistochemistry in the diagnosis of bioterrorism agents. J Histochem Cytochem. 54:3–11, 2006. 288. Ezzell JW, Abshire TG, Little SF, et al: Identification of Bacillus anthracis by using monoclonal antibodies to cell wall galactoseN-acetylglucosamine polysaccharide. J Clin Microbiol. 28:223– 231, 1990. 289. Shieh WJ, Guarner J, Paddock C, et al: The critical role of pathology in the investigation of bioterrorism-related cutaneous anthrax. Am J Pathol. 163:1901–1910, 2003. 290. Guarner J, Jernigan JA, Shieh WJ, et al: Pathology and pathogenesis of bioterrorism-related inhalational anthrax. Am J Pathol. 163:701–709, 2003. 291. Jernigan JA, Stephens DS, Ashford DA, et al: Bioterrorismrelated inhalational anthrax: The first 10 cases reported in the United States. Emerg Infect Dis. 7:933–944, 2001.
72.e7
292. Jernigan DB, Raghunathan PL, Bell BP, et al: Investigation of bioterrorism-related anthrax, United States, 2001: Epidemiologic findings. Emerg Infect Dis. 8:1019–1028, 2002. 293. Tatti KM, Greer P, White E, et al: Morphologic, immunologic, and molecular methods to detect Bacillus anthracis in formalinfixed tissues. Appl Immunohistochem Mol Morphol. 14:234–243, 2006. 294. Grinberg LM, Abramova FA, Yampolskaya OV, et al: Quantitative pathology of inhalational anthrax I: Quantitative microscopic findings. Mod Pathol. 14:482–495, 2001. 295. Guarner J, Greer PW, Bartlett J, et al: Immunohistochemical detection of Francisella tularensis in formalin-fixed paraffinembedded tissue. Appl Immun Mol Morphol. 7:122–126, 1999. 296. DeBey BM, Andrews GA, Chard-Bergstrom C, et al: Immunohistochemical demonstration of Francisella tularensis in lesions of cats with tularaemia. J Vet Diagn Invest. 14:162–164, 2002. 297. Guarner J, Shieh WJ, Greer PW, et al: Immunohistochemical detection of Yersinia pestis in formalin-fixed paraffin-embedded tissue. Am J Clin Pathol. 117:205–209, 2002. 298. Davis KJ, Vogel P, Fritz DL, et al: Bacterial filamentation of Yersinia pestis by β-lactam antibiotics in experimentally infected mice. Arch Pathol Lab Med. 121:865–868, 1997. 299. Davis KJ, Fritz DL, Pitt ML, et al: Pathology of experimental pneumonic plague produced by fraction 1-positive and fraction 1-negative Yersinia pestis in African green monkeys (Cercopithecus aethiops). Arch Pathol Lab Med. 120:156–163, 1996. 300. Williams ES, Mills K, Kwiatkowski DR, et al: Plague in black-footed ferret (Mustela nigripes). J Wild Dis. 30:581–585, 1994. 301. Gabastou JM, Proaño J, Vimos A, et al: An outbreak of plague including cases with probable pneumonic infection, Ecuador, 1998. Trans R Soc Tro Med Hyg. 94:387–391, 2000. 302. Guarner J, Shieh WJ, Chu M, et al: Persistent Yersinia pestis antigens in ischemic tissues of a patient with septicemic plague. Hum Pathol. 36:850–853, 2005. 303. Figueroa ME, Rasheed S: Molecular pathology and diagnosis of infectious diseases. Am J Clin Pathol. 95:S8–S21, 1991. 304. Fredricks DN, Relman DA: Application of polymerase chain reaction to the diagnosis of infectious diseases. Clin Infect Dis. 29:475–486; quiz 487–488, 1999. 305. McNicol AM, Farquharson MA: In situ hybridization and its diagnostic applications in pathology. J Pathol. 182: 250–261, 1997. 306. Mothershed EA, Whitney AM: Nucleic acid-based methods for the detection of bacterial pathogens: Present and future considerations for the clinical laboratory. Clin Chim Acta. 363:206– 220, 2006. 307. Procop GW, Wilson M: Infectious disease pathology. Clin Infect Dis. 32:1589–1601, 2001. 308. Sklar J: DNA hybridization in diagnostic pathology. Hum Pathol. 16:654–658, 1985. 309. Tang YW, Procop GW, Persing DH: Molecular diagnostics of infectious diseases. Clin Chem. 43:2021–2038, 1997. 310. Andrade ZR, Garippo AL, Saldiva PH, et al: Immunohistochemical and in situ detection of cytomegalovirus in lung autopsies of children immunocompromised by secondary interstitial pneumonia. Pathol Res Pract. 200:25–32, 2004. 311. Burt FJ, Swanepoel R, Shieh WJ, et al: Immunohistochemical and in situ localization of Crimean-Congo hemorrhagic fever (CCHF) virus in human tissues and implications for CCHF pathogenesis. Arch Pathol Lab Med. 121:839–846, 1997. 312. Fredricks DN, Relman DA: Localization of Tropheryma whippelii rRNA in tissues from patients with Whipple’s disease. J Infect Dis. 183:1229–1237, 2001. 313. Gentilomi G, Musiani M, Zerbini M, et al: Double in situ hybridization for detection of herpes simplex virus and cytomegalovirus DNA using non-radioactive probes. J Histochem Cytochem. 40:421–425, 1992. 314. Gentilomi G, Zerbini M, Musiani M, et al: In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labelled probe. Mol Cell Probes. 7:19–24, 1993. 315. Hayden RT, Qian X, Procop GW, et al: In situ hybridization for the identification of filamentous fungi in tissue section. Diagn Mol Pathol. 11:119–126, 2002.
72.e8
Immunohistology of Infectious Diseases
316. Hayden RT, Qian X, Roberts GD, et al: In situ hybridization for the identification of yeastlike organisms in tissue section. Diagn Mol Pathol. 10:15–23, 2001. 317. Hulette CM, Downey BT, Burger PC: Progressive multifocal leukoencephalopathy. Diagnosis by in situ hybridization with a biotinylated JC virus DNA probe using an automated Histomatic Code-On slide stainer. Am J Surg Pathol. 15: 791–797, 1991. 318. Krimmer V, Merkert H, von Eiff C, et al: Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization. J Clin Microbiol. 37:2667–2673, 1999. 319. Matsuse T, Matsui H, Shu CY, et al: Adenovirus pulmonary infections identified by PCR and in situ hybridisation in bone marrow transplant recipients. J Clin Pathol. 47:973–977, 1994. 320. Montone KT, Litzky LA: Rapid method for detection of Aspergillus 5S ribosomal RNA using a genus-specific oligonucleotide probe. Am J Clin Pathol. 103:48–51, 1995. 321. Morey AL, Keeling JW, Porter HJ, et al: Clinical and histopathological features of parvovirus B19 infection in the human fetus. Br J Obstet Gynaecol. 99:566–574, 1992. 322. Morey AL, Porter HJ, Keeling JW, et al: Non-isotopic in situ hybridisation and immunophenotyping of infected cells in the investigation of human fetal parvovirus infection. J Clin Pathol. 45:673–678, 1992. 323. Moter A, Göbel UB: Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J Microbiol Methods. 41:85–112, 2000. 324. Musiani M, Zerbini M, Venturoli S, et al: Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation. J Clin Pathol. 47:886–891, 1994. 325. Naoumov NV, Daniels HM, Davison F, et al: Identification of hepatitis B virus-DNA in the liver by in situ hybridization using a biotinylated probe. Relation to HBcAg expression and histology. J Hepatol. 19:204–210, 1993. 326. Porter HJ, Padfield CJ, Peres LC, et al: Adenovirus and intranuclear inclusions in appendices in intussusception. J Clin Pathol. 46:154–158, 1993. 327. Schmidbauer M, Budka H, Ambros P: Herpes simplex virus (HSV) DNA in microglial nodular brainstem encephalitis. J Neuropathol Exp Neurol. 48:645–652, 1989. 328. Schmidbauer M, Budka H, Pilz P, et al: Presence, distribution and spread of productive varicella zoster virus infection in nervous tissues. Brain. 115(Pt 2):383–398, 1992. 329. Thompson CH, Biggs IM, de Zwart-Steffe RT: Detection of molluscum contagiosum virus DNA by in situ hybridization. Pathology. 22:181–186, 1990. 330. Unger ER: In situ diagnosis of human papillomaviruses. Clin Lab Med. 20:289–301, 2000. 331. Wu TC, Mann RB, Epstein JI, et al: Abundant expression of EBER1 small nuclear RNA in nasopharyngeal carcinoma. A morphologically distinctive target for detection of Epstein-Barr
virus in formalin-fixed paraffin-embedded carcinoma specimens. Am J Pathol. 138:1461–1469, 1991. 332. Zaki SR, Judd R, Coffield LM, et al: Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction. Am J Pathol. 140:1345–1355, 1992. 333. Akhtar N, Ni J, Langston C, et al: PCR diagnosis of viral pneumonitis from fixed-lung tissue in children. Biochem Mol Med. 58:66–76, 1996. 334. Amaker BH, Chandler FW, Jr, Huey LO, et al: Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J Forensic Sci. 42:1157–1159, 1997. 335. Beqaj SH, Flesher R, Walker GR, et al: Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens. Diagn Mol Pathol. 16:169–173, 2007. 336. Bhatnagar J, Guarner J, Paddock CD, et al: Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: A useful adjunct to conventional tissue-based diagnostic methods. J Clin Virol. 38:106–111, 2007. 337. Clavel C, Binninger I, Polette M, et al: Polymerase chain reaction (PCR) and pathology. Technical principles and application. Ann Pathol. 13:88–96, 1993. 338. Guarner J, Bhatnagar J, Shieh WJ, et al: Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis. Hum Pathol. 38:1412–1419, 2007. 339. Lamps LW, Madhusudhan KT, Greenson JK, et al: The role of Yersinia enterocolitica and Yersinia pseudotuberculosis in granulomatous appendicitis: A histologic and molecular study. Am J Surg Pathol. 25:508–515, 2001. 340. Paddock CD, Sanden GN, Cherry JD, et al: Pathology and pathogenesis of fatal Bordetella pertussis infection in infants. Clin Infect Dis. 47:328–338, 2008. 341. Qian X, Jin L, Hayden RT, et al: Diagnosis of cat scratch disease with Bartonella henselae infection in formalin-fixed paraffinembedded tissues by two different PCR assays. Diagn Mol Pathol. 14:146–151, 2005. 342. Schild M, Gianinazzi C, Gottstein B, et al: PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffinembedded brain sections. J Clin Microbiol. 45:564–567, 2007. 343. Singh HB, Katoch VM, Natrajan M, et al: Improved protocol for PCR detection of Mycobacterium leprae in buffered formalinfixed skin biopsies. Int J Lepr Other Mycobact Dis. 72:175–178, 2004. 344. Tatti KM, Wu KH, Sanden GN, et al: Molecular diagnosis of Bordetella pertussis infection by evaluation of formalin-fixed tissue specimens. J Clin Microbiol. 44:1074–1076, 2006. 345. Wilson DA, Reischl U, Hall GS, et al: Use of partial 16S rRNA gene sequencing for identification of Legionella pneumophila and non-pneumophila Legionella spp. J Clin Microbiol. 45:257–258, 2007.
C H A P T E R 4
IMMUNOHISTOLOGY OF NEOPLASMS OF SOFT TISSUE AND BONE LEONA A. DOYLE, JASON L. HORNICK
Overview 73 Biology of Antigens and Antibodies 73 Specific Soft Tissue and Bone Tumors 89 Summary 129
Overview Tumors of soft tissue and bone are a diverse family, and categorization continues to evolve as more insight is gained into their patterns of differentiation and the underlying molecular pathogenesis of many distinct tumor types. As such, the classification of these neoplasms is an increasingly complex subject that requires at least a basic understanding of the biochemical attributes of the lesions in question. This chapter presents a practical summary of this topic; however, we do not purport it to be an encyclopedic or all-inclusive treatise. In particular, Chapter 13 discusses tumors that are most common in the skin and subcutis, and we have excluded select lesions of soft tissues and bone that are morphologically distinct (i.e., they do not require immunohistochemistry for diagnosis). In the same vein, we emphasize that immunohistochemical (IHC) evaluation of mesenchymal neoplasms is merely an adjunct to thorough histologic evaluation, not a substitute for it. Furthermore, clinical and radiologic correlation and molecular studies all play significant roles in the evaluation of many soft tissue and bone lesions.
Biology of Antigens and Antibodies Intermediate Filament Proteins Intermediate filament proteins (IFPs) are structural components of all human cells, together with microfilaments and microtubules.1,2 They are 7 to 10 nm in diameter and are often arranged in skeins or bundles in the cytoplasm. Parallel aggregation of IFPs often is
observed in epithelial cells that are rich in highmolecular-weight keratins, yielding the structures known to electron microscopists as tonofilaments or tonofibrils.3 Otherwise, the IFPs as a family are not morphologically distinguishable from one another at the ultrastructural level. Based on biochemical and functional grounds, they are composed of at least six distinct moieties: keratins, vimentin, desmin, neurofilament proteins (NFPs), glial fibrillary acidic protein (GFAP), and the lamins (nuclear envelope proteins).4 In this chapter, we will discuss the first five of these entities because they have been well characterized in diagnostic pathology. All IFPs share structural homologies,5 but their precise natures vary considerably. Their molecular weights vary between 40 and 200 kD. IFPs also have dissimilar isoelectric pH values, and, more important, characteristic distribution patterns in nonneoplastic cells and human tumors.2 Two members of the IFP family—the keratins and NFPs—appear to be composed of heteropolymeric aggregates of two or more proteins, whereas the other members are homopolymers that contain only one protein isoform.6 The IFPs are encoded by multiple genes on various chromosomes (e.g., chromosomes 12q and 17q for the keratins, chromosome 2q for desmin, chromosome 10p for vimentin, and chromosome 17q for GFAP).7-10 In keeping with their proposed cytoskeletal nature, IFPs initially were thought to serve a purely structural role in muscle cells. It was hypothesized that the function of these proteins was to keep other cytoplasmic proteins in proper relationship to one another, as well as to anchor the cytoplasmic contractile apparatus to the cell membrane. However, subsequent developments in cell biology cast considerable doubt on this premise.11 The intermediate filaments are now known to serve a nucleic acid–binding function; moreover, they are susceptible to processing by calcium-activated proteases and are substrates for cyclic adenosine monophosphate– dependent protein kinases. Thus it has been proposed that all IFPs serve as modulators between extracellular influences that govern calcium flux into the cell, subsequent protease activation, and nuclear function at a transcriptional or translational level.12 Their morphologic associations with cell membranes and the 73
74
Immunohistology of Neoplasms of Soft Tissue and Bone
perinuclear cytoplasm are consistent with this theory and relegate a cytoskeletal role to a secondary level. Current opinion favors the view that fibrils of the IFPs are formed to restrict the availability of their nucleic acid–binding domains in accord with cell-cycle activity, not as cellular “buttresses.” Although some IFPs insert into intercellular desmoplakin-containing desmosomes, which are responsible for maintaining tissue integrity, this fact does not necessarily assign a structural biologic role to these filaments. Intercellular junctions may be points of biochemical communication within the extracellular milieu.13 The distribution of each of the IFPs will be described in some detail; desmin will be discussed later in the section on markers of muscle differentiation. KERATINS
As the essential IFPs of epithelial cells and epithelial neoplasms, keratins have a high degree of specificity and sensitivity for the diagnosis of carcinoma among malignant tumors. Cytokeratins typically are expressed by any given cell type in pairs, representing an acidic (type I) and a basic (type II) keratin.14 These vary in molecular weight from 40 to 67 kD and have been given catalog designations by Moll and colleagues15 such that they are numbered within each respective type grouping from lowest to highest molecular weight. There are 12 type I keratins with acidic isoelectric points and 8 type II proteins with basic biochemical attributes. As described by Miettinen,14 cytokeratins tend to pair themselves during cell development so that a type I keratin is associated with a type II keratin that is 7 to 9 kD larger. The particular keratin types that can be detected in given tissues or neoplasms follow predictable, known patterns of gene expression that serve, in part, to identify the cells containing them. With particular reference to nonepithelial cells, selected cytokeratins—CK8, CK18, and occasionally CK19—are demonstrable in the physiologic state, but special techniques such as acetone fixation, frozen section immunohistology, or amplified immunodetection methods are usually necessary to preserve or detect extremely low densities of these IFPs. Selected mesenchymal neoplasms may likewise exhibit keratin reactivity, which can be broader in scope. For example, CK1, CK7, CK8, CK13, CK14, CK18, and CK19 have all been observed in synovial sarcomas.14,16 Other soft tissue or bone tumors that are regularly cytokeratin-reactive include epithelioid sarcoma (CK8, CK18, and CK19); chordoma (CK8, CK18, and CK19, with or without CK4 and CK5); myoepithelioma/ myoepithelial carcinoma, including tumors previously known as parachordoma (CK8, among others); and adamantinoma (CK5 and CK19).14-19 In reference to still other mesenchymal neoplasms, experience since the 1990s has shown that under some circumstances, selected tumors that are typically devoid of keratins may synthesize those IFPs in an aberrant fashion. Indeed, at this point, virtually all sarcomas have been reported to show this potential. Nevertheless, we would like to emphasize that aberrant keratin reactivity
is most common in a narrow spectrum of malignant mesenchymal tumors, principally leiomyosarcoma (LMS); angiosarcoma, particularly epithelioid examples; and, to a lesser degree, Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) and alveolar rhabdomyosarcoma (RMS).20,21 In keeping with the comments mentioned earlier in this chapter, CK8, CK18, and CK19 are most often expected in this setting. It should be noted that aberrant IFP expression may also represent idiosyncrasies or flaws in IHC technique, wherein antikeratin antibodies are used at inappropriately high concentration or with especially sensitive detection procedures. Inasmuch as IFPs are structurally interrelated, cross-labeling of vimentin, desmin, NFPs, or GFAP can be obtained spuriously with many antikeratin reagents. A large body of clinical literature exists on the IFP profiles of human neoplasms. To benefit from the value of its contents in a diagnostic setting, the techniques used in obtaining these data must be retained. Thus in a practical sense, it is not advisable to substitute a new and improved immunodetection protocol for an old one without considering the effect the step has on the final interpretation. The diagnostic pathologist’s goal should not be to identify every molecule of a particular IFP, no matter how sparse, in any given tumor but rather to establish and maintain the windows of immunodetection to minimize the overlap between related moieties and maximize diagnostic utility. Attention to these maxims will lower the incidence of aberrant keratin expression in mesenchymal neoplasia when using routinely processed (formalin-fixed paraffin-embedded [FFPE]) clinical material. NEUROFILAMENT PROTEINS
Neurofilament proteins are composed of three basic subunits with molecular weights of 68, 150, and 200 kD22,23; hence, they are larger than all other IFPs. Each NFP appears to be a separate gene product, rather than a derivative of the other two.24 Expression of this family of IFPs is correlated with differentiation of neurogenic blast cells into committed neurons in the developing embryo or in neoplasia, and each isoform is differentially expressed in different types of neurons.25,26 Another peculiarity of NFPs that is shared only by GFAP, another intermediate filament, is that each of the three neurofilament isoforms may be either phosphorylated or nonphosphorylated in vivo.27 Correspondingly, antibodies to the NFPs may be specific for only one of those two configurations.28,29 Neurofilament proteins form a major component of the cytoskeleton of neurons and their axons. Expression is not seen in normal epithelium. Practically speaking, NFPs are generally not well detected in FFPE tissue, even with modern IHC methods and commercial antibodies. Among these, the SMI series of monoclonal antibodies30 and clone 2F11 are probably the most consistently active against routinely processed surgical pathology specimens. Among soft tissue neoplasms, NFP staining demonstrates axons in neurofibroma and ganglioneuroma; in the latter tumor, ganglion cells will also be positive for NFP. Neuroblastoma and its variants
Biology of Antigens and Antibodies
also express NFP, as do paragangliomas/pheochromocytomas and a subset of neuroendocrine tumors; the presence of a perinuclear dotlike pattern of staining is characteristically found in Merkel cell carcinoma, in which it may be a helpful diagnostic feature.31-34 NFP expression in other sarcomas is very limited. GLIAL FIBRILLARY ACIDIC PROTEIN
GFAP is another intermediate filament protein that plays a somewhat limited role in the evaluation of soft tissue tumors. This 51-kD protein is the major component of astrocytes, ependymal cells, and retinal Müller cells and typically is not expressed by mature oligodendroglia.35,36 Nonglial tissues with putative GFAP reactivity include Schwann cells, myoepithelial cells, Kupffer cells, and some chondrocytes.37 It is therefore expected that selected neoplasms that include such elements— peripheral nerve sheath, myoepithelial, and cartilaginous tumors38-43—may occasionally demonstrate immunolabeling for GFAP. As such, GFAP may be used as a second-line marker for malignant peripheral nerve sheath tumor (MPNST), although it shows only focal labeling in at most 30% of cases. Less frequently, GFAP is used to identify a component of Schwann cells in benign nerve sheath tumors.42 In addition, GFAP expression may help support the diagnosis of soft tissue myoepithelioma or myoepithelial carcinoma, being present in as many as 50% of myoepithelial tumors.19,44,45 VIMENTIN
Vimentin is a 57-kD protein that was initially isolated from a mouse fibroblast culture.2,46 Its name derives from the Latin vimentum, describing an array of flexible rods. This IFP is considered to be the primordial member of the intermediate filament family, because it is present in most, if not all, fetal cells early in development. Moreover, when two or more IFPs are coexpressed by a cell line or neoplasm, vimentin is virtually always one of them.47 Accordingly, vimentin is not considered to be cell-type specific. From the perspective of mesenchymal tumor pathology, it is of interest that vimentin shows a greater amino acid homology to desmin, NFPs, and GFAP than it does to the keratins.46,48 The ubiquity of vimentin in soft tissues limits its diagnostic use in the setting of tumor pathology. However, it does serve a useful control marker function—to ensure that the tissue has been properly preserved and processed, although other markers can also be used for this purpose.49 If vimentin cannot be easily detected in nonneoplastic endothelial cells, fibroblasts, and other mesenchymal elements routinely present in any tissue section, the reactivity or nonreactivity of accompanying neoplastic cells cannot be properly determined. Occasionally, the pattern of vimentin expression is also distinctive. For example, in malignant rhabdoid tumors, this IFP usually assumes a densely globular cytoplasmic configuration, indenting the nuclei of the neoplastic cells.50 The utility of vimentin in current clinical practice is almost obsolete because of its low specificity.
75
Epithelial Markers EPITHELIAL MEMBRANE ANTIGEN
Epithelial membrane antigen (EMA; MUC1) is a transmembrane glycoprotein encoded for by the MUC1 gene on chromosome 1. EMA is one of several human milk fat globule proteins (HMFGPs) derived from mammary epithelium. The HMFGPs vary greatly in molecular weight (51 kD to >1000 kD). They are glycoproteins51 that compose part of the plasmalemma of epithelial cells in areas of the cell membrane overlying tight junctions.52 In addition, because HMFGPs are packaged in the Golgi apparatus, globular labeling of this structure may be seen immunohistologically.52 The function of HMFGPs, including EMA, is still not absolutely certain. It is thought that they serve a role in secretion or, alternatively, that they provide a protective function for the cell.51 The distribution of HMFGPs is such that many, but not all, nonneoplastic human epithelial cells express at least one member of this protein family. EMA expression is found in a variety of normal epithelial cells (breast, eccrine and apocrine glands, pancreas, urothelium, and renal collecting tubules) and nonepithelial cells (perineurial cells, plasma cells, arachnoid cells, ependyma, and choroid plexus).53,54 Exceptions include the gastrointestinal (GI) surface epithelium, endocervical epithelium, prostatic acinar epithelium, epididymis, germ cells, hepatocytes, adrenal cortical cells, rete testis, squamous cells of the epidermis, and thyroid follicular epithelium.55 The most well-established monoclonal antibody to EMA, which is the most widely used HMFGP, is E29. It labels a glycoprotein of approximately 450 kD. Neoplastic processes that may be EMA-immunoreactive are numerous. Synovial sarcoma, epithelioid sarcoma, lowgrade fibromyxoid sarcoma, perineuriomas, angiomatoid fibrous histiocytoma, epithelioid benign fibrous histiocytoma, chordoma, myoepithelioma/myoepithelial carcinoma, and some plasmacytomas are commonly labeled.19,56-62 Note that true EMA reactivity (i.e., that which generally equates with epithelial differentiation) must be localized to the cell membrane. Purely cytoplasmic labeling without a membrane component is a spurious pattern that should generally be ignored for diagnostic purposes. Expression of EMA is also seen in various carcinomas, mesothelioma, meningioma, and a subset of B-cell and T-cell lymphomas.54,63 OTHER EPITHELIAL MARKERS
When stains for standard epithelial determinants (e.g., keratin and EMA) yield equivocal results, it may be desirable to assess additional potential indicators of epithelial differentiation, especially if morphologic features suggest aberrant reactivity. The best known among the adjunctive epithelial markers are desmoplakin, desmoglein, and E-cadherin. The first two are desmocollins or elements of desmosomal complexes, which represent specialized intercellular anchoring structures.64,65 On the other hand, cadherins are calcium-dependent
76
Immunohistology of Neoplasms of Soft Tissue and Bone
transmembranous intercellular adhesion molecules that are divided into three subclasses—E-, P-, and Ncadherin—and have distinctive immunologic specificities and tissue distributions.66,67 These molecules have subclass specificities in cell-cell binding and are involved in selective cellular adhesion. E-cadherin is typically associated with epithelial differentiation. Analysis of amino acid sequences, as deduced from the nucleotide sequences of complementary DNAs (cDNAs) that encode cadherins, has demonstrated that they share common sequences and are therefore regarded as a family of adhesion molecules with differential specifi cities. Although there are exceptions, concurrent immunoreactivity for desmoplakin or desmoglein and E-cadherin is generally restricted to epithelial neoplasms and mesenchymal tumors with epithelial characteristics, such as synovial sarcoma. One large study of epithelialtype and neural-type cadherin expression in soft tissue tumors showed expression of E-cadherin in 100% of biphasic synovial sarcomas, 50% of monophasic synovial sarcomas, and 8% of epithelioid sarcomas. N-cadherin expression was present in 100% of chordomas, 86% of biphasic synovial sarcomas, and 38% of epithelioid sarcomas.66 However, these markers are not widely used in the diagnosis of soft tissue tumors.
Markers of Muscle Differentiation DESMIN
Desmin is a cytoplasmic IFP that is characteristically found in muscle cells and in neoplasms with myogenic differentiation.68,69 In smooth muscle cells, desmin is seen with cytoplasmic dense bodies and subplasmalemmal dense plaques; in striated muscle, desmin filaments are linked to sarcomeric Z disks. In both muscle types, desmin helps bind myofilaments into bundles. In 1977, Small and Sobieszek70 were the first to recognize desmin as a distinct biochemical moiety. They found that it represented a residual filamentous protein in muscle cells that had been depleted of actin and myosin in vitro, and they assigned the provisional designation “skeletin” to it. It was observed to have an isoelectric point of approximately 4 and to be heat stable and insoluble in salt-rich solutions. Amino acid analysis revealed a high concentration of glutamate and aspartate and a significant chemical homology with glial filaments and NFPs. A notable finding in this study was that muscle cells depleted of skeletin (desmin) were still able to contract in response to adenosine triphosphate (ATP) and calcium. This point led the authors to conclude that the protein in question played no role in contractility but rather served to keep actin and myosin filaments associated and to anchor them to the plasmalemma. These observations have been confirmed and expanded by others.71,72 Desmin is now known to have a molecular weight of 53 kD and a mass per unit of 36 to 37 kD/nm. It is composed of an N-terminal headpiece and a C-terminal tailpiece, both of which are nonhelical in conformation. These bracket an α-helical middle domain of approximately 300 amino acid residues. The former are greatly variable in biochemical
constitution from species to species, but the helical segment is highly conserved, meaning that interspecies homology in this domain is striking. Like other intermediate filaments, desmin displays a 20 to 22 nm axial periodicity. Ip and Heuser73 showed that it forms heteropolymers that aggregate in a cross-linked, fibrillar, tetrameric fashion. The helical segment composition of desmin allows it to form coils with respect to the tertiary structure of the molecule. Hydrophobic amino acid residues are thereby exposed, which explains the ability of desmin to associate with nonhydrophilic nuclear and plasmalemmal membranes. Desmin appears in developing striated muscle cells at the myotube-forming stage, in which myoblasts fuse with one another.74 It replaces vimentin, at least in large measure, because the latter is the intermediate filament first expressed by virtually all embryonic mesenchymal cells. Desmin filaments are oriented in a longitudinal fashion initially, but as the muscle cell matures, they become concentrated around Z disks.75 Not all muscle cells contain desmin, however; for example, mammalian aortic smooth muscle is typically negative for desmin, unlike the smooth muscle wall of most other blood vessels.76 Immunoelectron microscopic analyses have documented the binding of suitably specific antidesmins to the intermediate filaments of muscle cells and their neoplasms.77 There should be no cross-reactivity of such reagents with associated contractile proteins, such as actin and myosin; this is particularly important, because these three proteins appear to share some epitopes. The three best-characterized monoclonal antibodies to desmin are D33, DER-11, and DEB-5. By the Western blot technique, they have been shown to recognize desmin epitopes between residues 324 and 415 and to have no cross-reactivity with other IFPs. These reagents show tissue specificity but species nonspecificity. In general, desmin is, as expected, a specific marker for myogenic differentiation among soft tissue tumors. As such, it is seen in the majority of rhabdomyoma, leiomyoma, RMS, and LMS.68,69,78-80 Heterologous rhabdomyoblastic differentiation in other tumor types—such as MPNST (malignant Triton tumor), dedifferentiated liposarcoma (DDLPS), or carcinoma— will also show desmin positivity. Although the vast majority of leiomyomas express desmin, expression is seen in a smaller percentage of LMS (~70%), and expression in LMS can be limited in extent.81 Approximately 30% of cellular, benign, fibrous histiocytomas express desmin, presumably reflecting the myofibroblastic nature of fibrous histiocytoma.82 Other tumors that may have myofibroblastic features, and therefore may contain a subset of tumor cells that express desmin, include inflammatory myofibroblastic tumor, desmoid fibromatosis, angiomyofibroblastoma, and deep (“aggressive”) angiomyxoma.83-85 Reactivity for desmin may also be observed in neoplasms with divergent or uncertain phenotypes, including variable expression in perivascular epithelioid cell (PEC) tumors, so-called PEComas86,87; as much as 40% of ossifying fibromyxoid tumors88; 50% of angiomatoid fibrous histiocytomas89,90; and approximately 80% of desmoplastic small round cell tumors (DSRCTs), often with a dotlike pattern.91
Biology of Antigens and Antibodies
Interdigitating reticulum cells of lymph nodes and a subset of reactive mesothelial cells are also positive for desmin.92,93 Other tumor types that occasionally express desmin include diffuse malignant mesothelioma and Wilms tumor.94,95 ACTINS
77
(molecular weight 460 kD), actin, tropomyosin (molecular weight 70 kD), and troponin. The troponin molecule has three subunits—troponin I, troponin T, and troponin C—with molecular weights between 18 kD and 35 kD.105 Myosin is an actin-binding protein that has two globular heads and an elongated tail. In particular, myosin II is composed of two heavy chains and four light chains (two phosphorylatable and two basic). These combine with N-terminal portions of the myosin heavy chains to form globular heads, each of which has an actin-binding site and an enzymatic locus that hydrolyzes ATP. The heads of the myosin molecules form cross-bridges to actin.106 Myosin molecules are configured in a symmetric fashion on either side of the center of the sarcomere. Sarcomeric thin filaments are polymers composed of two actin chains arranged in a double helix. Tropomyosin molecules, in turn, are situated in the groove between the two chains of actin, and troponins are interspersed along the tropomyosin.107 Troponin T melds other troponin components with tropomyosin; troponin I inhibits the interaction of myosin and actin, and troponin C contains binding sites for calcium in the initiation of muscle contraction. Actinin, a 190-kD moiety, binds actin to the Z lines of the sarcomere. Another protein, titin, connects Z lines to M lines and provides the base on which thick filaments may form.108 Because of their relatively poor sensitivity, the markers discussed in this section are used only in rare circumstances for diagnostic purposes.
Aside from desmin, the next most useful group of cytoplasmic determinants for defining myogenic differentiation is the protein family of the actins.69,96 The six major isoforms of these microfilamentous contractile polypeptides are designated skeletal muscle-α, smooth muscle-α, smooth muscle-γ, cardiac muscle-α, nonmyogenous-β, and nonmyogenous-γ.81,96 Alpha and gamma muscle isoforms are seen in tissues with pure myogenic differentiation, but they are also demonstrable in cells with myofibroblastic or myoepithelial features.97-100 The molecular weights of all these biochemical moieties cluster around 45 kD, and they may be labeled with antibodies that recognize conserved amino acid sequences or, alternatively, with isoform-selective reagents.81,96,97 From a diagnostic perspective, only the latter method is desirable. However, because of inevitable problems that arise in the immunohistologic detection of heteropolymeric proteins, even some of these antiactins are not truly specific for pure myogenic differentiation. This is particularly true of one commonly used commercial reagent, clone 1A4, which is widely known as anti–(α) smooth muscle actin (SMA).98 In practice, it is expressed in cell types other than smooth muscle, including myofibroblasts, myoepithelia, and others. In fact, nearly any neoplasm that shows spindle cell morphology may express SMA, including spindle cell (sarcomatoid) carcinomas. However, SMA is not detected in normal skeletal muscle; rarely, limited reactivity for SMA is seen in RMS.101 Myoepithelial tumors frequently express SMA, as do tumors of the PEComa family, which are characterized by dual myoid and melanocytic differentiation.19,45,86,102 Another antibody, designated HHF-35, or anti–musclespecific actin, shows more muscle-restricted immunoreactivity in routinely processed specimens.69,97 HHF-35 is expressed in both smooth muscle and skeletal muscle, because it is targeted against the isoforms skeletal muscle-α, smooth muscle-α, and smooth muscle-γ. RMS, LMS, and benign smooth muscle and skeletal muscle tumors all express HHF-35.97 Myofibroblastic cells and tumors with myofibroblastic features, such as nodular fasciitis, show variable reactivity for HHF-35.103 Smooth muscle myosin shows a similar staining profile to that of SMA, but the sensitivity appears to be lower, thus limiting its diagnostic utility.104 Sarcomeric actin is expressed in skeletal muscle tumors and to a lesser extent in smooth muscle tumors, but its use in clinical practice is limited because of low sensitivity and specificity.101
Caldesmon is a cytoplasmic protein with two isoform classes, one of which is found predominantly in smooth muscle cells and other cell types with partial myogenic differentiation. High-molecular-weight isoforms, those with molecular weights between 89 and 93 kD, are capable of binding to actin, tropomyosin, calmodulin, myosin, and phospholipids, and they function to counteract actin-tropomyosin–activated myosin adenosine triphosphatase (ATPase). As such, they are mediators for the inhibition of calcium-dependent smooth muscle contraction.109 Commercial antibodies to (“heavy”) h-caldesmon are of clinical utility in diagnostic surgical pathology. They appear to be relatively specific for smooth muscle differentiation and, as such, are useful adjuncts to desmin and actin immunostains in the evaluation of leiomyoma and LMS.109,110 The vast majority of leiomyomas express h-caldesmon, but expression in LMS is more variable, with one study showing reactivity for h-caldesmon in only 36% of LMS.111 Caldesmon expression is not entirely specific to smooth muscle tumors, however, because reactivity is also observed in the majority of gastrointestinal stromal tumors (GISTs), glomus tumors, and myopericytomas.112,113 In contrast, myofibroblastic cells do not express caldesmon.114,115
OTHER SARCOMERIC CONTRACTILE PROTEINS
CALPONIN
The contractile mechanism in skeletal muscle is transduced by a complex of proteins that includes myosin II
Calponin is an actin- and tropomyosin-binding cytoskeleton-associated protein involved in the
CALDESMON
78
Immunohistology of Neoplasms of Soft Tissue and Bone
regulation of smooth muscle contraction. It is expressed in smooth muscle and myoepithelial cells and myofibroblasts. Reactivity for calponin is observed in leiomyoma, LMS, nodular fasciitis and other myofibroblastic tumors, and myoepithelial neoplasms, but GISTs tend to be negative for calponin.19,112
Markers of Skeletal Muscle Differentiation MYOGLOBIN
Myoglobin is a 17.8-kD protein found exclusively in skeletal muscle, where it forms complexes with iron molecules to transport oxygen.116 The concentration of this molecule is highest in muscles that undergo sustained contraction. Because myoglobin appears relatively late in the maturational sequence of striated muscle, it is typically undetectable immunohistologically in embryonic neoplasms that show differentiation toward that tissue. Accordingly, pleomorphic (adulttype) RMS and rhabdomyoma are the soft tissue tumors in which myoglobin is identified most often.117-120 Staining for myoglobin is cytoplasmic. Most available antibodies are polyclonal, and background staining often limits the clinical utility of this marker, as does its limited expression in RMS subtypes. MYOD1 AND MYOGENIN
A superfamily of transcription factors that regulates cell lineage–specific proliferation is represented in striated muscle by several moieties collectively known as the MyoD family.121,122 They are encoded by genes that reside on chromosomes 1, 11, and 12 and are part of a polypeptide complex called the basic helix-loop-helix (BHLH) motif, small proteins composed of 220 to 320 amino acids. Two members of this intranuclear protein group, MYOD1 (46 kD; MYF3) and myogenin (32 kD; MYF4), have been used since the 1990s as specific markers of striated muscle differentiation in human neoplasms.121-123 They activate their own transcription and that of other BHLH proteins and, in concert with the retinoblastoma gene, govern exodus from the cell cycle and initiation of striated muscle differentiation. MYOD1 and myogenin are expressed by fetal skeletal muscle cells and regenerating skeletal muscle cells, but normal adult skeletal muscle is negative for both markers. Because antibodies to MYOD1 and myogenin must gain access to the nucleus, they have been difficult to use in routine surgical specimens. However, modification of antigen retrieval solutions and utilization of heat-mediated epitope enhancement have allowed these reagents to enter diagnostic use. MYOD1 immunostaining is best performed on freshly cut tissue sections, because antigen reactivity decreases with exposure to room temperature. It is important to note that MYOD1 and myogenin are strictly localized to cellular nuclei; therefore background cytoplasmic labeling, a consistent problem with antibodies to MYOD1 especially, must be ignored as a spurious pattern of staining.
In contrast, stains for myogenin do not generally show this problem. In contrast to myoglobin, expression of MYOD1 and myogenin is greatest in less differentiated RMS, such as alveolar (greatest amount) and embryonal subtypes, and typically less in spindle cell and pleomorphic subtypes or in tumors that show cytodifferentiation after chemotherapy. The specificity of these markers for skeletal muscle differentiation is high. In other tumor types, expression is limited to heterologous rhabdomyoblastic differentiation in other tumors (e.g., MPNST and DDLPS). Care should be taken when interpreting MYOD1 and myogenin immunostains, because positivity limited to reactive skeletal muscle cells may lead to an erroneous diagnosis of RMS. S-100 PROTEIN
S-100 protein derives its name from the fact that it is soluble in saturated (100%) ammonium sulfate solution. It was first isolated from the central nervous system (CNS) but is now known to have a wide distribution in human tissues, including glia, neurons, chondrocytes, Schwann cells, melanocytes, fixed phagocytic or antigenpresenting cells, Langerhans cells, myoepithelial cells, notochord, and various epithelia but especially in the breast, salivary glands, sweat glands, and the female genital system. S-100 protein is dimeric in nature, with α and β subunits. Hence, it has three isoforms: S-100ao (α-dimer), S-100a (α-β isoform), and S-100b (β-dimer). Each of the two subunits of this protein has a molecular weight approximating 10.5 kD, and the function of S-100 protein is essentially that of a calcium flux regulator.124 Both monoclonal antibodies and heteroantisera to S-100 protein are available for diagnostic use. Some of the former reagents are monospecific for the α or β subunits, therefore they exhibit relatively narrower spectra of reactivity that than seen with polyclonal antisera. For example, β-subunit–specific antibodies preferentially label glial cells and Schwann cells.125 However, β-subunit– specific antibodies have not enjoyed widespread use among diagnostic pathologists, and hetero-antisera to S-100 protein are most commonly used in clinical practice. In the proper context, as part of panels of antibodies designed to evaluate several possible lineages in a morphologically indeterminate neoplasm, reagents against S-100 protein are still valuable indicators of schwannian or melanocytic differentiation in tumors of the soft tissues and bone.126 Expression of S-100 protein is usually both nuclear and cytoplasmic. S-100 protein is detected in more than 90% of clear cell sarcomas of tendons and aponeuroses, although expression may sometimes be limited in extent.127 Schwannomas are diffusely and strongly positive for S-100 protein, whereas neurofibromas show more variable reactivity (i.e., usually in 50% to 80% of constituent cells). S-100 protein expression in MPNST is usually limited, and only 30% to 70% of cases show demonstrable staining.128,129 S-100 protein expression in normal adipocytes is variable; although S-100 protein may sometimes highlight lipoblasts in liposarcomas, its utility in this regard is limited. Other soft tissue tumors that express S-100
Biology of Antigens and Antibodies
protein include ossifying fibromyxoid tumor (73% to 94%),88,130,131 extraskeletal myxoid chondrosarcomas (at most 20%),132 synovial sarcoma (30%),133 and 5% to 10% of GISTs (most often observed in duodenal tumors).134,135 S-100 protein expression is found in 90% of soft tissue myoepitheliomas19 and in around 75% of myoepithelial carcinomas.44 Among bone tumors, S-100 protein is consistently detected in well-differentiated cartilaginous neoplasms and in as much as 80% of chordomas.136,137 Reactivity for S-100 protein is also seen in as much as 40% of breast carcinomas and less frequently in renal cell carcinomas and carcinomas of müllerian origin. S-100 protein is also useful in supporting the diagnosis of some histiocytic and dendritic cell lesions. In Rosai-Dorfman disease, S-100 protein highlights the lesional cells, with particularly strong staining in the cytoplasm, and it can make emperipolesis more easily appreciable.138 Langerhans cell histiocytosis is also consistently positive for S-100 protein.139 Similarly, the tumor cells of histiocytic sarcoma often express S-100 protein (in ~50% of cases),140 as do the extremely rare interdigitating dendritic cell sarcomas.141
Potential Markers of Nerve Sheath Differentiation CD56 (NEURAL CELL ADHESION MOLECULE) AND CD57 (NKH1/LEU19 AND LEU7/HNK-1)
CD56 and CD57 are membrane antigens expressed in peripheral blood mononuclear leukocytes, a proportion of which have natural killer (NK) activity. Their respective molecular weights are 140 kD and 95 kD. Antibodies in these cluster designations also react with several neural molecules that have a variety of molecular weights.142,143 Some of these moieties are associated with 5′-nucleotidase activity, whereas others are the neural cell adhesion molecule and myelin-associated glycoproteins (MAGs).144 The largest of the MAGs, MAG-72, is related structurally to the immunoglobulin superfamily gene products and to neural adhesion molecules and the autophosphorylation site of epidermal growth factor receptor (EGFR). MAGs are integral cell membrane proteins normally found in oligodendroglia. It is believed that their function involves the mediation of interaxonal or axonal-glial interaction during myelination. As such, their additional association with Schwann cells and neural neoplasms should not be surprising. Nevertheless, CD57 reactivity has also been documented in perineurial (non-schwannian) peripheral nerve sheath lesions.145 In general, CD56 and CD57 have been used as potential markers not only of peripheral nerve sheath tumors (PNSTs) but also of Ewing sarcoma, in which matrical proteins of neurosecretory granules and synaptic vesicles are thought to contain a target protein for the antibodies in question.146,147 However, CD56 and CD57 are not restricted to nerve sheath cells or neuroectodermal elements among soft tissue tumors but rather are most often observed in those cell types. Synovial sarcoma, LMS, and some metastatic carcinomas may also express both of these
79
markers.60,148,149 Because of their lack of specificity, the authors of this chapter do not use these markers in clinical practice for diagnosing soft tissue tumors. COLLAGEN TYPE IV AND LAMININ
Volumetrically, collagen type IV is the predominant component of basement membranes, regardless of which cell types they invest. This triple-helical molecule weighs 550 kD and has globular end regions and two noncollagenous domains. One of the latter is located 330 nm from the carboxy end of the molecule, where a bending point gives the moiety a hockey stick configuration overall.150 Type IV collagen differs from other collagen types in that it does not form fibrils, it shows interruptions of its helical structure, and it has a different amino acid constituency. Genes coding for the helical chains of this molecule are located on chromosome 13q.151 Laminin is another important component of basement membranes. It is a 1000-kD molecule that binds to glycosaminoglycans, acting as a bridge for attachment of collagen type IV in basement membranes to the surrounding matrix.152 The exact location of laminin in basement membranes has been contentious. Some investigators claim that it is part of the lamina densa, others suggest that it resides in the lamina lucida, and yet others believe that it is codistributed between these two compartments.153 Beyond simple boundary and anchoring functions, laminin probably also influences intercellular interactions and contributes to alterations in cellular morphology.154 In soft tissues, complete basement membranes are formed around endothelial, smooth muscle, and Schwann cells.155 Thus reagents directed against collagen type IV and laminin were traditionally included in antibody panels aimed at detecting those lineages, in particular, to help distinguish MPNSTs from some other spindle cell sarcomas. The use of both of these markers in current practice is diminishing as a result of the availability of more sensitive and specific markers. CLAUDIN-1
The claudins are a family of approximately 18 proteins that play important structural and functional roles in tight junctions. They are transmembrane proteins that interact with other transmembrane proteins, such as junctional adhesion molecule (JAM) and occludin, as well as the scaffolding proteins ZO-1, ZO-2, and ZO-3. Members of the claudin family are differentially expressed in various cell types: claudin-3 is expressed primarily in lung and liver epithelia, whereas claudin-5 is expressed principally in endothelial cells; claudin-1 expression is widespread among epithelial cells, but in mesenchymal tissues, expression appears limited to perineurial cells. Claudin-1 expression has been reported in perineuriomas and occurs in 29% to 92% of tumors.156,157 Claudin-1–positive (perineurial) cells may also be found in neurofibromas, often at the periphery of the tumor, and in the capsule of schwannomas. Endothelial cells variably expressed claudin-1 without a
80
Immunohistology of Neoplasms of Soft Tissue and Bone
consistent staining pattern; expression is not seen in normal Schwann cells, fat, smooth muscle, skeletal muscle, or fibroblasts. Claudin-1 is consistently negative in desmoplastic fibroblastoma, dermatofibrosarcoma protuberans (DFSP), and fibromatosis. Although claudin-1 expression has been reported in cases of lowgrade fibromyxoid sarcoma (LGFMS) with perineuriomalike features,158 in the authors’ experience, LGFMS is consistently negative for claudin-1, irrespective of the histologic appearances (unpublished data); lack of demonstrable claudin-1 expression in LGFMS was also reported in the initial paper describing claudin-1 expression in perineurioma.156 SOX10
SOX10 is a neural crest transcription factor that plays a critical role in the differentiation, maturation, and maintenance of Schwann cells and melanocytes. Recent studies have indicated that SOX10 shows similar sensitivity for MPNST as S-100 protein, with nuclear expression observed in 30% to 50% of MPNSTs.159,160 SOX10 is also detected in more than 90% of melanocytic neoplasms; expression of this marker should therefore be interpreted in context.159,160 However, unlike S-100 protein, expression of SOX10 in other mesenchymal and epithelial tumors is limited; expression is found in myoepitheliomas and diffuse astrocytomas. Sustentacular cells of paraganglioma/pheochromocytoma and a subset of carcinoid tumors also express SOX10.159
Markers of Endothelial Differentiation Several markers associated with endothelial cells have been applied to the recognition of vascular neoplasms of soft tissue. The varying degrees of sensitivity and specificity of these markers are discussed below. FACTOR VIII–RELATED ANTIGEN (VON WILLEBRAND FACTOR)
Factor VIII–related antigen, or von Willebrand factor (vWF), is a very large polymeric protein synthesized exclusively by endothelial cells and megakaryocytes. It consists of three multimeric subunits more than 10,000 kD in molecular weight; physiologically, they undergo proteolysis to yield substantially smaller fragments that can be found in plasma. The function of vWF is twofold. First, it forms circulating complexes with factor VIII coagulant protein, a 265-kD protein that effects the activation of factor X in the intrinsic coagulation pathway. Second, vWF plays a crucial role in platelet aggregation such that patients with low levels or dysfunctional variants of this protein have the clinical bleeding diathesis known as von Willebrand syndrome.161 In the context of soft tissue pathology, vWF is used principally to distinguish vascular neoplasms from their morphologic simulants.162,163 Because vWF is packaged within Weibel-Palade bodies (WPBs) in endothelial cells, it is logical to expect that immunoreactivity for that analyte would parallel the ultrastructural presence
of such organelles. This is indeed the case, and because WPBs are rare in poorly differentiated endothelial neoplasms, this explains why the sensitivity of vWF is low (~10% to 15%) for the recognition of morphologically high-grade angiosarcoma. Accordingly, this marker is more consistently expressed in the spectrum of benign and locally aggressive endothelial tumors, such as hemangioma variants and hemangioendotheliomas.164 As would be expected given the cellular location of WPBs, expression of vWF is typically granular and cytoplasmic. Although expression of vWF is considered to be 100% specific for endothelial cells, because vWF is secreted into serum and can be found in fibrin thrombi and areas of hemorrhage, interpretation of this stain can be difficult; in fact, vWF has largely been abandoned in clinical practice because of the availability of more sensitive and more easily interpretable markers of endothelial differentiation, such as CD31 and V-ETS avian erythroblastosis virus E26 oncogene homolog (ERG). CD34
CD34, the hematopoietic progenitor cell antigen, is recognized by several monoclonal antibodies that include My10/HPCA1, QBEND-10, and BI-3C5.165,166 CD34 is a 110-kD transmembrane glycoprotein that is expressed by embryonic cells of the hematopoietic system,167 including both lymphoid and myeloid cells and endothelial cells. Correspondingly, again in the setting of soft tissue tumors, CD34 is a potential indicator of vascular differentiation. It is a relatively sensitive marker for the endothelial lineage and recognizes more than 85% of angiosarcomas and Kaposi sarcomas.165,166 However, in the authors’ experience, reactivity for CD34 is observed in only approximately 60% of poorly differentiated angiosarcomas, which limits its diagnostic utility in this setting. Moreover, the specificity of CD34 is low, inasmuch as CD34 expression is seen in some LMS, PNSTs, and epithelioid sarcomas,167-169 which could potentially simulate variants of angiosarcoma or hemangioendothelioma. In addition, CD34 is so commonly detected in DFSP and its variants and in spindle cell lipoma, GIST, and solitary fibrous tumor that it is regularly used as an adjunct to the diagnosis of these tumor types.170 Although 70% to 90% of spindle cell GISTs express CD34, most frequently in rectal and esophageal tumors, at most only 50% of epithelioid GISTs and GISTs of the small intestine are CD34 positive.135 Approximately 20% to 30% of retroperitoneal LMS expresses CD34.171 Diffuse CD34 expression is characteristic of DFSP and can help distinguish this tumor type from benign fibrous histiocytoma/dermatofibroma, which is typically negative for CD34 (although around 5% of benign fibrous histiocytomas may show focal staining). However, when fibrosarcomatous transformation of DFSP occurs, CD34 expression is often lost in the higher-grade component.172,173 CD34 expression is observed in 65% of soft tissue perineuriomas and highlights the delicate cytoplasmic processes of the lesional cells.157 Thus, as endothelial markers, antibodies to CD34 are best used in a panel of reagents designed to
Biology of Antigens and Antibodies
account for these other diagnostic possibilities when appropriate. CD31
CD31, also known as platelet–endothelial cell adhesion molecule 1 (PECAM-1)174 is a 130-kD transmembrane glycoprotein expressed by endothelial cells, megakaryocytes, platelets, and histiocytes. CD31 expression can also be found on some myeloblasts, plasma cells, and lymphocytes, and is recognized by the monoclonal antibody JC/70A. This marker is highly restricted to endothelial neoplasms among soft tissue tumors, where it shows a membranous pattern of staining, and its sensitivity is generally excellent.175,176 In our hands, more than 90% of angiosarcomas are CD31 positive, regardless of morphologic grade or subtype, and CD31 expression is consistently detected in hemangioma and hemangioendothelioma variants. It must be acknowledged, however, that Kaposi sarcoma is labeled more consistently for CD34 than for CD31.175,176 The greatest pitfall in the interpretation of CD31 expression in tumors is due to the fact that CD31 also commonly labels macrophages, which may be present in large numbers, dispersed among tumor cells.177 Not surprisingly, CD31 expression is also seen in histiocytic sarcomas.140 Less specifically, limited expression of CD31 can occasionally be found in otherwise undifferentiated pleomorphic sarcomas and very rarely in carcinomas and mesotheliomas.178 FRIEND LEUKEMIA VIRUS INTEGRATION 1
Human friend leukemia virus integration 1 (FLI1) is a member of a family of transcription factor proteins that share a conserved DNA-binding region, the E26 transformation-specific (ETS) domain. That peptide sequence is 98 amino acid residues long, and it bears a molecular resemblance to the helix-turn-helix motif of DNA-binding proteins.179 FLI1 is a sequence-specific transcriptional activator involved in cell proliferation. It recognizes the DNA sequence 5′-C(CA)GGAAGT-3′ and is encoded by a gene on the long arm of chromosome 11 (11q24).180 In rodents, Fli1 is a common region for viral nucleic acid integration in virus-driven leukemogenesis.181 In all vertebrates, this gene also appears to act at the top of the transcriptional network that governs the development of hematopoietic precursors and endothelial cells.182 Landry and colleagues have shown that this effect occurs by regulation of the proximal promoter of the LMO2 gene in endothelia.183 FLI1 is usually expressed in the nuclei of lymphocytes and endothelial cells, thus care must be taken in the interpretation of this stain so as not to overinterpret the presence of FLI1-positive cells. The best-known association between FLI1 and human tumors relates to its fusion with the EWSR1 gene on chromosome 22 (22q12) in most cases of ES/ PNET.184 The resultant fusion protein may play a role in the evasion of cellular senescence.185 FLI1 positivity has also been found in melanoma, synovial sarcoma, Merkel cell carcinoma, and lung adenocarcinoma.186
81
Folpe and colleagues187 were the first to study FLI1 immunoreactivity in human endothelial neoplasms, including hemangiomas, hemangioendotheliomas, angiosarcoma, and Kaposi sarcoma. They observed a sensitivity of 94% in that specific context and found no labeling of nonvascular tumors. However, more recently, Mhawech-Fauceglia and colleagues188 have documented FLI1 positivity in some carcinomas, lymphomas, and RMS as well. In a study of cutaneous tumors of the head and neck, all angiosarcomas examined showed strong diffuse nuclear expression of FLI1, but FLI1 reactivity of variable intensity was observed in 87% of squamous cell carcinomas, 92% of atypical fibroxanthomas, 59% of melanomas, and 20% of atypical intradermal smooth muscle neoplasms.189 In that study, the sensitivity of FLI1 for angiosarcoma was 100% with a specificity of only 29% if any staining in other tumors was considered positive and 76% if only moderate or strong staining was used. In aggregate, these data do not support the use of FLI1 as an adjuvant to the diagnoses of either Ewing sarcoma or vascular neoplasms. V-ETS AVIAN ERYTHROBLASTOSIS VIRUS E26 ONCOGENE HOMOLOG (ERG)
Similar to FLI1, ERG is a member of the ETS family of transcription factors. In human fetal tissues, ERG is also expressed in a nuclear pattern in a subset of primitive mesenchymal cells but subsequently becomes limited to vascular endothelium and a subset of normal hematopoietic stem cells/immature myeloid cells. ERG expression is an extremely sensitive and specific marker of endothelial differentiation, and a rabbit anti-ERG monoclonal antibody is commercially available. ERG is ubiquitously expressed in the nuclei of normal endothelium, which serves as an internal control. Nuclear expression of ERG has recently been reported in 100% of hemangiomas, 98% of epithelioid hemangioendotheliomas (EHEs), and 96% of angiosarcomas.191 All cutaneous angiosarcomas expressed ERG in two large studies, regardless of the degree of morphologic differentiation, usually in a strong and diffuse pattern.189,191 The majority (90%) of deeply situated angiosarcomas were also reactive for ERG.191 With regard to nonvascular tumors, nuclear ERG expression is observed in approximately 50% of prostatic adenocarcinomas as a result of the presence of the TMPRSS2ERG fusion gene in these carcinomas. Of note, ERG is not expressed by normal prostatic epithelium. Nuclear expression of ERG in other epithelial and mesenchymal tumors is very rare; in the largest study to date of ERG expression, one large cell carcinoma of the lung and one pleural mesothelioma showed focal nuclear staining for ERG, out of 643 (549 nonprostatic) epithelial tumors examined.191 In another study, all cutaneous tumors examined—atypical fibroxanthoma, squamous cell carcinoma, melanoma, and atypical intradermal smooth muscle neoplasms—were negative for ERG.189 Other tumors that may show nuclear expression of ERG include Ewing sarcoma, typically reflecting the presence of EWSR1-ERG fusion gene, and blastic extramedullary myeloid tumors.191,192 Although cytoplasmic
82
Immunohistology of Neoplasms of Soft Tissue and Bone
ERG staining has been described in some nonendothelial tumors, we have not observed this pattern in our practice. The sensitivity and specificity of ERG for detecting endothelial differentiation are therefore greater than those of CD34, CD31, and FLI1. KEY DIAGNOSTIC POINTS ERG • ERG is more sensitive and specific for vascular tumors than CD31, CD34, and FLI1. • More than 90% of all angiosarcomas are positive. • ERG is positive in approximately 50% of prostatic adenocarcinomas and, rarely, in other tumors that have an ERG-gene fusion (e.g., Ewing sarcoma).
GLUCOSE TRANSPORTER TYPE 1
Glucose transporter type 1 (GLUT-1) is an erythrocytetype glucose transporter protein and a member of the facilitative cell-surface glucose transporter family. GLUT-1 is present on brain capillary endothelium, where it functions in the transport of glucose across the blood-brain barrier.193 GLUT-1 also plays an important role in the cellular response to hypoxia, as a downstream target of hypoxia-inducible factor 1-α (HIF1-α). GLUT-1 upregulation and subsequent overexpression of GLUT-1 receptors on the plasma membrane of various tumor cells are thought to allow escape from the apoptosis-inducing effects of a hypoxic environment.194 A rabbit polyclonal antihuman GLUT-1 is commercially available. Constitutive expression of GLUT-1 has been documented in a variety of normal cell types, including placental trophoblast and perineurial cells, and upregulation of GLUT-1 expression has been reported in various carcinomas, including those of urothelial, breast, colonic, pancreaticobiliary, esophageal, renal, pulmonary, and ovarian surface epithelial origin, among others.194 In mesenchymal neoplasms, expression of GLUT-1 has been shown to be a constant feature of juvenile capillary hemangiomas, and its expression may be useful in distinguishing such tumors from various mimics, such as vascular malformations and kaposiform hemangioendothelioma. GLUT-1 expression has also been noted in a subset of soft tissue perineuriomas.195 However, expression appears to be widespread in mesenchymal neoplasms. In a large study of various mesenchymal tumors, Ahrens and colleagues194 found GLUT-1 expression in chordoma (100%), epithelioid sarcoma (63%), GIST (14%), LMS (40%), Ewing sarcoma (27%), synovial sarcoma (30%), and undifferentiated pleomorphic sarcoma (60%). Of note, staining in tumor cells adjacent to areas of necrosis was a common finding; therefore use of GLUT-1 as a discriminatory biomarker appears to be limited, but it may be helpful in selected settings. PROSPERO-RELATED HOMEOBOX 1
Prospero-related homeobox 1 (PROX1) is a nuclear transcription factor encoded for by the homebox gene
PROX1, which functions in lymphatic development during embryogenesis. A monoclonal antihuman antibody against PROX1 has been reported in two studies of endothelial neoplasms.196,197 PROX1 was expressed in 58% of all vascular tumors examined, including 42% of hemangiomas, 100% of lymphangiomas, 47% of EHEs, 92% of retiform hemangioendotheliomas, 86% of cutaneous angiosarcomas of the head and neck, and 20% to 36% of deep soft tissue or visceral angiosarcomas. Reactivity for PROX1 was also found in 25% of Ewing sarcomas, 33% of paragangliomas, and 19% of synovial sarcomas, as well as in various carcinomas.197 The utility of PROX1 as a discriminatory marker for endothelial differentiation therefore appears to be limited by a lack of specificity and sensitivity; however, it may be of use in suggesting lymphatic differentiation in endothelial lesions. CLAUDIN-5
Claudin-5 is a transmembrane tight-junction protein that functions as a barrier at epithelial and endothelial cell junctions. Expression of claudin-5 is seen in endothelium and in some epithelial cell types, such as glomerular podocytes.198 Although claudin-5 is a sensitive marker for detecting tumors with endothelial differentiation, it is nonspecific, because expression is also seen in many different epithelial tumors.199 Available antihuman antibodies for claudin-5 include a rabbit polyclonal antibody and a mouse monoclonal antibody (clone 4C3C2). In normal tissues, claudin-5 is expressed by endothelial cells of vessels of varying caliber, but it tends to be strongest in capillaries, lymph node sinusoidal endothelium, and high endothelial venules. Lymphatics of the intestinal mucosa may stain weakly for claudin-5.200 Miettenin and colleagues200 evaluated expression patterns of claudin-5 in a large study of normal tissues, vascular tumors, mesenchymal neoplasms, and epithelial tumors. Nonendothelial tissues that express claudin-5 include hair follicle epithelium, sweat glands, surface epithelium of the luminal GI tract and pancreatic ducts, bile ducts, prostatic glandular epithelium, thyroid follicular epithelium, glomerular capsular and tubular epithelium, tonsillar crypt epithelium, and ductal epithelium of the breast. Histiocytic and lymphoid cells are negative for claudin-5. The pattern of staining for claudin-5 is cytoplasmic and/or membranous, although epithelioid vascular tumors produce a predominantly membranous pattern of staining. Expression of claudin-5 is found in 96% of angiosarcomas, 97% of Kaposi sarcomas, 88% of EHEs, 100% of retiform hemangioendotheliomas, and 100% of hemangiomas (capillary, cavernous, spindle cell, verrucous, intramuscular, capillary, juvenile, and venous) and lymphangiomas. In spindle cell hemangioma, the spindle cells are negative for claudin-5. In juvenile capillary hemangioma, staining is usually limited in extent; in EHE, reactivity may be variable within an individual tumor. Angiosarcoma typically shows strong diffuse expression in tumor cells regardless of the degree of differentiation, but in occasional cases, staining may be weak or focal.200
Biology of Antigens and Antibodies
Claudin-5 expression is also found to varying degrees in lung, gastric, colonic, pancreatic, prostatic, ovarian, and endometrial adenocarcinomas; low-grade ductal breast carcinomas; well-differentiated cutaneous squamous cell carcinomas; esophageal squamous cell carcinomas; large cell undifferentiated lung carcinoma; and in the epithelial component of biphasic synovial sarcoma.200 THROMBOMODULIN
Thrombomodulin (CD141) is a 75-kD cytoplasmic glycoprotein that is distributed among endothelial cells, mesothelial cells, osteoblasts, mononuclear phagocytic cells, and selected epithelia.201,202 Its physiologic role is to convert thrombin from a coagulant protein to an anticoagulant. It has been shown that thrombomodulin is a sensitive indicator of endothelial differentiation, particularly in poorly differentiated malignant vascular neoplasms.203 Kaposi sarcoma is likewise immunoreactive for this marker.204 However, because of the potential presence of thrombomodulin in some metastatic carcinomas and most mesotheliomas,205 both of which may be confused with epithelioid angiosarcomas, it cannot be used as a single marker for vascular neoplasms. ULEX EUROPAEUS I AGGLUTININ
Ulex europaeus I (UEAI) agglutinin is not an antibody reagent but instead represents a lectin produced by the gorse plant. It recognizes the Fuc-α-1-2-Gal linkage in fucosylated oligosaccharides, which compose portions of various glycoproteins.206 In particular, the H blood group antigen and carcinoembryonic antigen (CEA) regularly bind to UEAI, as does another fucosylated protein expressed by endothelial cells. Biotinylated Ulex may be used as a histochemical reagent in surgical pathology or, alternatively, unlabeled lectin can be used, with its binding to tissue subsequently detected by application of biotinylated antiUlex and avidin-biotin-peroxidase complex. Because of the low specificity of UEAI for endothelial differentiation, as mentioned earlier in this discussion, and because in addition to vascular neoplasms, epithelioid sarcoma and various metastatic carcinomas may also bind Ulex,206 UEAI is now rarely used in clinical practice. PODOPLANIN
The antibody D2-40 recognizes podoplanin, also known as AGGRUS, gp36, M2A, and T1A-2.207 It is a transmembrane glycoprotein encoded by a gene on the short arm of chromosome 1 (1p36.21) and is expressed in various tissues. Breiteneder-Geleff and colleagues208 originally described this moiety in glomerular podocytes of the rat, but it has since been observed in lymphatic endothelium, mesothelium, various epithelia, follicular dendritic cells, and germ cells in several species, including humans. Tumors that show differentiation toward these lineages may therefore also be podoplanin-positive, such as mesothelioma, seminoma, follicular dendritic
83
cell sarcoma, and tumors of skin adnexa.207,209-214 Podoplanin is principally expressed during vertebrate development in lymphatic endothelial cells and is therefore thought to be a selective marker for lymphatic channels.215 Overexpression of podoplanin significantly increases endothelial cell adhesion, migration, and vascular lumen formation.209 In the context of soft tissue neoplasia, podoplanin may be used as a determinant of lymphatic differentiation in the evaluation of vascular neoplasms, although expression is also found in a subset of angiosarcomas, kaposiform hemangioendothelioma, and Kaposi sarcoma.207,216 This finding may reflect lymphatic differentiation within these tumors. Given the relative lack of specificity of D2-40 for vascular tumors, the utility of this marker is somewhat limited. However, immunolabeling with D2-40 may be helpful in delineating angiolymphatic invasion by carcinomas and melanomas. WILMS TUMOR 1
The Wilms tumor 1 gene, WT1, is located on the short arm of chromosome 11 (11p13). It encodes a protein that is a critical determinant of urogenital development and is expressed in more than 80% of nephroblastomas.217 With regard to osseous and soft tissue tumors, WT1 protein is not a specific marker and is potentially present in angiosarcoma, MPNST, synovial sarcoma, osteosarcoma, myxoid liposarcoma, and clear cell sarcoma (CCS).218 Nevertheless, it is occasionally used in diagnostic practice, in a structured panel with other immunostains, as an adjunctive indicator of endothelial differentiation.219 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3
Vasular endothelial growth factor receptor 3 (VEGFR3) is a transmembrane protein also known as FMS-like tyrosine kinase 4 (FLT4). VEGFR3 is encoded by a gene at chromosomal locus 5q33.220 Initially thought to be expressed predominantly in lymphatic endothelia, VEGFR3 positivity is found in more than 90% of Kaposi sarcoma, papillary intralymphatic angioendothelioma (Dabska tumor), and retiform and kaposiform hemangioendothelioma; 50% of angiosarcomas; and 15% of hemangiomas.221 VEGFR3 is also expressed by nonneoplastic capillaries within glomeruli and endocrine organs and in nonneoplastic capillaries in some carcinomas and sarcomas.
Fibrohistiocytic Markers A variety of monoclonal antibodies and heteroantisera have been identified as histiocytic or fibrohistiocytic markers in paraffin sections. They may be useful in the evaluation of potential histiocytic proliferations or neoplasms of skin and soft tissue, such as juvenile xanthogranuloma, reticulohistiocytoma, histiocytic sarcoma, and in tumors with a histiocytic component. The targets of these reagents include moieties such as α1-antitrypsin,
84
Immunohistology of Neoplasms of Soft Tissue and Bone
α1-antichymotrypsin, muramidase/lysozyme, cathepsin B, CD68, CD163, factor XIIIa, and the HAM 56 antigen.222-225 Although it is true that a majority of putative fibrohistiocytic neoplasms do label for some of the described markers, the specificity of many of these markers is poor. With the exception of CD163, which is highly specific for histiocytic differentiation,226 carcinomas, melanomas, and other sarcoma types also potentially express these markers with relatively high frequency.221,227-229 The current approach to the diagnosis of so-called fibrohistiocytic tumors is predominantly based on morphologic features, with the inclusion of an IHC panel as appropriate to exclude other diagnostic possibilities. CD68
CD68 is a 110-kD lysosomal protein. Antibodies against CD68 include KP1 and PG-M1, and the staining pattern is cytoplasmic.230 Expression is seen in any lysosomalrich cell and is therefore not limited to histiocytes. Normal cells that express CD68 therefore include histiocytes, osteoclasts, and other cells of monocytic lineage. CD68-positive cells are therefore frequently found within tumors, and care must be taken not to overinterpret positive staining in background tumor-infiltrating histiocytes. In addition to pure histiocytic tumors, it has been found that granular cell tumor, schwannoma, renal cell carcinoma (RCC), and melanoma may all express CD68.231-233 CD163
CD163 is the most specific histiocytic (monocyte and macrophage) marker currently available for diagnostic use. A glycoprotein belonging to a scavenger receptor superfamily, CD163 may play a role in immune response,226 and it also functions as a hemoglobin scavenger. The commercially available monoclonal antibody 10D6 performs well in paraffin-embedded tissue, showing a predominantly membranous pattern of staining. CD163 expression is found in the majority of cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease), histiocytic sarcoma, hemophagocytic lymphohistiocytosis, and tenosynovial giant cell tumor; a subset of cases of Langerhans cell histiocytosis; and some acute myeloid leukemias with monocytic differentiation.234 In the same study, all cases of littoral cell angioma showed staining for CD163, whereas normal splenic littoral cells lacked staining.234 Expression in other soft tissue tumors is extremely limited, and epithelial tissues and tumors are typically negative for CD163.234 KEY DIAGNOSTIC POINTS CD163 • Currently, clone 10D6 is the most specific histiocytic marker for paraffin tissue.
LYSOZYME (MURAMIDASE)
Lysozyme is a bacteriolytic enzyme present in granulocytes and histiocytes (monocytes/macrophages). Pure histiocytic lesions, such as juvenile xanthogranuloma and histiocytic sarcoma, show cytoplasmic expression of lysozyme in most cases, whereas the lesional cells of so-called fibrohistiocytic tumors, such as benign fibrous histiocytoma, are typically negative for lysozyme.235 FACTOR XIIIA
Factor XIIIa (FXIIIA) is a proenzyme involved in fibrin polymerization that is reportedly found on dermal dendritic phagocytic and antigen-presenting cells. The diagnostic utility of FXIIIa in surgical pathology is limited. Although once thought to be a useful marker in distinguishing benign fibrous histiocytoma from DFSP,236 it is now recognized that much of the staining seen in benign fibrous histiocytoma is due to expression in background nonneoplastic dermal cells. FXIIIa-positive stromal cells may be seen in DFSP but tend to be less prominent because of the greater cellularity of tumor cells in this lesion.237 Furthermore, whereas focal or multifocal reactivity for FXIIIa may be found in many cases of benign fibrous histiocytoma, the intensity of staining is usually greatest in background stromal cells, making interpretation difficult. NKI/C3
NKI/C3 is a monoclonal antibody that recognizes a melanoma-associated antigen in FFPE tissue samples. This melanoma-associated antigen is a glycoprotein located in cytoplasmic vacuoles, and it is strongly expressed in cells with a large population of melanosomes. Although NKI/C3 is positive in only a few normal tissues, it stains a wide spectrum of histiocytoid soft tissue neoplasms. Its greatest utility lies in its expression in cellular neurothekeoma, but NKI/C3 is also positive in many other cutaneous tumors, some of which may mimic cellular neurothekeoma, including juvenile xanthogranuloma, atypical fibroxanthoma, cellular benign fibrous histiocytoma, reticulohistiocytoma, and xanthoma. NKI/C3 is negative in epithelioid benign fibrous histiocytoma and Langerhans cell histiocytosis.238
Markers of Melanocytic Differentiation HUMAN MELANOMA BLACK 45
Human melanoma black 45 (HMB-45) is a 100-kD glycoprotein localized to immature melanosomes. Fetal melanocytes and activated adult melanocytes will therefore express HMB-45, whereas normal resting adult melanocytes and intradermal nevi are negative. The staining pattern is cytoplasmic and may be granular. Approximately 90% of primary melanomas are positive for HMB-45, but when spindled in morphology, the frequency of HMB-45 in melanoma is much lower; virtually all desmoplastic melanomas are negative for
Biology of Antigens and Antibodies
this marker.239 The lesional cells of blue nevi are also typically positive for HMB-45. Expression of HMB-45 in metastatic melanoma is variable, with reported rates of positivity between 70% and 90%. Among soft tissue tumors, HMB-45 is a useful marker for identifying neoplasms of the PEComa family. Tumors in the PEComa family, including angiomyolipoma of kidney, are characterized by dual myoid and melanocytic differentiation; the latter can be identified by staining for melanocytic markers such as HMB-45, melan A (see below), and MITF. The frequency and percentage of HMB-45–positive cells in these tumors is quite variable, and some cases show only scattered positive cells.87 HMB-45 is also expressed in CCS of tendons and aponeuroses, often with a stronger intensity of staining than that for S-100 protein.240 However, confirmatory molecular studies may be needed to confirm the diagnosis of CCS over either primary or metastatic melanoma. HMB-45 positivity is also seen in melanotic schwannoma but not in MPNST. Expression of HMB-45 appears to be limited to the tumors listed above, making it a highly specific marker for melanocytic differentiation. MELAN A
Melan A (also known as MART1, melanoma antigen recognized by T cells 1), is a protein coded for by the gene MLANA. MART 1 (clone M2-7C10) and melan A (clone A103) are two different antibody clones that recognize the same antigen. Melan A is expressed in the cytoplasm of normal melanocytes and adrenal cortical cells; the latter is only detectable with clone A103. Similar to HMB-45, expression of melan A is frequently seen in PEComas, which accounts for much of its use in soft tissue pathology.241 The other main use of melan A in soft tissue pathology is in the evaluation of possible melanocytic neoplasms. Melan A expression is found in approximately 80% of metastatic melanomas but is negative in desmoplastic melanomas.242,243 Expression of this marker is less specific for melanocytic differentiation than HMB-45; tumors of the adrenal cortex and ovarian and testicular Leydig cell tumors also express melan A (clone A103).244 MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTOR
Microophthalmia-associated transcription factor (MITF) is a nuclear protein that functions as a transcriptional regulator of melanocytic genes, and it has a functional role in osteoclasts. The clinical utility of MITF in surgical pathology is limited by its low specificity. Nuclear expression of MITF is seen in melanocytes, primary and metastatic melanomas, and in some PEComas, CCSs, and histiocytic proliferations or neoplasms.240,245 TYROSINASE
In soft tissue pathology, use of tyrosinase, a 75-kD glycoprotein that catalyzes the incorporation of tyrosine
85
into melanin pigment, is for the most part limited to identification of melanoma. As much as 90% of metastatic melanomas are reported to show diffuse cytoplasmic staining for this marker.240 CCS expresses tyrosinase in 40% of cases.240 Unlike melan A and HMB-45, expression of tyrosinase is not frequently seen in PEComas.240
KEY DIAGNOSTIC POINTS Soft Tissue Antigens and Antibodies • Desmin is a specific marker for myogenic differentiation, detected in almost all RMS and LMS. • Desmin expression may be seen in myofibroblastic tumors and in other tumors of uncertain lineage such as DSRCT. • α–SMA is less specific for myogenic differentiation than h-caldesmon or HHF-35/muscle-specific actin. • MYOD1 and myogenin/MYF4 are nuclear transcription factors highly specific for striated muscle differentiation. • Vimentin is ubiquitous in soft tissue tumors, and as such, its utility in diagnostic surgical pathology is limited. • Glial fibrillary acidic protein may be found in Schwann cells and chondrocytes. • Keratins are regularly detected in synovial sarcoma, chordoma, myoepithelial tumors, epithelioid sarcoma, DSRCT, and adamantinoma. They are seen less commonly in LMS, epithelioid angiosarcoma, Ewing sarcoma, and alveolar RMS. • Epithelial membrane antigen is frequently expressed in synovial sarcoma, epithelioid sarcoma, perineuriomas, chordoma, myoepithelial tumors, and plasmacytomas. • CD31 is highly restricted to endothelial neoplasms, with excellent sensitivity. • Although sensitive for detection of endothelial differentiation, CD34 lacks specificity, because it reacts with a wide variety of mesenchymal cells and tumor types. • ERG is a highly sensitive and specific nuclear marker for endothelial differentiation.
Other Useful Markers in Evaluation of Soft Tissue and Bone Tumors β-CATENIN
Nuclear localization of β-catenin, which is encoded by a gene at chromosomal locus 3p21 (CTNNB1), reflects a mutation in that moiety or in the APC gene that regulates it in an upstream fashion. Nuclear β-catenin, as recognized by the 14/β-catenin monoclonal antibody clone, functions as a transcriptional activator when complexed with members of the lymphocyte-enhancer– factor family of proteins. β-catenin is a key component of two separate cell-signaling pathways. First, in the Wnt pathway, β-catenin is released from a complex that involves the adenomatous polyposis coli (APC)
86
Immunohistology of Neoplasms of Soft Tissue and Bone
protein, allowing it to accumulate in the cytoplasm. As the levels of free β-catenin increase, the protein translocates into the nucleus, where it acts to induce expression of genes involved in cell proliferation and differentiation. In addition, β-catenin is also involved in cell adhesion through binding to type I cadherins, where it links them to the actin cytoskeleton, binds to the APC protein, and serves to negatively regulate proliferation through Wnt signaling.246 Patients with familial adenomatous polyposis (FAP) have germline mutations in APC and an increased incidence of desmoid fibromatosis. Approximately 10% of patients with FAP will develop desmoid fibromatosis, at a frequency correlating somewhat with the precise site of the APC mutation.247,248 In sporadic desmoid fibromatosis, somatic mutations in APC are identified in approximately 10% of tumors, and mutations in CTNNB1 are found in as much as 90% of tumors.249-251 Nuclear β-catenin expression is present in 80% of cases of sporadic desmoid fibromatosis and in 67% of desmoid tumors in patients with FAP.252-254 The tumor cells of Gardner fibroma show nuclear reactivity for β-catenin in 64% of cases,255 and nuclear staining is also observed in approximately 50% of superficial fibromatoses, 30% of low-grade myofibroblastic sarcomas, and 20% of solitary fibrous tumors. Occasional nuclear staining has been reported in infantile fibrosarcoma, desmoplastic fibroblastoma, and rarely in GISTs. Neurofibroma, schwannoma, nodular fasciitis, LMS, inflammatory myofibroblastic tumor, fibroma of tendon sheath, lipofibromatosis, and calcifying aponeurotic fibroma consistently lack nuclear reactivity for β-catenin. Nuclear staining for β-catenin is therefore supportive, but not diagnostic, of desmoid fibromatosis. ANAPLASTIC LYMPHOMA KINASE
Anaplastic lymphoma receptor tyrosine kinase (ALK) also known as p80, is a receptor tyrosine kinase first identified as the protein product of the translation of an abnormal ALK-NPM fusion gene produced by a t(2;5) (p23;q35) chromosomal translocation in anaplastic large cell lymphoma (ALCL).256 Corresponding overexpression of ALK that can be detected immunohistochemically is identified in the majority of cases of ALCL. In soft tissue neoplasms, the neoplastic cells of approximately 50% of inflammatory myofibroblastic tumors (IMTs) overexpress ALK as a result of abnormalities at the 2p23 chromosomal locus.257,258 Overexpression of ALK can result from chromosomal rearrangements with various other gene partners, all of which, similar to nucleophosmin (NPM), act as active promoters to the fusion protein. The resulting fusion tyrosine-kinase oncoproteins share a common structure: the N-terminal sequence encoded by the nonreceptor tyrosine kinase member of the pair replaces the extracellular and transmembrane domains of ALK and contributes proteinbinding sites that allow for oligomerization that mimics ligand binding and ultimately leads to constitutive, ligand-independent ALK autophosphorylation and activation.259
Expression of ALK fusion proteins in IMT can be detected by IHC, and, interestingly, the pattern of ALK staining seems to reflect the fusion partner: diffuse cytoplasmic staining when the fusion involves the cytoplasmic proteins TPM3, TPM4, CARS, ATIC, and SEC31a; granular cytoplasmic staining when the fusion partner is CLTC, a main component of the coated vesicles involved in selective protein transport; and nuclear membrane staining with RANBP2, a large protein found at the nuclear pores.260-262 The latter, distinct pattern of ALK immunoreactivity is usually seen in the aggressive IMT variant known as epithelioid inflammatory myofibroblastic sarcoma, which frequently harbors an ALK-RANBP2 fusion.263 ALK expression and corresponding genetic abnormalities have to date also been identified in small subsets of lung adenocarcinoma and RCC.264-266 In contrast to the translocation-associated ALK aberrations associated with these tumor types, point mutations in ALK have been described in neuroblastoma,267 and amplification of ALK has been identified in alveolar RMS.268 The study by Gaal and colleagues268 reported overexpression of ALK in 81% of alveolar RMS and 32% of embryonal RMS; copy-number gain of ALK was found in 88% of alveolar RMS and in 52% of embryonal RMS. ALK expression by IHC has also been reported in some cases of MPNST, LMS, and Ewing sarcoma.257,269 The level of overexpression of ALK in ALCL is significantly greater than that seen in other tumors with ALK gene aberrations; the ALK-1 antibody shows relatively low sensitivity in paraffin-embedded sections and is often negative in lung adenocarcinomas with ALK rearrangements and some cases of IMT. More recently, two new, more sensitive monoclonal antibodies, 5A4 and D5F3, have become commercially available. CD99
Also known as p30/32 glycoprotein or MIC2 protein,270,271 CD99 is a cell surface protein that is encoded by genes on the X and Y chromosomes272 and is expressed in a diffuse membranous pattern in the vast majority of Ewing sarcomas, but it may also be expressed in a less distinct pattern in T-lymphoblastic lymphomas, solitary fibrous tumors, synovial sarcomas, neuroblastomas, mesenchymal chondrosarcomas, and some epithelial tumors, particularly high-grade neuroendocrine carcinomas.147,273-277 Available commercial monoclonal antibodies to this determinant include 12E7, O13, and HBA-71. NEUROBLASTOMA MARKER (NB84)
Neuroblastoma marker, also known as NB84, is a monoclonal antibody directed against an uncharacterized antigen present within tumor cells of neuroblastoma. Cytoplasmic expression of NB84 is present in the majority of neuroblastomas, but it also stains approximately 20% of Ewing sarcomas, 20% of medulloblastomas, and 50% of DSRCTs, although expression is
Biology of Antigens and Antibodies
often less extensive in these tumors compared with neuroblastoma.278,279 TRANSCRIPTION FACTOR E3
Transcription factor E3 (TFE3) is a transcription factor with a basic helix-loop-helix DNA binding domain and a leucine zipper dimerization domain. TFE3 is ubiquitously expressed in humans and is presumed to regulate many genes, but native TFE3 protein is usually not detected by IHC. Tumors characterized by TFE3 gene fusions (alveolar soft-part sarcoma, Xp11 translocation RCCs) demonstrate nuclear TFE3 staining in almost all cases. A polyclonal antibody (P-16) against the C-terminus of TFE3 is commercially available.280,281 However, as a cautionary note, this antibody appears to have become less reliable over time; recently, a rabbit anti-TFE3 monoclonal antibody (clone MRQ-37) has become available that shows more consistent results. Reactivity for TFE3 has also been identified in a small subset of PEComas, which express TFE3 in a mutually exclusive pattern to MITF, often, although not invariably, associated with TFE3 rearrangements.282,283 MURINE DOUBLE-MINUTE 2 HOMOLOG
Murine double-minute type 2 (MDM2) protein is encoded by a gene at chromosome 12q14.3-q15 that acts as an inhibitor of the tumor-suppressor effects of p53. Amplification of MDM2 can be detected by fluorescence in situ hybridization (FISH), and overexpression of MDM2 protein can be detected with mouse monoclonal antibodies (clones 2A10 and 1F2). Amplification and overexpression of MDM2 is characteristic of well-differentiated and dedifferentiated liposarcomas, and both FISH and IHC have become extremely useful tests for confirming the diagnosis of these tumor types in conjunction with evaluation of CDK4 expression.284 CDK4 is typically overexpressed along with MDM2, and for both, only nuclear staining is considered positive. Staining in well-differentiated liposarcoma may be limited in extent (only observed in scattered nuclei), whereas in dedifferentiated liposarcoma (DDLPS), nuclear staining is usually more diffuse.284,285 Benign lipomatous lesions are negative for both MDM2 and CDK4, as are pleomorphic liposarcoma and myxoid liposarcoma. However, it is important to note that overexpression of MDM2 may also be seen in as many as 60% of MPNSTs, 40% of myxofibrosarcoma, and 29% of embryonal RMS, but when combined with coexpression of CDK4, only 3%, 2%, and 5% of each of these tumor types, respectively, express both of these markers.284 Therefore evaluation of MDM2 and CDK4 together with IHC is most useful. CYCLIN-DEPENDENT KINASE 4
Cyclin-dependent kinase 4 (CDK4) is one of the proteins involved with cell-cycle progression and is encoded by a gene at chromosome 12q13. CDK4 inhibits the retinoblastoma 1 (RB1) gene and is overexpressed in
87
well-differentiated and dedifferentiated liposarcomas, often with MDM2. This phenomenon reflects the common presence of ring or giant marker chromosomes that contain amplified material from the q13-15 region of chromosome 12, where both the CDK4 and MDM2 genes are located. The best diagnostic use of these determinants is in the recognition of DDLPS, as opposed to other high-grade sarcomas, and in the distinction between benign lipomas and atypical lipomatous tumors (well-differentiated liposarcomas), when the latter show very minimal atypical histologic features.284-286 RETINOBLASTOMA
Retinoblastoma (Rb) is a tumor suppressor protein that plays a crucial role in cell-cycle progression. Disruptions to the Rb protein and to the pathway controlled by Rb confer a proliferative advantage to tumor cells, which has been documented in many human cancers, including retinoblastoma. In addition, several recent reports have suggested that Rb also has context-specific functions, including a role in adipocytic differentiation and lineage commitment. A commercially available mouse anti-Rb monoclonal antibody, clone G3-245, may be of utility in diagnostic surgical pathology, in that loss of nuclear Rb expression associated with rearrangements of chromosome 13q, including the RB1 gene locus, have been identified in spindle cell and pleomorphic lipoma, mammary-type myofibroblastoma, and cellular angiofibroma.287 In the same study, nuclear reactivity for Rb was preserved in other tumor types that may be considered in the differential diagnosis with these tumors, including atypical lipomatous tumors, solitary fibrous tumors, myxoid liposarcomas, hibernomas, deep (“aggressive”) angiomyxomas, angiomyofibroblastomas, and vulval fibroepithelial stromal polyps. Of note, Rb staining may be difficult to interpret in cellular angiofibromas with reactive stromal changes. Nuclear reactivity for Rb was also deficient in 9% of conventional lipomas. SMARCB1
SWI/SNF-related, matrix associated, actin-dependent regular of chromatin, subfamily B, member 1 (SMARCB1) is the protein product of the gene SMARCB1 (also known as INI1 and SNF5) located on chromosome 22q11.2. INI1 is a core subunit of the SWI/SNF ATP-dependent chromatin remodeling complex and is ubiquitously expressed in the nuclei of normal cells; it is thought to function as a tumor suppressor. Loss of SMARCB1 function and expression can result from mutations or deletions of the SMARCB1 gene.288 Loss of SMARCB1 expression is observed in several tumor types. SMARCB1 abnormalities were first described in malignant rhabdoid tumors (renal, extrarenal, and atypical teratoid/rhabdoid tumor of the CNS), and loss of SMARCB1 expression is found in 98% of these tumors. A subset of affected infants harbors germline mutations in SMARCB1.289 More recently, loss of SMARCB1 expression has been found in approximately
88
Immunohistology of Neoplasms of Soft Tissue and Bone
90% of epithelioid sarcomas, both conventional and proximal types.290 Point mutations in SMARCB1 are common in malignant rhabdoid tumors, whereas chromosomal deletions are usually identified in epithelioid sarcomas.291 Furthermore, loss of SMARCB1 expression is observed in 50% of epithelioid malignant PNSTs and in a subset of myoepithelial carcinomas, particularly those in children (40%, versus 10% in adults). Other tumor types reported to show variable loss of SMARCB1 expression include extraskeletal myxoid chondrosarcomas (17%),292 poorly differentiated chordoma, undifferentiated hepatoblastoma, and renal medullary carcinoma.293,294 Reduced but not complete loss of INI1 expression has been reported in synovial sarcoma, although the significance of this finding is uncertain. Germline mutations in SMARCB1 occur in approximately 60% of patients with the rare hereditary syndrome of familial schwannomatosis associated with a “mosaic” pattern of protein loss by IHC.295 B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA/ LYMPHOMA 2
B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), an antiapoptotic protein, shows high-level expression in various types of lymphoma, such as follicular lymphoma and a subset of diffuse large B-cell lymphomas, but it is also differentially expressed in some soft tissue neoplasms. Soft tissue tumors that are most often reactive for BCL2 include solitary fibrous tumor, spindle cell lipoma, Kaposi sarcoma, and monophasic synovial sarcoma.296-298 In addition, a subset of MPNST and low-grade myxofibrosarcomas is BCL2 positive.296 Given this wide distribution in soft tissue tumors, BCL2 is of limited utility in differential diagnosis.
Markers Identified through Gene-Expression Profiling TRANSDUCIN-LIKE ENHANCER OF SPLIT 1
Transducin-like enhancer of split 1 (TLE1) is a member of the groucho/TLE family of genes that encodes a transcriptional corepressor implicated in epithelial and neuronal differentiation, body patterning, and hematopoiesis.299-301 Gene-profiling studies have shown that TLE1 is significantly overexpressed in synovial sarcoma.302,303 TLE proteins act through the Wnt/βcatenin signaling pathway, which has been associated with synovial sarcoma.304 The synovial sarcoma fusion protein SS18-SSX appears to perform a bridging function between activating transcription factor 2 (ATF2) and TLE1, resulting in repression of ATF2 target genes.305 Blocking this pathway results in growth suppression and apoptosis, which supports a pathogenetic role for TLE1 and ATF2 in synovial sarcoma. In one study, nuclear immunoreactivity for TLE1, usually of moderate to strong intensity, was observed in 82% of synovial sarcomas, with a sensitivity and specificity of 82% and 92%, respectively.306 Nuclear reactivity for TLE1 was also observed in 15% of MPNSTs and
in 8% of solitary fibrous tumors but was usually only weak in these tumor types. Terry and colleagues reported 16 of 88 MPNSTs (18%) with any staining for TLE1, but only 4 tumors (5%) showed more than weak staining.304 Similarly, in a study that used whole tissue sections and a high threshold for a “positive” result (i.e., strong staining in >25% of cells), Jagdis and colleagues307 observed nuclear staining for TLE1 in only 1 of 43 MPNSTs (2%) without compromising sensitivity; all 35 synovial sarcomas were positive with this criterion. MUCIN 4
Mucin 4 (MUC4) is a transmembrane glycoprotein usually expressed on certain epithelial surfaces (e.g., colonic, breast, and pulmonary epithelium), and it is overexpressed or aberrantly expressed in some carcinomas, most notably those arising in the biliary tract and pancreas. Gene-expression profiling studies of a group of mesenchymal tumors identified overexpression of the MUC4 gene, located on the long arm of chromosome 3 (3q29), in low-grade fibromyxoid sarcoma (LGFMS) compared with other tumor types examined.308 Overexpression of MUC4 at the protein level is also observed in LGFMS. In fact, MUC4 is an extremely sensitive marker for LGFMS and is detected in 100% of cases with FUS gene rearrangement, usually with a strong and diffuse cytoplasmic staining pattern.309 Similarly, extensive reactivity for MUC4 is also observed in approximately 80% of sclerosing epithelioid fibrosarcomas (SEFs), irrespective of FUS gene-rearrangement status.310 Hybrid tumors that contain both LGFMS and SEF components also show diffuse staining for MUC4. Expression of MUC4 is limited in other soft tissue tumors; focal staining or rare positive cells are seen in a small subset of epithelioid GISTs and ossifying fibromyxoid tumors (OFMTs).310 The epithelial component of biphasic synovial sarcomas, however, frequently shows diffuse positivity for MUC4, whereas the spindle cell component often shows limited staining in a subset of cells. MUC4 interacts with the ERBB2 family of receptors and may play a role in tumor development through this pathway.
Immunohistochemical Markers for Evaluation of Bone Tumors: Proposed Markers of Osteoblastic Differentiation One of the greatest challenges in the realm of bone and soft tissue tumor pathology is the reliable recognition of osseous matrix production in malignant lesions. This is an important issue, because the contextual presence of true osteoid equates with a diagnosis of osteosarcoma or with heterologous osteoblastic differentiation in other tumor types. Since the late 1990s, a number of putatively osteoblast-specific markers have been described in the literature, including bone morphogenetic protein (BMP), type I collagen, COL-I-C peptide, decorin, osteocalcin, osteonectin, osteopontin, proteoglycans I and II, bone sialoprotein, and bone glycoprotein 75. Only two among these, osteocalcin and
Specific Soft Tissue and Bone Tumors
osteonectin, have been associated with applicability to paraffin sections in diagnostic immunohistologic studies, but we have not found either to be useful in clinical practice. A recent promising marker for identifying cells that produce osteoid matrix is special AT-rich sequence binding protein 2 (SATB2), which is discussed in the section on osteosarcoma.
89
high levels of brachyury, and this was also true at the protein level; IHC staining for brachyury was found to be a sensitive and specific marker of chordomas.317 This finding provided molecular evidence that axial chordomas are related histogenetically to the notochord. Brachyury is not expressed in chondrosarcoma, myoepithelioma, or in various other carcinomas that may mimic chordoma, most notably RCC.317-319
OSTEOCALCIN
Osteocalcin is one of the most prevalent noncollagenous intraosseous proteins and is predominantly localized to osteoblasts. This 9-kD cytoplasmic protein contains abundant γ-carboxyglutamic acid residues. Its expression is downregulated by helix-loop-helix–type transcription factors, and it is upregulated by vitamin D analogs, such as 1,25 dihydroxyvitamin D2 and 24epi-1,25 dihydroxyvitamin D2 in the final steps of osteoblastic differentiation and osteoid formation.311 Various heteroantisera and monoclonal antibodies to osteocalcin have been reported in IHC studies,311-314 which indicate that polyclonal antiosteocalcin reagents are inferior to monoclonal antibodies for diagnostic purposes because of problems with specificity. Although studies have shown that osteocalcin has a reasonable level of sensitivity for osteoblastic differentiation (~70%) and is, for practical purposes, apparently virtually completely specific for bone-forming cells,314 it is rarely used in clinical practice because of practical difficulties using the antibody. OSTEONECTIN
Osteonectin is a regulatory protein involved in the adhesion of osteoblasts and platelets to their extracellular matrix and in early stromal mineralization. Osteonectin is modified differentially at a posttranslational level in bone cells and megakaryocytes to yield molecules with different oligosaccharide substructures; sequences of osteonectin-related genomic DNA, intranuclear RNA, and messenger RNA (mRNA) are identical in those two cell types.313,315,316 It appears that several other cells may synthesize osteonectin-associated epitopes; fibroblasts, vascular pericytes, endothelia, chondrocytes, selected epithelial cells, and nerves are also immunoreactive for this determinant. Because of potential problems concerning cross-reactivity of available antisera to this marker, monoclonal antibodies should be used when considering this as a diagnostic test. Even then, because osteonectin does not demonstrate the selectivity of expression that has been reported with osteocalcin, its use is limited. BRACHYURY
Brachyury (T), a transcription factor protein product of a T-box gene, regulates the development of notochordderived tissues and neoplasms. It is first detected in the marginal zone of the blastocyst and subsequently is restricted to the notochord and tailbud. Gene-expression studies have demonstrated that axial chordomas express
KEY DIAGNOSTIC POINTS Genomic Applications of Immunohistology • CDK4 and MDM2 identify well-differentiated and dedifferentiated liposarcomas. MDM2 overexpression is a sensitive indicator for these tumor types and differentiates them from potential mimics, such as sclerosing mesenteritis, retroperitoneal fibrosis, and leiomyosarcoma. Overexpression can be confirmed by MDM2 fluorescence in situ hybridization; gene amplification is detected in more than 95% of cases. • TLE1 expression is a consistent finding in synovial sarcoma and is uncommon in potential mimics, such as malignant peripheral nerve sheath tumor and solitary fibrous tumor. • TFE3 expression is found in alveolar soft-part sarcoma, Xp11 translocation renal cell carcinoma, and a small subset of PEComas. • MUC4 is a sensitive and specific marker for low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma. • Brachyury is an extremely useful marker for chordoma. • ALK expression is detected in 50% of inflammatory myofibroblastic tumors as a result of translocations that involve the ALK locus; ALK expression is also detected in a subset of malignant peripheral nerve sheath tumors, neuroblastomas, and RMS (especially the alveolar variant).
Specific Soft Tissue and Bone Tumors An approach to the diagnosis of both bone and soft tissue tumors includes morphologic, IHC, molecular, and clinical/radiologic assessment and correlation. In this chapter we will focus predominantly on discussion of the IHC features of a variety of soft tissue and bone tumors, particularly those with distinctive IHC profiles. Helpful approaches to guide the evaluation of such tumors include consideration of the dominant cytomorphologic patterns, such as spindle cell, epithelioid, round cell, and pleomorphic patterns. Examples of tumors with each type of cytomorphology, along with their IHC profile, are illustrated in Tables 4-1 through 4-4. Furthermore, determination of the line of differentiation within a given tumor based on morphology and immunophenotype is helpful in arriving at a specific diagnosis. For this reason, tumors are discussed based on their line of differentiation, as outlined below. Those
90
Immunohistology of Neoplasms of Soft Tissue and Bone
TABLE 4-1 Antibody Reagents Used by the Authors in the Study of Soft Tissue and Bone Tumors Reagent
Principal Diagnostic Use
Antikeratins (M)
Recognition of epithelioid sarcoma, synovial sarcoma, chordoma, and desmoplastic small round cell tumor
Anti–epithelial membrane antigen (clone E29; M)
Recognition of epithelioid sarcoma, synovial sarcoma, perineurioma, and chordoma
Antidesmin (clone D33; M)
Recognition of smooth muscle, striated muscle, and myofibroblastic tumors; PEComas; and heterologous rhabdomyoblastic differentiation in malignant peripheral nerve sheath tumor and dedifferentiated liposarcoma
Anti–α-isoform smooth muscle actin (clone 1A4; M)
Recognition of smooth muscle tumors, PEComas, and myofibroblastic differentiation
Anti–muscle-specific actin (clone HHF-35; M)
Recognition of striated muscle tumors
Anti–h-caldesmon (clone h-CD; M)
Recognition of smooth muscle tumors
Anti-myogenin/MYF4 (clone LO26; M)
Recognition of striated muscle tumors and heterologous rhabdomyoblastic differentiation in malignant peripheral nerve sheath tumor and dedifferentiated liposarcoma
Anti–S-100 protein (P)
Recognition of melanocytic, schwannian, myoepithelial, and cartilaginous neoplasms
Anti–HMB-45 (clone HMB-45; M)
Recognition of melanoma, PEComa, and clear cell sarcoma
Anti–MART-1 (clone M2-7C10) and anti– melan A (clone A103)
Recognition of melanoma, PEComa, and clear cell sarcoma
Anti–GFAP protein (P)
Recognition of schwannian and myoepithelial neoplasms
Anti–claudin-1 protein (P)
Recognition of perineuriomas
Anti-CD34 (clone QBEnd10; M)
Recognition of endothelial tumors, dermatofibrosarcoma protuberans, solitary fibrous tumor, spindle cell lipoma, perineurioma, and epithelioid sarcoma
Anti-CD31 (clone JC/70A; M)
Recognition of endothelial tumors
Anti-ERG (clone EPR3864[2]; M)
Recognition of endothelial tumors
Anti-CD68 (clones PG-M1 and KP1; M)
Recognition of histiocytic differentiation, although not specific
Anti-CD163 (clone10D6; M)
Recognition of histiocytic differentiation
Anti–β-catenin 1 (clone 4/β-catenin; M)
Recognition of desmoid fibromatosis
Anti–ALK protein (clones 5A4 and D5F3; M)
Recognition of inflammatory myofibroblastic tumor
Anti-CD99 (clone O-13; M)
Recognition of Ewing sarcoma/peripheral primitive neuroectodermal tumor
Anti–TFE3 protein (clone MRQ-37; M)
Recognition of alveolar soft part sarcoma and a small subset of PEComas
Anti–MDM2 protein (clone 1F2; M)
Recognition of well-differentiated and dedifferentiated liposarcomas
Anti–CDK4 protein (clone DCS-31; M)
Recognition of well-differentiated and dedifferentiated liposarcomas
Anti–Rb protein (clone G3-245; M)
Recognition of tumors associated with rearrangements of chromosome 13q, resulting in loss of expression: spindle cell and pleomorphic lipoma, mammary-type myofibroblastoma, and cellular angiofibroma
Anti-synaptophysin (P)
Detection of neuroendocrine differentiation
Anti–SMARCB1 (INI1) protein (clone BAF47; M)
Recognition of epithelioid sarcoma, malignant rhabdoid tumor, and a subset of epithelioid malignant peripheral nerve sheath tumors and myoepithelial carcinomas
Anti–TLE1 protein (P)
Recognition of synovial sarcoma
Anti-MUC4 (clone 8G7; M)
Recognition of low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma
Anti-brachyury (P)
Recognition of chordoma
GFAP, Glial fibrillary acidic protein; M, monoclonal; P, polyclonal (heteroantiserum); PEComa, perivascular epithelioid cell tumor.
Specific Soft Tissue and Bone Tumors
91
TABLE 4-2 Percentages of Positivity for Pertinent Immunomarkers in Malignant Small Round Cell Tumors of Soft Tissue and Bone* Antigen Tumor
KER
EMA
DES
MYOG/MYF-4
SYN
S-100P
CD45
CD99
TLE1
A-RMS
30
0
>95
>95
20
E-RMS
<5
0
>95
>90
LD
<10
0
<10
<20
0
65
<10
0
>95
0
0
<5
0 (LD)
0
20
>90
0
80
0 (LD)
†
†
<10
0
10
<20
ES/PNET
20
0
<1
DSRCT
85
90
90
0
15
<5
PDSS
75
80
0
0
0
30
MCS
<5
0
>95
†
Lymphoma
0
Small cell carcinoma
>90
†
20
†
†
30
0
‡
0
0
0
0
0
70
<1
0
80
<1
>95
50
0
50
§
UNK 0 (LD)
*Data for this table were derived from the literature and from the authors’ experience. † Reactivity is usually focal. ‡ Reactivity in the cartilaginous component. § Typically present in lymphoblastic lymphoma. A-RMS, Alveolar rhabdomyosarcoma; DES, desmin; EMA, epithelial membrane antigen; E-RMS, embryonal rhabdomyosarcoma; ES/PNET, Ewing sarcoma/peripheral primitive neuroectodermal tumor; DSRCT, desmoplastic small round cell tumor; KER, keratin; LD, limited data; MCS, mesenchymal chondrosarcoma; MYOG, myogenin; PDSS, poorly differentiated synovial sarcoma; S-100P, S-100 protein; SYN, synaptophysin; UNK, unknown.
tumors of uncertain lineage are discussed in the final section on soft tissue tumors. Although hematopoietic neoplasms rarely present as soft tissue masses, consideration of such lesions is important in the evaluation of any soft tissue or bone lesion, particularly if the morphologic appearances are those of a round cell neoplasm. The most frequent hematopoietic lesions to involve soft tissue include diffuse large B-cell lymphoma, plasmablastic lymphoma, myeloid neoplasms (myeloid sarcoma), Burkitt lymphoma, and lymphoblastic lymphoma, the latter two tumors being relatively more common in children. In pediatric patients, other forms of small round cell tumors, such as neuroblastoma and Ewing sarcoma, are more common than lymphoma, but a basic workup should include this latter diagnostic possibility. Because of the virtually ubiquitous expression of CD45 (leukocyte common antigen) by hematopoietic cells and its extremely high degree of specificity, this marker is invaluable in this context. Not all antibodies raised against CD45 identify determinants that survive routine tissue processing, but the monoclonal antibody cocktail PD7/26+2B11 is active in paraffin sections. For practical purposes, reactivity for CD45 is diagnostic of a hematopoietic lineage; conversely, lymphomas and leukemias do not generally express markers of other lineages. It should be noted, however, that lymphoblastic lymphomas commonly label for CD99 (as seen in ES/PNET), thereby representing a potential pitfall in interpretation.320,321 Concurrent reactivity for terminal deoxynucleotidyl transferase is also observed in lymphoblastic lymphoma; it is generally, but not always,322 absent in other small round cell tumors. Lymphomas may rarely show a variety of unusual morphologic features that simulate those of sarcomas or carcinomas, such as one featuring
signet-ring cells,323 another with myxoid stroma,324 or the presence of a fibrillary matrix.325
Vascular Tumors BENIGN VASCULAR TUMORS Hemangioma/Lymphangioma
Benign vascular proliferations that include hemangiomas, lymphangiomas, and vascular malformations comprise a wide spectrum of clinical and pathologic entities. The role of IHC in the evaluation of such lesions is twofold: first, to highlight the architecture of the proliferation and therefore aid in evaluation of the biologic nature of the lesion (benign versus malignant), and second, to determine the presence of a lymphatic component to the lining endothelial cells. Some benign vascular lesions with dense cellularity and little overt canalization may also prove diagnostically troublesome. Cellular benign hemangiomas, for example, may mimic angiosarcoma, and epithelioid hemangioma can be confused with epithelioid angiosarcoma. Although to date there are no substantiated IHC methods to differentiate hemangiomas from histologically similar angiosarcomas, with regard to the former differential diagnosis, IHC with an endothelial marker and a marker of pericytes, such as SMA, is helpful to dissect the architecture of the lesion. Hemangiomas will show well-developed vessels with a uniform surrounding pericytic layer, in contrast to angiosarcoma, which has a more haphazard distribution of vessels and lacks the organized structure of benign vascular lesions. When evaluating an epithelioid vascular lesion, when the diagnosis of epithelioid sarcoma is under consideration, a panel of immunostains can almost always resolve
75
MSS
75
10‡ 10
<5
<5
<5
10-20
DDLPS
SCAS
0
0
<1
>95
MMEL
MCA
40 (LD)
<5
0
<1
<5
0
0
20-30*
<10
<5
10
10
10
20
80‡
<5 <1
10
0
0
0
0
0
0
0
0
0
0
0
<5
<5
>95
<5
0
0
<5‡
10‡
0
30
50
10
0
<1 85
S-100P
CALD
90
25
SMA
<5
15
5-60
60
10-80
§
66 (LD)
0 (LD)
>90
0 (LD)
‡
65
5
0
MDM2
UNK
UNK
10-80
§
0 (LD)
0 (LD)
>90
0 (LD)
UNK
0
10
2
0
CDK4
Variable
0
UNK
UNK
0
UNK
0
100
30
‡
0
0
UNK
MUC4
0
0
0
10
90
50
30
0
0
LD
0
0
0
>90 0
0
10
0
0 (LD)
80
15-30
5-20
20
TLE1
80
0
0
<5 0
0
0
0
0
CD31
0
10
30
UNK
CD34
50 (prostatic adenocarcinoma)
0
0
>95
>95
0
0
0
0
0
0
0
ERG
Data were derived from the literature and from the authors’ experience. *May suggest rhabdomyoblastic differentiation. ‡ Reactivity is usually focal. § High frequency of MDM2 and CDK4 overexpression in parosteal and central low-grade osteosarcomas; low frequency in conventional high-grade osteosarcomas CALD, h-caldesmon; DDLPS, dedifferentiated liposarcoma; DES, desmin; EMA, epithelial membrane antigen; FOS, fibroblastic osteosarcoma; KER, keratin; KS, Kaposi sarcoma; LD, limited data; LGFMS, low-grade fibromyxoid sarcoma; LMS, leiomyosarcoma; MCA, metastatic carcinoma; MMEL, metastatic malignant melanoma; MPNST, malignant peripheral nerve sheath tumor; MSS, monophasic spindle cell synovial sarcoma; S-100P, S-100 protein; SCAS, spindle cell angiosarcoma; SC-RMS, spindle cell rhabdomyosarcoma; SMA, smooth muscle actin (α-isoform); UNK, unknown.
80 (variable)
0
<5
<5
FOS 0 (LD)
0
0
0
KS
70
70
<5
0
40-60
0
<5 10-20*
95
10 ‡
DES
‡
β-catenin
<1
90
‡
25
40
0
EMA
Fibromatosis
LGFMS
10
MPNST
‡
40
0
SC-RMS
LMS
KER
Tumor
TABLE 4-3 Percentages of Positivity for Pertinent Immunomarkers in Malignant and Locally Aggressive Spindle Cell Tumors of Soft Tissue and Bone
92 Immunohistology of Neoplasms of Soft Tissue and Bone
20
10
0
30
>50
<5
<1
<1
30
0
0
EAS
EMPNST
CCS
SEF
ELMS
ASPS
PEComa
60
>95
Variable
0
0
0
0
0
80
0
UNK
<5
<5
<10 0
50
†
90
0
85
0
0
<5
0
45
33
SMA
†
<5
70
10
75
0
0
<5
0
0
<5
DES
0
0
0
60
0
75
0
0
0
0
0
0
CALD
10
>95
<5 †
0
80
0
90
0
<5 10
0
0
>90
0
0
0
0
HMB-45
10
30
>90
>95
<1
0
0
S-100P
Positive in adrenal cortical carcinomas (A103 clone)
70
0
60
0
0
0
80
0
0
0
0
MART-1 MELAN A
0 <1
<1
10
0
Positive in 50% of prostatic adenocarcinoma
0
0
0
0
0
15 0
UNK
0 (LD)
0
>95
>95
0
ERG
<1
<5
<10
50
50
50
CD34
0
0
0
0
0
0
0
0
90
>90
<1
CD31
<1
0
1
UNK
UNK
LD
UNK
0
50
0
0
95
SMARCB1 (INI1) Loss
Data were derived from the literature and from the authors’ experience. † Reactivity is usually focal. ASPS, Alveolar soft part sarcoma; CALD, h-caldesmon; CCS, clear cell sarcoma; DES, desmin; EAS, epithelioid angiosarcoma; EHE, epithelioid hemangioendothelioma; ELMS, epithelioid leiomyosarcoma; EMA, epithelial membrane antigen; EMPNST, epithelioid malignant peripheral nerve sheath tumor; EPS, epithelioid sarcoma; LD, limited data; KER, keratin; MCA, metastatic carcinoma; MMEL, metastatic malignant melanoma; OS, osteosarcoma; PEComa, perivascular epithelial cell tumor; SEF, sclerosing epithelioid fibrosarcoma; S-100P, S-100 protein; SMA, smooth muscle actin (α-isoform); UNK, unknown.
MCA
<5
<5
MMEL
†
20
†
20
OS
†
†
0
0
30
0
<5
30
EHE 0
0
>95
>95
EPS
MUC4
EMA
KER
Antigen Tumor
TABLE 4-4 Percentages of Positivity for Pertinent Immunomarkers in Malignant Epithelioid Tumors of Soft Tissue and Bone
Specific Soft Tissue and Bone Tumors
93
94
Immunohistology of Neoplasms of Soft Tissue and Bone
this differential diagnosis. The absence of podoplanin, FLI1, CD31, and ERG expression is typical of epithelioid sarcoma. Epithelial markers, which are characteristically positive in epithelioid sarcoma, are also typically absent in benign vascular proliferations, but reactivity for keratins is relatively common in angiosarcomas, especially in epithelioid examples. Expression of SMARCB1 is lost in the majority of epithelioid sarcomas, but expression is consistently retained in vascular tumors of all types.290 The lymphatic origin of lymphangioma and lymphangiomatosis can be documented with the use of the lymphatic markers podoplanin (D2-40) and VEGFR3. Lymphatic differentiation in other vascular lesions— such as hobnail hemangioma, papillary intralymphatic angioendothelioma (Dabska tumor), kaposiform hemangioendothelioma, and Kaposi sarcoma—can also be detected with these immunomarkers.207,221 However, neither marker is entirely specific for lymphatic endothelia.207,209-214 One study of vascular lesions found intense endothelial GLUT-1 reactivity in 97% (139 of 143) of juvenile capillary hemangiomas, and absence of expression was found in 66 vascular malformations.326 However, GLUT-1 is not a specific determinant; increased expression of GLUT-1 has been reported in a variety of solid human tumors.194,327-329 Recent studies have documented superior sensitivity and specificity of nuclear ERG expression for vascular endothelium over FLI1, CD34, and CD31. In our experience, ERG is an extremely useful marker of endothelium, but in benign vascular lesions, CD31, and sometimes CD34, often results in better delineation of architecture than ERG. KEY DIAGNOSTIC POINTS Vascular Tumors • Absence of podoplanin, FLI1, CD31, and ERG expression is typical of epithelioid sarcoma. • Reactivity for keratins is relatively common in angiosarcomas. • Recent studies have documented superior sensitivity and specificity of nuclear ERG expression for vascular endothelium over FLI1, CD31, and CD34.
LOCALLY AGGRESSIVE AND RARELY METASTASIZING VASCULAR TUMORS Kaposiform Hemangioendothelioma
Kaposiform hemangioendothelioma (KHE) was first thought to arise exclusively in children and infants330 but is now recognized as an unusual vascular tumor that may also affect adults.331 Many cases are associated with lymphangiomatosis and Kasabach-Merritt syndrome, a consumption coagulopathy syndrome; in fact, the latter condition accounts for a great deal of the morbidity caused by KHE,332 which may arise in superficial or deep soft tissues of the extremities and in the retroperitoneum. These tumors show a sheetlike to micronodular growth pattern that consists of spindle-shaped endothelial cells with slitlike vessels, reminiscent of
Kaposi sarcoma or spindle cell hemangioma. Hemosiderin deposition and hyaline droplets may also be seen. KHE has the potential for locally aggressive behavior, but distant metastases have not been described. The tumor cells of KHE show expression of various endothelial markers. ERG, CD34, podoplanin, and FLI1 are generally detected, but GLUT-1, factor VIII–related antigen, and UEAI are usually absent.191,330,333 Neither human herpesvirus 8 (HHV-8)–related nucleic acid or proteins have been found in KHE.332 VEGFR3 has been identified in this tumor, in common with other endothelial proliferations.221 Pseudomyogenic Hemangioendothelioma
Pseudomyogenic (epithelioid sarcoma–like) hemangioendothelioma is a recently described distinctive soft tissue tumor characterized by multifocality in different tissue planes of a limb.334-336 Local recurrence is relatively frequent, but distant metastasis is uncommon. Although this tumor type has some similarities to epithelioid sarcoma in that it also arises in skin and soft tissue of the distal extremities, affects mostly young adults, and shows diffuse keratin positivity (AE1/AE3), it differs by consisting predominantly of myoidappearing plump spindle cells with brightly eosinophilic cytoplasm that also express ERG and FLI1 in 100%, CD31 in 50%, show intact expression of SMARCB1, and are usually negative for EMA, CD34, and pancyto keratin MNF116 (Fig. 4-1). Tumor cells also lack expression of desmin and myogenin. A translocation at t(7;19)(q22;q13) has been identified in a subset of cases of pseudomyogenic (epithelioid sarcoma–like) hemangioendothelioma.337 MALIGNANT VASCULAR TUMORS Epithelioid Hemangioendothelioma
Epithelioid hemangioendothelioma (EHE) is a distinctive malignant vascular neoplasm with somewhat less aggressive behavior than angiosarcoma. EHE may arise in superficial or deep somatic soft tissues and in liver, lung, and bone. In some cases the tumor is centered on a vein. EHE is composed of strands, nests, and cords of epithelioid cells with eosinophilic cytoplasm and intracytoplasmic vacuoles embedded in a myxohyaline stroma (Fig. 4-2). Recent studies have identified a WWTR1-CAMTA1 gene fusion that results from the (1;3)(p36;q25) translocation in nearly all EHE cases examined, but this was not found in epithelioid hemangioma, epithelioid angiosarcoma, or epithelioid sarcoma–like (pseudomyogenic) hemangioendothelioma.338,339 Identification of endothelial differentiation in EHE can be achieved with various vascular markers such as vWF (granular cytoplasmic), CD31, CD34 (approximately 50%), and FLI1.340 Nuclear expression of ERG is observed in 98% of EHE cases,191 and podoplanin (D2-40) expression is absent in most examples of EHE.207 The intracellular vacuoles that typify EHE may mimic mucin-containing vacuoles of adenocarcinoma. Several other sarcomas with an epithelioid appearance
Specific Soft Tissue and Bone Tumors
A
C
may likewise be considered in the differential diagnosis with EHE, but perhaps the most troublesome mimic of EHE, particularly when located on the extremities, is epithelioid sarcoma. Both epithelioid sarcoma and EHE show a nodular growth pattern and are composed of plump, eosinophilic tumor cells arranged in cords and nests. Loss of nuclear labeling for SMARCB1 is highly specific for epithelioid sarcoma and is therefore an extremely useful marker in this differential diagnosis, because all evaluated cases of EHE have retained nuclear staining for this ubiquitously expressed component of the SWI/SNF chromatin remodeling complex.341 Reactivity for ERG and CD31 also favors a diagnosis of EHE over epithelioid sarcoma. Importantly, keratin expression is frequently detected in EHE.340 Immunolabeling for keratin is therefore of limited use in this differential diagnosis, given that expression will also be found in epithelioid sarcoma and adenocarcinoma. Furthermore, CD34 expression is found in around 50% of epithelioid sarcomas and therefore will not distinguish between these two tumor types.168,169 A panel of markers including endothelial and epithelial markers, as well as SMARCB1 (when relevant), are thus often used in the immunohistologic evaluation of EHE.
95
B
Figure 4-1 Pseudomyogenic (epithelioid sarcoma–like) hemangioendothelioma is composed of plump spindle cells with eosinophilic cytoplasm (A). Tumor cells express keratin AE1/AE3 (B) and endothelial transcription factor ERG (C).
Angiosarcoma
Angiosarcoma is an aggressive malignant vascular tumor that arises in many different clinical settings, which often reflect the underlying pathogenetic mechanism of tumor formation. The most common form of angiosarcoma is cutaneous angiosarcoma, which arises in sundamaged skin of the elderly, usually in the head and neck region. Angiosarcoma also occurs in the setting of radiation therapy, chronic lymphedema, after exposure to certain chemicals (e.g., hepatic angiosarcoma that occurs secondary to vinyl chloride exposure), and rarely as a secondary component of other tumors (e.g., germ cell tumors). Other clinical subtypes include angiosarcoma of deep soft tissue or other viscera, such as primary mammary angiosarcoma and cardiac angiosarcoma, in which the underlying etiology is not yet known. In general the morphologic appearances of angiosarcoma that arise in these settings fall within a spectrum that ranges from well-differentiated angiosarcoma, composed of complex anastomosing vascular spaces lined by single or multiple layers of atypical endothelial cells, to less differentiated forms that consist of predominantly spindle-shaped or epithelioid tumor cells.
96
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
C
Figure 4-2 Epithelioid hemangioendothelioma (A) can be identified by expression of endothelial markers CD34 (B), CD31 (C), and ERG (not shown).
Regardless of the degree of differentiation or morphologic grade, angiosarcoma is a high-grade tumor biologically and carries a uniformly poor prognosis. When the predominant pattern is spindled or epithelioid, other entities may enter the differential diagnosis with angiosarcoma, in which case IHC is extremely useful in identifying endothelial differentiation. ERG, a member of the ETS family of transcription factors, shows nuclear positivity in nearly 100% of angiosarcomas (Fig. 4-3). In two studies, all cutaneous angiosarcomas expressed ERG regardless of the degree of morphologic differentiation.189,191 The majority (90%) of deep-seated angiosarcomas also express ERG.191 CD31 expression is found in more than 90% of angiosarcomas and shows a membranous pattern of staining,178 whereas CD34 is less consistently expressed in angiosarcoma and is found in only 50% of cases.165,166 FLi1 expression is found in as many as 100% of angiosarcomas, but its utility is limited by its poor specificity for vascular lesions, given that it is also found in various carcinomas and other soft tissue tumors, many of which are included in the differential diagnosis with angiosarcoma. VEGFR3 expression is found in approximately 50% of angiosarcomas,221 and 30% of angiosarcomas express thrombomodulin.203
Expression of cytokeratins in angiosarcoma is not uncommon, particularly in epithelioid variants. CK18 is the most frequently found keratin in angiosarcoma, and it may be detected with broad-spectrum keratin markers.342 Limited reactivity for EMA is found in as many as 20% of angiosarcomas.343 Podoplanin/D2-40 expression, usually considered a marker of lymphatic differentiation, is found in a subset of angiosarcomas.207 Expression of KIT is found in 25% to 50% of angiosarcomas,344,345 but KIT mutations have not been identified. As mentioned above, histologic variants of angiosarcoma include those with a predominantly spindleshaped or epithelioid tumor cell morphology, so-called spindle cell angiosarcoma and epithelioid angiosarcoma, respectively. Angiosarcomas with spindle cell morphology have a similar immunophenotype to conventional angiosarcoma, although the extent of labeling with CD31 and CD34 may be limited. The high sensitivity of ERG for endothelial differentiation allows for recognition of this morphologic variant in almost all cases. Epithelioid angiosarcoma often involves the deep soft tissues, which suggests a differential diagnosis of malignant melanoma, poorly differentiated carcinoma, and other sarcomas with epithelioid morphology.
Specific Soft Tissue and Bone Tumors
A
97
B Figure 4-3 Angiosarcomas with spindle cell morphology (A) are nearly always positive for the endothelial marker ERG (B).
Immunoreactivity patterns vary somewhat in epithelioid angiosarcoma compared with classic forms. In particular, keratin reactivity is much more frequent in epithelioid angiosarcoma than in conventional angiosarcoma. However, CD31 and ERG, both sensitive and specific markers of endothelial differentiation, will usually allow for discrimination from carcinoma.191 Podoplanin is also seen in a subset of cases, as is WT1 (with a cytoplasmic staining pattern).346 The potential presence of CD34 in various epithelioid soft tissue tumors, including epithelioid LMS and epithelioid sarcoma, has diminished its potential utility as an indicator of endothelial differentiation in this context. Radiation-associated angiosarcoma, particularly cutaneous angiosarcoma occurring in the breast after radiation therapy, shows amplification of MYC by FISH, with corresponding overexpression at the protein level, which can be detected immunohistochemically. Strong
KEY DIAGNOSTIC POINTS Angiosarcomas • ERG shows nuclear positivity in nearly 100% of angiosarcomas. • Podoplanin/D2-40 expression is found in a subset of angiosarcomas. • FLI1 expression is found in as many as 100% of angiosarcomas, but utility is limited by poor specificity for vascular lesions. • Expression of cytokeratins in angiosarcoma is not uncommon, particularly in epithelioid variants, and limited epithelial membrane antigen reactivity is seen in as many as 20% of cases. • Strong nuclear staining for MYC is a consistent finding in postradiation cutaneous angiosarcoma, whereas postradiation atypical vascular proliferations and angiosarcomas not related to radiation therapy are negative for MYC.
nuclear staining for MYC is a consistent finding in postradiation cutaneous angiosarcoma, whereas postradiation atypical vascular proliferations and angiosarcomas not related to radiation therapy are negative for MYC.347,348 This finding has potential utility in mapping the extent of vascular lesions in surgical resections and has also gleaned some insight into the pathogenesis of radiation-associated angiosarcoma. Kaposi Sarcoma
Kaposi sarcoma (KS) occurs in four clinical forms— classic (Mediterranean), lymphadenopathic/endemic, transplantation-associated, and acquired immune deficiency syndrome (AIDS)–related—but its microscopic features are similar in each of these settings. In its fully developed state, KS may be confused morphologically with spindle cell angiosarcoma and kaposiform hemangioendothelioma. The tumor cells in all of these lesions are generally positive for ERG, CD31, CD34, and thrombomodulin, but only KS manifests nuclear reactivity for human herpesvirus 8 (HHV-8) latent nuclear antigen-1 (Fig. 4-4).349,350 Podoplanin (D2-40) positivity is also consistently found in KS, including those cases with a lymphangioma-like morphologic appearance.207,351
Skeletal Muscle Tumors BENIGN SKELETAL MUSCLE TUMORS Rhabdomyoma
Rhabdomyoma is a benign tumor that shows skeletal muscle differentiation. Rhabdomyomas are classically divided into those that occur in the heart and those occur outside the heart. Cardiac rhabdomyomas are frequently associated with tuberous sclerosis. Noncardiac rhabdomyomas are subdivided into adult, fetal, and the rare genital variant. Adult rhabdomyoma most commonly arises in the head and neck region of adults and is composed of mature rhabdomyoblasts with abundant,
98
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
Figure 4-4 Tumor cells of nodular Kaposi sarcoma are characteristically spindle shaped (A) and show diffuse expression of human herpesvirus 8 (B).
brightly eosinophilic cytoplasm and central or peripheral small nuclei.352 Cross-striations and rodlike inclusions are seen focally within the cytoplasm. Fetal rhabdomyoma consists of small, primitive spindle cells with delicate myotubules and more mature appearing straplike rhabdomyoblasts. The relative proportion of each cell type varies among tumors.353 Similar to the adult type, fetal rhabdomyoma shows a predilection for the head and neck region but occurs more commonly in young children. Genital rhabdomyomas usually occur in the female genital tract and have morphologic features similar to fetal rhabdomyoma.354 The differential diagnosis of rhabdomyoma may include granular cell tumor, hibernoma, paraganglioma, PEComa, histiocytic tumors, and RMS. All forms of rhabdomyoma stain for desmin, musclespecific actin, and myoglobin. SMA, vimentin, S-100 protein, GFAP, CD56, CD57, CD68, cytokeratin, and EMA are generally absent, although rare SMA-reactive cells are occasionally identified.352-354 S-100 protein has been described in some cases of rhabdomyoma, but if present, staining is usually very focal353 and therefore is not likely to be confused with the pattern of S-100 protein reactivity in paraganglioma (sustentacular cells) or granular cell tumor. The presence of myogenic markers and lack of CD68 expression is useful in the differential diagnosis with histiocytic tumors. As with vascular tumors, IHC distinction between benign skeletal muscle tumors and their malignant counterparts is impossible. In most cases, standard clinicopathologic evaluation is sufficient for their unequivocal separation. Rhabdomyosarcoma
Embryonal Rhabdomyosarcoma. Embryonal rhabdomyosarcoma (E-RMS) accounts for more than half of all RMS, but recognition of this tumor can be difficult. The most commonly involved sites include the head and neck region and genitourinary tract, and children are typically affected. The morphologic features of E-RMS vary widely, depending on the degree of
differentiation, cellularity, stromal quality and quantity, and pattern of growth. The tumor cells of E-RMS may vary considerably in their degree of differentiation.355 The spectrum includes nondistinctive round cells with scant cytoplasm, primitive stellate cells with pale cytoplasm, strap cells, and large eosinophilic myoblasts, which may be present focally in E-RMS but which are not seen in most of its histologic mimics. Botyroid RMS consists of linearly arranged tumor cells closely associated with an epithelial surface. A significant number of E-RMSs consist only of densely apposed, undifferentiated, small spindled or ovoid cells (Fig. 4-5) that evoke a broad differential diagnosis that includes neuroblastoma, Ewing sarcoma, poorly differentiated synovial sarcoma, melanoma, and lymphoma. Furthermore, E-RMS may be confused with the solid variant of alveolar RMS (Fig. 4-6), which has a significantly worse prognosis. In adult patients, small cell carcinoma and poorly differentiated angiosarcoma may also be considerations. The most frequent cytogenetic finding in E-RMS is allelic losses in chromosomal region 11p15, and it is thought that loss of a tumor suppressor gene in this region may contribute to tumorigenesis.356 Other complex structural changes and chromosomal gains and losses are frequently found in E-RMS,357 and prognosis in general depends on age, site, stage, and histologic classification.358 It is with the poorly differentiated variants of E-RMS that IHC analysis proves to be most helpful. RMS expresses striated muscle markers in a cumulative and consistent sequence that recapitulates the pattern of normal myogenesis (myogenin/MYF4, MYOD1, desmin, fast myosin, and myoglobin), as well as that of muscle-specific actin (HHF-35), which is also found in other myogenic tumors. Among myogenic markers, myogenin/MYF4 and desmin are the most consistently detectable in paraffin sections, showing appreciable staining in virtually all cases of E-RMS and in alveolar and pleomorphic subtypes of RMS (see Fig. 4-5).68 The extent of staining with skeletal muscle
Specific Soft Tissue and Bone Tumors
A
C
markers correlates with the degree of differentiation, and therefore some of the most primitive cells may be desmin negative. The extent of staining with myogenin/ MYF-4 is also helpful in subclassification, because the alveolar variant of RMS usually shows extensive nuclear labeling in nearly all tumor cells, whereas E-RMS shows more heterogeneous reactivity (see Figs. 4-5 and 4-6). Other small round cell tumors lack expression of striated muscle markers, with the notable exception of DSRCT, which is usually positive for desmin but negative for myogenin/MYF4. MYOD1 is a highly specific and sensitive marker for RMS, showing nuclear expression in approximately 90% of cases.123,359 MYOD1 is a DNA-binding nuclear regulatory protein that initiates myogenesis in mesenchymal stem cells. The staining pattern of MYOD1 is heterogeneous among tumor cells of E-RMS: nuclear labeling is most intense in small, primitive tumor cells, whereas larger cells with more obvious skeletal muscle differentiation are generally nonreactive. On occasion, MYOD1 cross-reacts with unknown cytoplasmic antigens in some small round cell tumors, showing variable fibrillary immunoreactivity in neuroblastoma and Ewing
99
B
Figure 4-5 Primitive round to short spindled cells of embryonal rhabdomyosarcoma with occasional larger eosinophilic rhabdomyoblasts (A). Tumor cells show diffuse expression of desmin (B) but more limited nuclear reactivity for myogenin (C).
sarcoma.359,360 Among other muscle-related determinants, myogenin/MYF4 is probably the one most frequently detected in E-RMS, and it is also highly specific for true rhabdomyoblastic differentiation. Nuclear staining for myogenin is stronger than that seen with anti-MYOD1. Ewing sarcoma lacks myogenin/MYF4 reactivity123 and may be excluded if myogenin expression is present in tumor cells. SMA staining occurs in approximately 10% of cases of E-RMS.119 Occasional aberrant staining for cytokeratins, S-100 protein, and neurofilaments has also been reported.361 Therapy often causes cytodifferentiation to occur in RMS and decreases mitotic activity.362 In addition, unchanged or increased posttherapeutic proliferative activity, as assessed by Ki-67 immunostaining, has been equated with aggressive biologic potential in E-RMS. Myogenous marker expression has not been found to change after therapy, other than detecting greater numbers of fast myosin–positive differentiated rhabdomyoblasts, correlating with the presence of more abundant, brightly eosinophilic cytoplasm. Reactivity for neuroectodermal markers such as CD56, CD57, and synaptophysin is not uncommon in treated RMS.
100
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
C
Figure 4-6 Uniform round cells of alveolar rhabdomyosarcoma (A). Tumor cells show diffuse expression of desmin (B) and extensive staining for myogenin (C).
Alveolar Rhabdomyosarcoma. Alveolar rhabdomyosarcoma (A-RMS) is a primitive round cell malignant neoplasm that shows partial skeletal muscle differentiation. A-RMS most often arises in the extremities of young adults. Morphologic patterns include dyshesive growth within a fibrovascular stroma, which imparts an alveolar appearance; a solid variant lacks a fibrovascular stroma and instead forms sheets of tumor cells (see Fig. 4-6).355 This tumor is characterized by the recurrent translocations t(2;13)(q35;q14) and less commonly t(1;13) (q36;q14), which involve the FOXO1A gene on chromosome 13 with either PAX3 on chromosome 2 or PAX7 on chromosome 1.363-365 The resultant fusion proteins function as transcriptional activators. The most common morphologic differential diagnosis for A-RMS is with lymphoma, but any small round blue cell tumor might be considered in the differential diagnosis. Similar to E-RMS, A-RMS stains with myogenic markers and with the specific markers of skeletal muscle differentiation, myogenin/MYF4 and MYOD1, both of which show more extensive staining in this type of RMS than in E-RMS (see Fig. 4-6). Expression of cytokeratins and synaptophysin is relatively common and may lead to misdiagnosis as neuroendocrine carcinoma.21 Evaluation of FOXO1A gene rearrangement by FISH or identification of the fusion transcripts by reverse
transcription polymerase chain reaction (RT-PCR) may be warranted as confirmatory studies in some cases. Pleomorphic Rhabdomyosarcoma. Among the various forms of RMS, pleomorphic rhabdomyosarcoma (P-RMS) is the least common type and is seen almost exclusively in the extremities of older adults. Morphologically, tumors consist of undifferentiated round, spindled, and pleomorphic cells with brightly eosinophilic cytoplasm.366 IHC is usually necessary to confirm the diagnosis. Tumor cells show uniform positivity for desmin, muscle-specific or sarcomeric actins, myosin, or myoglobin.367 These proteins can be detected in both characteristic large strap cells and in spindle cell or epithelioid foci. Myogenin/MYF4 is typically positive in scattered cells only, unlike in pediatric RMS, which generally shows more extensive reactivity for this marker. MYOD1 also tends to show more limited staining in P-RMS than that seen in pediatric RMS. P-RMS lacks S-100 protein, CD56, CD57, and myelin basic protein expression and shows a complex karyotype without recurrent translocations.368 Spindle Cell Rhabdomyosarcoma. Spindle cell rhabdomyosarcoma (S-RMS) most commonly arises in the paratesticular region of children but may occasionally
Specific Soft Tissue and Bone Tumors
A
C
occur in adults with a wider anatomic distribution. S-RMS consists of fascicles of spindle cells, which may mimic LMS or other fascicular spindle cell tumors but which often show cytoplasmic cross-striations, at least focally, and evidence of rhabdomyoblastic differentiation immunohistochemically (Fig. 4-7).369 Mentzel and colleagues369 reported variable positivity for desmin and myogenin in all tumors evaluated, whereas fast myosin was positive in only 28% of cases; 71% of tumors showed focal positivity for α-SMA. In contrast, h-caldesmon, S-100 protein, CD34, pancytokeratin, and EMA were all negative. This particular subtype of RMS, when occurring in children, tends to have a better prognosis than other subtypes, but in adults, the clinical course is often aggressive. Sclerosing Rhabdomyosarcoma. This variant is a relatively recently described type of RMS composed of spindle-shaped or more rounded cells with rhabdomyoblastic differentiation embedded within a densely sclerotic stroma. Tumor cells show strong expression of MYOD1 and variable expression of myogenin/MYF4 and desmin.370,371 Recurrrent chromosomal translocations have not been identified in this subtype to date. Because some RMS shows areas with both spindle cell
101
B
Figure 4-7 Spindle cell rhabdomyosarcoma may mimic other fascicular spindle cell sarcomas (A), but diffuse reactivity for desmin (B) and variable myogenin expression (C) aids in differential diagnosis.
and sclerosing features, it is believed that these variants are likely related.336 Epithelioid Rhabdomyosarcoma. Epithelioid RMS is a morphologically distinct variant of RMS with prominent epithelioid cytomorphology that closely mimics carcinoma or melanoma. It primarily affects older patients and shows a male predilection.372 The clinical course is typically aggressive. In the original series of 16 cases, all tumors examined showed diffuse desmin expression and diffuse or multifocal reactivity for myogenin/MYF4.372 Cytokeratin was very focally positive in four cases, and S-100 protein was absent in all cases.
KEY DIAGNOSTIC POINTS Rhabdomyosarcoma • Striated muscle differentiation markers are expressed in a consistent sequence: myogenin/MYOD1, desmin, fast myosin, and myoglobin. • Alveolar rhabdomyosarcoma shows more extensive reactivity for myogenin than embryonal rhabdomyosarcoma.
102
Immunohistology of Neoplasms of Soft Tissue and Bone
Fibroblastic Tumors FIBROMATOSES
Several forms of fibromatosis are currently recognized that include desmoid, plantar, palmar, and penile. Other rare forms include hyaline, gingival, and digital fibromatoses. Desmoid fibromatosis may arise in the abdominal wall, abdominal cavity, or at extraabdominal sites. The morphologic appearance of fibromatosis is relatively uniform and consists of long, sweeping fascicles of spindled fibroblastic/myofibroblastic cells in a variably collagenous stroma. The lesional cells have indistinct cytoplasm, fine nuclear chromatin, and small nucleoli (Fig. 4-8). There is at most minimal atypia, and mitotic activity is usually low. Mesenteric desmoid tumors often have a fasciitislike appearance. Approximately 15% of desmoid fibromatosis, particularly at intraabdominal locations, arises in the setting of FAP syndrome.373 Fibromatosis may appear histologically similar to leiomyoma, GIST, and PNST. Approximately 70% of cases of desmoid fibromatosis show nuclear expression of β-catenin (see Fig. 4-8). Nuclear localization of β-catenin, which is encoded by a gene at chromosomal locus 3p21 (CTNNB1), reflects a mutation in that moiety or in the APC gene on chromosome 5q that regulates it in an upstream fashion.246 Nuclear β-catenin, which can be recognized by the 14/β-catenin monoclonal antibody clone, functions as a transcriptional activator when complexed with members of the lymphocyte enhancer factor family of proteins.246 An absence of nuclear β-catenin staining does not preclude the diagnosis of fibromatosis, however, because as many as 30% of desmoid tumors lack this pattern of expression.252 Similarly, nuclear β-catenin is not specific to desmoid fibromatosis. Potential histologic mimics such as LGFMS, myxofibrosarcoma, solitary fibrous tumor, myofibroma, nodular fasciitis, and hypertrophic scars demonstrate this staining pattern in as many as 25% of cases.252,253,374
A
Desmoid fibromatosis usually shows patchy SMA expression, may show focal S-100 protein expression, and is rarely reactive for desmin. Expression of calretinin has recently been demonstrated in 75% of desmoid fibromatosis, but WT1 and cytokeratins are consistently negative.375 It is important to be aware of this finding because of the potential diagnostic confusion with sarcomatoid or desmoplastic mesothelioma. CD34 and BCL2 are usually negative in desmoid fibromatosis, which can help in the distinction from solitary fibrous tumor. More extensive reactivity for S-100 protein would favor a PNST (neurofibroma, schwannoma) over a fibroblastic or myofibroblastic lesion. In the differential diagnosis with LGFMS, MUC4 is the most helpful stain, because desmoid fibromatosis is consistently negative for this marker.310 Fibroma of tendon sheath and collagenous fibroma (desmoplastic fibroblastoma) are usually sufficiently distinctive clinically and histologically to separate them from fibromatosis. They often react either diffusely or focally for SMA. Desmin, however, is typically negative.376,377 In addition to sharing a similar immunophenotypic profile, fibroma of tendon sheath and collagenous fibroma have been observed to harbor the same abnormality of chromosome 11q12.378 Gardner fibroma, a benign fibroblastic tumor that typically arises in children, shows nuclear β-catenin expression in as many as 64% of cases.255 Most patients with Gardner fibroma are found to have FAP syndrome, and approximately 45% will subsequently develop desmoid fibromatosis. ANGIOMYOFIBROBLASTOMA
Angiomyofibroblastoma is a distinctive lesion of the superficial soft tissues that shows a marked predilection for the vulvar region. This tumor shows strong diffuse reactivity for desmin and focal reactivity for actin and estrogen receptor protein.85,379 Desmin reactivity may be weaker (or occasionally negative) in postmenopausal
B
Figure 4-8 Desmoid fibromatosis, with characteristic long fascicles of bland fibroblastic/myofibroblastic spindle cells (A), which show nuclear expression of β-catenin in as many as 70% of cases (B).
Specific Soft Tissue and Bone Tumors
patients. Although the clinical features of angiomyofibroblastoma may overlap considerably with those of deep (“aggressive”) angiomyxoma, morphologic differences are usually sufficient for their separation. Angiomyofibroblastomas are small, well-circumscribed lesions, contrasting with the more infiltrative and deeply seated deep angiomyxoma. The perivascular accentuation of stromal cells typical of angiomyofibroblastoma is not found in deep angiomyxoma. Desmin reactivity may be seen in both angiomyofibroblastoma and deep angiomyxoma, so IHC is generally not helpful in this distinction, and the diagnosis ultimately rests on morphology. The separation of such lesions is important, because deep angiomyxoma has a significant potential for recurrence that is not shared by angiomyofibroblastoma. Some studies have also elucidated a subset of angiomyofibroblastomas with CD34 expression and have shown consistent estrogen and progesterone receptor positivity.379,380 There is no staining for factor XIIIa, keratin, S-100 protein, CD57, GFAP, or CD68. These findings are shared with smooth muscle tumors but not with most other myxoid tumors, including myxoid liposarcoma, myxofibrosarcoma, myxoid neurofibroma, and myxoid MPNST.
A
103
KEY DIAGNOSTIC POINTS Angiomyofibroblastoma • Angiomyofibroblastoma is usually separated from deep (“aggressive”) angiomyxoma on morphologic grounds, because these tumor types have a similar immunoprofile that includes desmin expression.
SOLITARY FIBROUS TUMOR
Although it was originally described in 1931 as a pleural lesion,381 solitary fibrous tumor (SFT) was recognized increasingly in various extrapulmonary sites during the 1990s. Many reported cases of hemangiopericytoma likely represent SFTs that were previously unrecognized as such outside the thorax. SFT consists of a variably cellular proliferation of typically bland spindle cells with a haphazard growth pattern, variably collagenous stroma, and branching “staghorn” or hemangiopericytoma-like blood vessels (Fig. 4-9). Lipomatous differentiation is occasionally present in a form known as lipomatous or fat-forming SFT.382 Rare cases show increased cellularity or atypia. A diagnosis of
B
C Figure 4-9 Solitary fibrous tumor is composed of short spindle cells in a haphazard growth pattern within a collagenous stroma (A). Tumor cells express CD34 (B) and BCL2 (C).
104
Immunohistology of Neoplasms of Soft Tissue and Bone
malignant SFT is made when mitoses number four or more per 10 high power fields, which correlates well with a significant risk of metastasis. The main histologic differential diagnoses include synovial sarcoma, cellular angiofibroma, neurofibroma, and spindle cell lipoma. The neoplastic cells of SFT are strongly positive for CD34 and frequently show BCL2 reactivity (see Fig. 4-9), and focal positivity for SMA is occasionally seen. SFT is typically negative for cytokeratins, S-100 protein, desmin, and CD31 but often shows strong diffuse cytoplasmic CD99 reactivity.297,383,384 CD10 is present in approximately 65% of SFTs,385 and nuclear positivity for β-catenin has been reported in as many as 40%.386 A panel of immunostains may be necessary to exclude other spindle cell proliferations that can be confused with SFT. Neurofibromas may also be reactive for BCL2 and CD34, but they express S-100 protein, unlike SFT. BCL2 reactivity is also common in synovial sarcoma,297 but synovial sarcoma usually lacks CD34 expression and usually labels at least focally for epithelial antigens and diffusely for TLE1, the latter of which is uncommon in SFT and is usually weak in intensity when present.304 Spindle cell lipoma, another CD34-positive tumor, usually differs histologically from SFT but may occasionally show similar morphologic features to the latter lesion. Reactivity for S-100 protein is seen in adipocytic elements of spindle cell lipoma, which usually can be found even in cellular variants. Evaluating expression of Rb may be helpful, because loss of expression of this protein is found in spindle cell lipomas, whereas expression is retained in SFT.287 The myxoid variant of SFT may mimic other myxoid lesions such as LGFMS, myxoid liposarcoma, and myxoid MPNST. Myxoid liposarcoma and LGFMS are negative for CD34, and MUC4 is positive in LGFMS but is consistently negative in SFT.309 Labeling for S-100 protein, CD56, or CD57 may assist in the recognition of myxoid MPNST; however, negativity for those markers is observed in roughly 50% of cases and does not necessarily exclude the latter diagnosis.
A
No consistent chromosomal alterations have been identified in SFT,387 but losses in 13q are seen in a subset of cases.388 Overexpression of insulin-like growth factor 2 (IGF-2) is found in SFT and may play a role in producing tumor-related hypoglycemia.389 The IGF-2 peptide can be detected by IHC means in such cases, but this finding is not diagnostically discriminatory. LOW-GRADE FIBROMYXOID SARCOMA
Low-grade fibromyxoid sarcoma (LGFMS) is a fibroblastic neoplasm that usually occurs in the deep soft tissues of the proximal extremities or trunk of young adults.390 Despite its bland cytomorphology, LGFMS has a tendency for a protracted clinical course characterized by local recurrences and late, distant metastases.390-392 LGFMS harbors the oncogenic chimeric fusion gene FUSCREB3L2, or rarely FUS-CREB3L1, resulting from the translocations t(7;16)(q34;p11) or t(11;16)(p11;p11), respectively, which are present in virtually all cases.393-395 LGFMS is classically composed of alternating fibrous and myxoid areas with bland spindle or stellate cells in a whorled growth pattern (Fig. 4-10), but the morphologic spectrum of LGFMS is quite variable. Hyalinizing spindle cell tumor with giant rosettes is a variant of LGFMS with collagen pseudorosettes.396 Given its bland cytology and variable morphology, LGFMS has traditionally been difficult to distinguish from some benign mesenchymal tumors and other low-grade sarcomas. A recently identified novel marker for LGFMS, MUC4, has made evaluation of these differential diagnoses more straightforward. MUC4 overexpression in LGFMS was identified through gene-expression profiling of a series of myxoid and fibrous tumors.308 All cases in a large cohort of LGFMS, all with FUS gene rearrangement confirmed by FISH, showed cytoplasmic staining for MUC4, usually in a strong and diffuse manner (see Fig. 4-10).310 Apart from patchy staining in 30% of monophasic synovial sarcomas, all other tumor types examined were negative for MUC4, including
B
Figure 4-10 Low-grade fibromyxoid sarcoma consists of bland spindle or stellate cells in a whorled growth pattern (A). Diffuse strong expression of MUC4 is seen in nearly all cases (B).
Specific Soft Tissue and Bone Tumors
soft tissue perineurioma, myxofibrosarcoma, cellular myxoma, SFT, low-grade MPNST, desmoid fibromatosis, neurofibroma, schwannoma, DFSP, myxoid liposarcoma, and extraskeletal myxoid chondrosarcoma. EMA expression is also a frequent finding in LGFMS and is present in as many as 90% of cases.395 However, the utility of EMA as a diagnostic marker for LGFMS is relatively limited, because the extent of expression of EMA is often limited in LGFMS, and EMA positivity is also observed in tumors that may mimic LGFMS, such as soft tissue perineurioma and some SFTs. SCLEROSING EPITHELIOID FIBROSARCOMA
Sclerosing epithelioid fibrosarcoma (SEF) is a rare aggressive fibroblastic neoplasm that arises in deep soft tissue sites, where it is often intimately associated with fascial planes, periosteum, or skeletal muscle.397,398 SEF is composed of cords and nests of epithelioid cells with clear or eosinophilic cytoplasm, embedded within a densely collagenous and hyalinized stroma that may have an osteoidlike appearance (Fig. 4-11). Some cases of SEF show both morphologic and molecular overlap with LGFMS and contain FUS-CREB3L2 fusion transcripts or FUS gene rearrangements.62,399 Because of its epithelioid cytomorphology, SEF can be difficult to distinguish from some other epithelioid mesenchymal tumors and from metastatic melanoma and carcinoma. IHC was traditionally of limited value in the diagnosis of SEF, because the immunoprofile of SEF was notoriously nonspecific; IHC was primarily useful to exclude histologic mimics. Similar to LGFMS, focal expression of EMA is a frequent finding in these tumors, and it is detected in as many as 50% of cases. S-100 protein is reported to be present in 29% of cases, usually demonstrating a similarly focal pattern of immunoreactivity, whereas cytokeratin, neuron-specific enolase (NSE) CD45, HMB-45, CD68, and desmin are typically absent.397,398,400 Recently, strong diffuse cytoplasmic staining for MUC4 has been reported in 78% (32 of 41) of cases of
A
105
SEF, including 12 with hybrid LGFMS features (see Fig. 4-11).310 FUS rearrangement was found in only 38% of MUC4-positive cases of SEF, suggesting that FUS rearrangement is not the driving mechanism behind MUC4 overexpression. In that study, it was found that two cases of hybrid LGFMS-SEF had EWSR1 and CREB3L1 rearrangements, identifying EWSR1 as an alternate fusion partner to FUS for the first time in this class of tumors. MUC4-positive SEFs that lack FUS rearrangement may be related to LGFMS but could therefore have alternate fusion partners, including EWSR1. SEF without MUC4 expression may represent a distinct group of tumors. With regard to potential morphologic mimics, MUC4 expression was found in the glandular component of biphasic synovial sarcomas (90%). Focal staining, usually as only scattered cells, was also seen in a subset of ossifying fibromyxoid tumors (29%), epithelioid GISTs (20%), and myoepithelial carcinomas (10%), whereas all other epithelioid soft tissue tumors— including CCS, epithelioid sarcoma, EHE, PEComa and melanoma—were negative. It should be noted that various carcinomas may express MUC4, and therefore additional keratins or lineage-specific markers may be needed to exclude this possibility in some cases.401-403 MYXOINFLAMMATORY FIBROBLASTIC SARCOMA
Myxoinflammatory fibroblastic sarcoma (MIFS) occurs principally in the subcutaneous tissue of the distal extremities, mainly hands and wrists. MIFS tends to be slow growing and shows a propensity for repeated local recurrence, which may necessitate amputation; however, metastasis is rare.404 The majority of MIFS show the translocation t(1;10)(p22;q24), in addition to aberrations of chromosome 3.405 Histologically, MIFS is poorly demarcated, with a multinodular architecture and alternating myxoid and cellular areas. The tumor is composed of a polymorphous population of plump fibroblastic cells, a prominent inflammatory infiltrate, and a variable number of large atypical cells with vesicular nuclei and prominent viral inclusion–like nucleoli
B
Figure 4-11 Linearly arranged cords of tumor cells with an epithelioid appearance are characteristic of sclerosing epithelioid fibrosarcoma (A). Tumor cells show diffuse cytoplasmic reactivity for MUC4 in 80% of cases (B).
106
Immunohistology of Neoplasms of Soft Tissue and Bone
and multinucleate Reed-Sternberg–like cells. The IHC profile of MIFS is nonspecific. SMA positivity may be seen in the larger atypical cells, and variable CD34 positivity may also be evident. The atypical mononuclear cells are negative for CD68, but background staining of histiocytes may be seen. S-100 protein, HMB-45, EMA, and cytokeratins are negative in tumor cells.406,407 Myxoinflammatory fibroblastic sarcoma and hemosiderotic fibrolipomatous tumor (HFLT), a tumor that is most common around the ankle and consists of lobules of mature adipocytes separated by cellular fibrous septa containing cytologically bland spindle cells with eosinophilic cytoplasm and abundant hemosiderin deposition, likely fall on a morphologic spectrum defined by the translocation t(1;10)(p22;q24) and aberrations of chromosome 3, which are seen in the majority of MIFS cases and in tumors showing hybrid features of both MIFS and HFLT.405,407,408 ANGIOMATOID FIBROUS HISTIOCYTOMA
Angiomatoid fibrous histiocytoma (AFH) typically arises in the deep dermis or subcutis of the extremities of children and young adults. It recurs in approximately 10% of cases but has a low metastatic potential, with metastasis occurring in less than 2% of patients. AFH harbors the fusion genes EWSR1-CREB1 (most commonly), EWSR1-ATF1, or FUS-ATF1.409,410 Karyotypic analysis has demonstrated a (2;22)(q33;q12) translocation that corresponds to the EWSR1-CREB1 fusion product. Morphologically, AFH shows a multinodular pattern of growth, often with dilated, pseudoangiomatoid spaces. The tumor is composed of histiocytoid and/ or myoid cells, which may appear spindled or epithelioid. A fibrous pseudocapsule and/or peritumoral lymphoplasmacytic infiltrates may be present. Although it shows no specific line of differentiation, it seems to have a partial myoid or myofibroblastic phenotype. Expression of desmin is present in more than 50% of cases, whereas variable expression of HHF-35 and SMA has been reported in occasional cases.89,90 Approximately
A
40% of tumors show expression of EMA, and staining for CD99 is found in 50% of cases. Tumor cells are negative for S-100 protein, cytokeratins, myoglobin, HMB-45, CD21, CD35, CD34, and CD31.90,411 INFLAMMATORY MYOFIBROBLASTIC TUMOR
Inflammatory myofibroblastic tumor (IMT) is a mesenchymal neoplasm composed of myofibroblastic spindle cells in a myxoid or collagenous stroma with an inflammatory infiltrate composed chiefly of plasma cells and lymphocytes (Fig. 4-12). IMT most often arises in lung or abdominal soft tissues of children and young adults, although a wide anatomic distribution and age range have been documented.412 IMT is considered a neoplasm of intermediate biologic potential because of its tendency to recur locally, but metastasis is rare and occurs in less than 5% of cases.413 Immunohistochemically, the tumor cells of IMT variably express SMA (80% to 90%), HHF-35 and desmin (as much as 60%), and approximately one third of cases are positive, usually focally, for cytokeratins AE1/AE3 and CAM5.2.412,414 S-100 protein, myogenin/MYF4, MYOD1, and KIT are negative.414 MDM2 expression is common in IMT, and therefore care should be taken in the evaluation of this marker, because the differential diagnosis of IMT with inflammatory liposarcoma may occasionally be difficult.415 Approximately 50% of IMTs show clonal rearrangements of the ALK gene on chromosome 2 at band 2p23 with corresponding overexpression at the protein level that can be detected immunohistochemically (see Fig. 4-12).416 ALK is a receptor tyrosine kinase first identified as a component of the NPM-ALK fusion oncoprotein, which is aberrantly expressed in anaplastic large cell lymphomas (ALCLs) that harbor a (2;5) translocation.417 Other ALK partner genes have since been described in ALCL as a result of alternative chromosomal rearrangements, and similar to ALCL, a variety of gene partners can be fused to ALK in IMT as a result of various chromosomal rearrangements, including tropomyosin 3 (TPM3),
B
Figure 4-12 Inflammatory myofibroblastic tumor is composed of myofibroblastic spindle cells with an admixed inflammatory infiltrate composed of plasma cells and lymphocytes (A). Anaplastic lymphoma receptor tyrosine kinase expression is found in approximately 50% of cases (B).
Specific Soft Tissue and Bone Tumors
tropomyosin 4 (TPM4), CLTC, RANBP2, CARS, ATIC, and SEC31A (formerly SEC31L1). Expression of ALK fusion proteins in IMT can be detected by IHC, and the pattern of ALK staining seems to reflect the fusion partner260-262: diffuse cytoplasmic staining is seen when the fusion involves the cytoplasmic proteins TPM3, TPM4, CARS, ATIC, and SEC31A; granular cytoplasmic staining occurs when the fusion partner is CLTC, a main component of the coated vesicles involved in selective protein transport; and nuclear membrane staining is seen with RANBP2, a large protein found at the nuclear pores. A variant of IMT known as epithelioid inflammatory myofibroblastic sarcoma, is a distinctive intraabdominal sarcoma with a predilection for male patients that shows nuclear membrane or perinuclear ALK expression, usually the result of a RANBP2-ALK fusion that is commonly present in this variant.263 Unlike conventional IMT, abundant myxoid stroma and prominent neutrophils are commonly seen in this variant. These tumors pursue an aggressive course, with rapid local recurrences and frequent death as a result of the disease. In addition to ALK, tumor cells of this variant express desmin in approximately 90% of cases, SMA in 50%, and generally show weak CD30 expression. Myogenin, caldesmon, keratins, EMA, and S-100 protein are consistently negative. Expression of desmin and negativity for EMA is helpful in distinguishing this tumor type from ALCL.
107
stroma. Consistent with its purported fibroblastic/ myofibroblastic differentiation, infantile fibrosarcoma shows variable positivity for actins, whereas desmin, keratins, and EMA are typically negative.418 MYXOFIBROSARCOMA
Myxofibrosarcoma, a malignant fibroblastic neoplasm, is the most common sarcoma of older adults and most often arises in subcutaneous tissue of the limbs and limb girdles. This entity has also been referred to as myxoid malignant fibrous histiocytoma (MFH). Myxofibrosarcoma characteristically has a multinodular growth pattern with alternating hypercellular and hypocellular (myxoid) areas that contain spindled to pleomorphic cells with varying degrees of atypia, curvilinear blood vessels, and mucin-containing pseudolipoblasts. Some high-grade myxofibrosarcomas have predominantly epithelioid cytomorphology and may therefore mimic carcinoma or melanoma.420 Immunohistochemically, the stains most frequently positive are CD34 and occasionally SMA, both of which usually show only limited expression.421 Myxofibrosarcoma commonly has a “null” phenotype that lacks expression of routinely used stains for the evaluation of mesenchymal neoplasms. The tumor cells of myxofibrosarcoma are negative for cytokeratins, S-100 protein, CD68, and desmin.421 Cytogenetically, myxofibrosarcoma is characterized by complex chromosomal aberrations, none of which, however, is specific for this tumor type.422,423
INFANTILE FIBROSARCOMA
In the late 1960s, fibrosarcoma was perhaps the most commonly diagnosed malignant neoplasm of soft tissue in adults. The morphologic diagnosis of classic fibrosarcoma is predicated on finding a herringbone pattern of intersecting spindle-cell fascicles and a variably collagenous stroma, with no other specific signs of differentiation. However, the diagnostic criteria for that lesion have evolved considerably over time, now making it one of the rarest sarcomas in adults. The recognition of other entities such as monophasic synovial sarcoma, MPNST, desmoid fibromatosis, and nodular fasciitis (among others) largely accounts for this change. Many tumors that are histologically indistinguishable from classic fibrosarcoma in fact represent fibrosarcomatous transformation of DFSP; when a fibrosarcoma-like lesion is encountered in superficial soft tissues, areas of conventional DFSP should be carefully sought. Although the diagnosis of fibrosarcoma is now rarely made in adults, a clinically and genetically distinct fibrosarcoma that occurs in infants, namely infantile fibrosarcoma, remains. Infantile fibrosarcoma typically presents in the first year of life as a rapidly growing mass on the distal aspect of the upper or lower extremities.418 A reciprocal translocation t(12;15)(p13;q25) is found in the vast majority of cases and results in the fusion gene ETV6-NTRK3.419 The same translocation is found in cellular mesoblastic nephroma. The morphologic features of infantile fibrosarcoma are those of a mitotically active fascicular spindle cell neoplasm, often with a herringbone pattern and with a variably collagenous
Smooth Muscle Tumors BENIGN SMOOTH MUSCLE TUMORS
Benign smooth muscle tumors (leiomyoma) that arise in soft tissues include angioleiomyoma and leiomyoma of deep soft tissue and, rarely, benign smooth muscle tumors that arise in the retroperitoneum of females. Angioleiomyoma most frequently arises on the lower extremities with a predilection for women. Angioleiomyoma is well circumscribed and composed of smooth muscle bundles intimately associated with variably sized vascular channels. Leiomyoma of deep soft tissues is also well circumscribed and composed of bland smooth muscle cells with no nuclear atypia, no (or very low) mitotic activity, and no necrosis. Leiomyomas, as expected, show expression of desmin, SMAs, and h-caldesmon, usually in a strong diffuse pattern. Certain other markers with specificity for muscle differentiation may be used in recognizing leiomyomas, but their application in diagnostic IHC is not generally considered routine. For example, smooth muscle myosin and Z-band protein have been advocated by some, particularly when either the histologic pattern is unusual, such as with myxoid or hyalinized lesions, or the interpretation of a myogenous lesion is not corroborated by other stains. Among retroperitoneal leiomyomas, Billings and colleagues424 reported frequent expression of estrogen and progesterone receptors in tumor cells, whereas somatic leiomyomas were negative for both of these markers.
108
Immunohistology of Neoplasms of Soft Tissue and Bone
Leiomyosarcoma
LMS of soft tissue most commonly arises in the retroperitoneum of older adults. Some retroperitoneal LMS arises in association with large blood vessels, particularly the inferior vena cava. Less frequently, LMS occurs in the deep soft tissues of the extremities, but it may also be seen in more superficial sites, particularly in the dermis and subcutis. When confined to the dermis, cutaneous LMS has no metastatic potential, and therefore the term atypical intradermal smooth muscle neoplasm has been proposed as a more appropriate designation for these tumors.425 Regardless of location, conventional LMS is a fascicular spindle cell neoplasm, and tumor cells have brightly eosinophilic cytoplasm and cigarshaped nuclei, recapitulating normal smooth muscle (Fig. 4-13). As tumors become less differentiated, they show less resemblance to normal smooth muscle. The differential diagnosis of LMS traditionally includes other fascicular spindle cell neoplasms, such as MPNST, synovial sarcoma, spindle cell RMS, and inflammatory myofibroblastic and solitary fibrous tumors. Currently, IHC confirmation of smooth muscle differentiation in LMS is based on the demonstration of desmin, α–SMA, muscle actin (HHF-35), and
h-caldesmon (see Fig. 4-13). Some studies have indicated that actins and h-caldesmon may be more sensitive than desmin in detecting myogenic differentiation in smooth muscle neoplasms, with desmin expression occurring in 70% to 80% of cases.426 None of these markers are specific for smooth muscle tumors, and reactivity for at least one of these markers is usually expected to support the diagnosis in the presence of appropriate morphologic features of LMS. Desminpositive spindle cell RMS may be differentiated from LMS by the former’s negativity for h-caldesmon and SMA and reactivity for MYOD1 and myogenin/MYF4. Partial immunophenotypic similarity to other soft tissue tumors is not uncommon in LMS. For example, labeling for S-100 protein may occasionally be encountered in smooth muscle tumors, similar to MPNST. The presence of keratin and EMA expression in LMS occurs in 30% to 40% of cases and appears to be most common in uterine and cutaneous LMS, but it may also occur in retroperitoneal cases.427,428 CD34 may also show focal positivity in tumor cells in as many as 30% of cases.171 LMS is consistently KIT negative, which is of discriminatory diagnostic use in the differential diagnosis with GIST.429 Calponin is another smooth muscle–related protein that is developmentally expressed in as many as
A
B
C
Figure 4-13 Leiomyosarcoma composed of fascicles of spindle cells with eosinophilic cytoplasm (A). Tumor cells show diffuse positivity for smooth muscle actin (B) and desmin (C).
Specific Soft Tissue and Bone Tumors
four isoforms and binds strongly to actin in a calciumindependent manner.430 It is expressed in parenchymal and vascular smooth muscle cells and is also present in myofibroblasts and myoepithelial cells, and this low specificity limits its diagnostic utility. Synovial sarcoma may also show calponin positivity.112 LMS occasionally may show strong positivity for CD99 but with a dotlike cytoplasmic staining pattern.431 In addition, LMS can demonstrate reactivity for estrogen and progesterone receptors, both when they arise in viscera and in the somatic soft tissues.432 Increased expression of the GLUT-1 protein has been reported in some examples of soft-tissue LMS, whereas leiomyomas are uniformly negative.432 Pleomorphic Leiomyosarcoma. Some LMS, particularly those that arise in the retroperitoneum, show extensive pleomorphism. In some cases the tumor may be recognized as LMS only after identification of focal areas with identifiable smooth muscle differentiation. Although desmin and/or SMA immunoreactivity may be present in the pleomorphic tumor cells, staining for desmin is often less diffuse than in areas with morphologically recognizable smooth muscle differentiation.433 In dedifferentiated LMS, areas of abrupt transition from conventional LMS to a high-grade, otherwise unclassified sarcoma correlates with loss of expression of all muscle markers in the dedifferentiated component.434,435 Other Morphologic Variants of Leiomyosarcoma. A distinct morphologic variant of LMS is inflammatory LMS. In this tumor type, spindle-shaped tumor cells are found scattered within a sea of mixed inflammatory cells.436 Recognition of the neoplastic spindle cells may be difficult, but the presence of atypia is often helpful in this regard. The differential diagnosis of this subtype, particularly when it arises in the retroperitoneum, includes dedifferentiated liposarcoma, and, as such, a panel of immunostains that include SMA, desmin, MDM2, and CDK4 is helpful. Epithelioid features are known to occur in smooth muscle tumors, usually as a focal finding but very rarely as the predominant pattern. Many tumors previously called epithelioid LMS are now increasingly being recognized as either PEComa or epithelioid type GIST. Epithelioid features in either leiomyoma or LMS are most commonly found in tumors that arise in the female genital tract. Another rare appearance to LMS is round cell morphology. In such cases, the differential diagnosis is broad, and IHC is necessary to confirm the diagnosis. Epstein-Barr Virus–associated Smooth Muscle Tumors
Both benign and malignant smooth muscle tumors arise at a greater frequency in immunosuppressed patients, particularly in the setting of human immunodeficiency virus (HIV) infection or following solid organ transplantation. The morphology of these tumors may be that of a well-differentiated smooth muscle tumor or a more cellular tumor with a round cell component and prominent infiltrating lymphocytes; this is often classified as
109
LMS, although clinical behavior does not correlate well with histologic appearances in this distinctive subset (Fig. 4-14). Such lesions in immunocompromised individuals may occur in unusual locations and show evidence of latent infection by clonal Epstein-Barr virus (EBV).437 Several EBV antigens are expressed in HIVrelated smooth muscle tumors, including latent antigen EBNA-1, immediate-early antigen BZFL1, early antigen EA-D, and viral capsid antigen p160, gp125, and membrane antigen gp350.438 These findings show that EBV is capable of lytic infection of selected mesenchymal cells, and they support a role for EBV in smooth-muscle tumorigenesis in that context. EBV RNA in situ hybridization (EBER) shows positivity within lesional cells in this subtype of smooth muscle tumor and is widely used clinically to confirm the diagnosis (see Fig. 4-14). Metastasis and death as a result of EBV-associated smooth muscle tumors are uncommon. Perivascular Epithelioid Cell Family of Tumors
Perivascular epithelioid cell (PEC) tumors, also known as PEComas, are a peculiar family of neoplasms that often show dual smooth muscle and melanocytic differentiation. They can arise at any anatomic site, in visceral soft tissue (clear cell “sugar” tumor, lymphangioleiomyomatosis), kidney (angiomyolipoma), gynecologic and gastrointestinal sites, and also in the urinary tract, liver, pancreas, soft tissue of the extremities, and skin.439 Most PEComas that arise in the kidney or liver with classic appearances of angiomyolipoma are still referred to as angiomyolipoma. The morphologic appearance of PEComas can vary. Typically, epithelioid cells with clear to palely eosinophilic, granular cytoplasm predominate, with focal radial arrangement around small blood vessels (Fig. 4-15). A small subset of tumors contains a prominent spindle cell component. The nested growth pattern often seen is due to the presence of a fine capillary network. As such, differential diagnostic considerations may include pure smooth muscle tumors, clear cell sarcoma (CCS), and renal cell carcinoma (RCC), among others. A sclerosing variant composed of cords of epithelioid cells in a densely hyalinized stroma occurs most often in the pararenal retroperitoneum.87 Angiomyolipoma of the kidney or liver consists of thick-walled vessels, admixed epithelioid or spindled tumor cells, and variable amounts of mature fat. The epithelioid variant is cellular with minimal fat and often with marked atypia, and prior to recognition of this entity, most of these tumors were diagnosed as RCC. In deep soft tissues, these lesions show a range of clinical behavior that ranges from benign to very aggressive; aggressive behavior may be suggested by the presence of marked atypia, mitotic activity, or necrosis. Given the potentially wide differential diagnosis for these tumors, IHC is required for confirming the diagnosis of PEComa. Given their reported myoid and melanocytic differentiation, PEComas are expected to show IHC evidence of differentiation along both of these lines. Smooth muscle markers—α-SMA, musclespecific actin, desmin, h-caldesmon, calponin, and smooth-muscle myosin—all show variable expression in terms of intensity and extent, and in some cases
110
Immunohistology of Neoplasms of Soft Tissue and Bone
A
C
expression of only one muscle marker is present (see Fig. 4-15).86 Epithelioid examples with clear cell morphology may be entirely negative for smooth muscle markers. The same variability of staining is also true of markers of melanocytic differentiation (HMB-45, melan A, tyrosinase, microphthalmia transcription factor [MITF]); HMB-45 is the most frequently positive melanocytic marker (see Fig. 4-15). S-100 protein expression is uncommon in PEComa but is usually cytoplasmic and limited in extent when present.87 Nuclear staining for TFE3 protein is observed in a subset of PEComas, some with TFE3 gene fusions; TFE3 expression in PEComa is mutually exclusive to MITF expression.282 KIT may also rarely be detected; keratin and CD34 are typically absent.440
B
Figure 4-14 Epstein-Barr virus (EBV)–associated smooth muscle tumor (A) with diffuse expression of smooth muscle actin (B), confirmed by positive EBV RNA (EBER) in situ hybridization (C).
Nerve Sheath Tumors BENIGN PERIPHERAL NERVE SHEATH TUMORS Neurofibroma and Schwannoma
The most common benign PNSTs in both children and adults are neurofibromas and schwannomas. The differential diagnostic considerations differ somewhat between neurofibroma and schwannoma. Neurofibromas are commonly confused with myxomas, nonpigmented spindle cell or neurotizing melanocytic nevi, or cellular and organizing scar tissue; schwannomas are more likely to be confused with smooth muscle or myofibroblastic tumors. Occasionally, schwannomas and neurofibromas can show somewhat similar features.
Specific Soft Tissue and Bone Tumors
A
B
C
D
111
Figure 4-15 Epithelioid tumor cells of a malignant perivascular epithelioid cell tumors with granular cytoplasm and arrangement around a blood vessel (A). Tumor cells show diffuse expression of desmin (B) and multifocal staining for HMB-45 (C) and melan A (D).
S-100 protein is extremely useful in this context, because it is strongly expressed by schwannomas (Fig. 4-16) and more variably expressed by neurofibromas.441 In addition, the presence of scattered neurofilament protein (NFP)–positive axons is typical of neurofibroma, whereas axons are rare within schwannomas. Fine and colleagues442 have also found that calretinin is typically diffusely present in schwannomas, whereas neurofibromas lack that marker or are labeled only weakly for it. The presence of myogenic markers, including desmin and muscle-associated actins, is useful for recognizing smooth muscle tumors. Second-line markers of melanocytic differentiation such as HMB-45 or melan A can help with the differential diagnosis of melanocytic lesions. A potential pitfall in the use of HMB-45 to recognize melanocytic lesions is that melanotic schwannoma is an HMB-45–positive tumor that arises in retroperitoneal, mediastinal, and paraspinal soft tissue and bone and is composed of melanosome-laden cells with ovoid to spindled nuclei, often with longitudinal nuclear grooves.443 Cytokeratin expression is often seen in retroperitoneal schwannoma, and expression of AE1/ AE3 occurs in as many as 70% of cases, unlike
peripheral schwannomas, which are negative for cytokeratins.444 The perineurial cells in neurofibroma express EMA, which may be detected immunohistochemically in a small population of cells in these tumors. EMA may also be of some value in the diagnosis of nerve sheath myxoma because of the existence of perineurial cells around most tumor lobules. EMA highlights the capsule in schwannomas, but schwannomas of the GI tract are usually unencapsulated. Nerve sheath tumors are sometimes labeled by antibodies to GFAP, with a reported frequency of as many as 62%,441 as are 50% of soft tissue myoepitheliomas, whereas other benign soft tissue tumors lack GFAP expression. Similar to keratin, the frequency of GFAP expression is greater in retroperitoneal schwannomas than in peripheral schwannomas.444 Perineurioma
Perineuriomas are benign tumors that arise from perineurial cells, which form the outer lining of nerve fascicles. Perineurial cells are typically slender spindle cells with long, delicate, palely eosinophilic cytoplasmic processes (Fig. 4-17). Rare tumors arise within nerves
112
A
Immunohistology of Neoplasms of Soft Tissue and Bone
B Figure 4-16 Schwannoma (A) shows diffuse strong expression of S100 protein (B).
A
B
C
D
Figure 4-17 Soft tissue perineurioma is composed of slender spindle cells with long, delicate, palely eosinophilic cytoplasmic processes (A) and shows expression of epithelial membrane antigen (B) and CD34 (C) and variable reactivity for claudin-1 (D).
Specific Soft Tissue and Bone Tumors
(intraneural perineurioma). Other distinctive variants of soft tissue perineurioma include sclerosing perineurioma— which shows a predilection for fingers and hands, is often superficial and involves dermis, and consists of bland, plump, ovoid to spindled cells in a dense collagenous stroma—and the rare reticular perineurioma, in which tumor cells form a netlike pattern. Otherwise, soft tissue perineuriomas show a wide anatomic distribution and display relatively uniform cytomorphologic features. A myxoid stroma is not uncommonly encountered.157 Perineurial cells, whether neoplastic or nonneoplastic, express EMA, which highlights their long, delicate cytoplasmic processes. Reactivity for CD34 is found in around 60% of cases. Claudin-1 shows variable positivity in perineurioma, and expression is reported in 30% to 92% of cases (see Fig. 4-17).156 GLUT-1 has been shown to be positive in some perineuriomas,195 but S-100 protein and cytokeratins are negative in perineurioma. Soft tissue perineurioma may mimic cellular myxoma or LGFMS and occasionally DFSP. Unlike perineurioma, both cellular myxoma and DFSP are typically negative for EMA. MUC4 is helpful in distinguishing perineurioma from LGFMS, because MUC4 is negative in perineurioma and positive in LGFMS.309 A subset of perineuriomas shows hybrid features with schwannoma and consists of an intimate admixture of Schwann cells and perineurial cells, without Antoni A and B zonation, called hybrid schwannoma/ perineurioma. Immunohistochemically, EMA highlights the perineurial cells, whereas S-100 protein is positive in the Schwann cell component. Immunoreactivity for claudin-1 is present in approximately 80% of tumors, usually in a similar distribution as EMA, and CD34 expression is seen in almost all tumors. Expression of GFAP is found in approximately 80% of cases and tends to be more limited in distribution than S-100 protein.445 Granular Cell Tumor
Granular cell tumor, a benign schwannian neoplasm with a very rare malignant counterpart, has been intensely studied by IHC methods. In addition to resembling histiocytic lesions, granular cytoplasmic change is a recognized change in smooth muscle tumors,446 melanocytic lesions, and certain carcinomas such as RCC. Granular cell tumors of the adult type show consistent diffuse positivity for S-100 protein (nuclear and cytoplasmic), NSE, α-inhibin, calretinin, and CD68.447-450 A rare, histologically similar tumor, albeit often with spindle cell morphology and mild nuclear atypia and variability, lacks expression of S-100 protein and is known as primitive nonneural granular cell tumor. This tumor type shows strong expression of the lysosomal marker NKI-C3, as do all tumors with granular cell change, and it often shows reactivity for CD68 and NSE but lacks lineage-associated markers.451,452 A histologically identical lesion occurs almost exclusively in female newborns or infants and occurs only along the alveolar ridge, designated congenital granular cell tumor. It differs from the adult tumor by a complete lack of S-100 protein and NSE expression.453 Both the adult and congenital types share positivity for CD68 and NKI-C3. Although traditionally regarded as a
113
histiocytic marker, CD68 positivity is related to the abundance of cytoplasmic phagolysosomes, a distinctive feature of granular cell tumors. Therefore it is not possible to distinguish true granular cell tumors from reactive histiocytic granular cell proliferations with only CD68, and CD163 must be used for that purpose. MALIGNANT PERIPHERAL NERVE SHEATH TUMORS
Malignant peripheral nerve sheath tumor (MPNST)— formerly also known as neurofibrosarcoma, malignant schwannoma, and neurogenic sarcoma—is a tumor with highly variable morphologic features. It may be indistinguishable from classic fibrosarcoma, or it may exhibit divergent differentiation with the presence of glandlike structures, or, more commonly, it may show nonschwannian mesenchymal elements. However, many MPNSTs are recognizable on morphologic grounds alone, especially if they show an anatomic association with a nerve or a neurofibroma. The classic morphologic appearance is that of a fascicular spindle cell tumor with tapering or buckled nuclei, variable cellularity (usually hypercellular), and perivascular accentuation of tumor cells. MPNST has complex clonal abnormalities in most cases and often shows homozygous deletion of the CDKN2A (INK4A) gene on 9p, which encodes the cell-cycle regulators p16 and p19.454,455 Immunomarkers associated with Schwann cells and benign PNSTs are frequently detected in MPNSTs, although MPNST shows reactivity for S-100 protein or GFAP in only approximately 50% of cases, usually with labeling of only a small proportion of tumor cells (Fig. 4-18).128 CD56 and CD57 are likewise observed in approximately 50% of MPNSTs, but these markers lack specificity (see Fig. 4-17).41 Myelin basic protein is much less frequently encountered. Note that none of these nerve sheath–associated determinants by themselves is definitive in the identification of MPNST. Reactivity for cytokeratins 8 and 18, but not cytokeratins 7 or 19, is occasionally present.456 Thus immunohistologic support for a diagnosis of MPNST can be difficult to obtain in many instances. Moreover, MPNST has the potential for divergent differentiation, most frequently myogenic differentiation, but this also includes osteosarcomatous, chondrosarcomatous, and very rarely epithelial differentiation. As such, expression of corresponding lineage markers may be identified in such cases. Most notably, when strong desmin expression is observed, the possibilty of rhabdomyosarcomatous differentiation (malignant Triton tumor) should be considered; myogenin reactivity will confirm this finding. Recent studies have suggested that the neural crest transcription factor SOX10 can be used to support a diagnosis of MPNST; however, SOX10 is also detected in melanocytic lesions and must therefore be interpreted in context, similar to other markers.159,160 Another spindle cell tumor that shares immunophenotypic features with MPNST is monophasic synovial sarcoma. Roughly 30% of cases of synovial sarcoma also show reactivity for S-100 protein. Cytokeratin (CK) subset analysis may be useful in difficult cases, because
114
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
Figure 4-18 Malignant peripheral nerve sheath tumor (A) shows reactivity for S-100 protein in approximately 50% of cases, often with labeling of only a small proportion of tumor cells (B).
most synovial sarcomas are reactive for CK7 or CK19, or both. In contrast, MPNST typically lacks expression of those proteins. Biphasic synovial sarcoma (BSS) should not be confused with the extremely rare glandular variant of MPNST; differential keratin expression can be helpful in such cases, and the epithelial elements in BSS express CK7 or CK19, which is typically negative in the glandular component of MPNST. Although strong nuclear TLE1 expression is characteristic of synovial sarcoma, a subset of MPNST is also positive for this marker, albeit usually with only weak staining. Molecular studies to identify the presence of t(X;18) or the SS18-SSX fusions are helpful to confirm the diagnosis of synovial sarcoma. One particularly difficult issue is the distinction between spindle cell malignant melanoma and MPNST, either in the skin or in metastatic sites.457 As a practical approach, cytologically malignant spindle cell neoplasms with strong and diffuse S-100 protein reactivity generally should be considered to be melanomas until proven otherwise. This is especially true if concomitant positivity for HMB-45, tyrosinase, or melan A is obtained, because the latter three markers are only exceptionally associated with nerve sheath tumors. However, spindle cell melanomas only rarely express these determinants. Another consideration in the presence of diffuse S-100 protein expression and a morphologically lowgrade spindle cell tumor is cellular schwannoma. Despite the production of collagen type IV by nerve sheath cells, the IHC detection of this marker has limited value in the diagnosis of schwannian neoplasms, because cells with smooth muscle, endothelial, and myofibroblastic differentiation may also synthesize it. Reports of MPNST with angiosarcomatous differentiation have also been described.458 IHC analysis has confirmed the presence of endothelial differentiation in those exceptional cases. Epithelioid Malignant Peripheral Nerve Sheath Tumor
Epithelioid MPNST is an extremely rare variant that shows a histologic resemblance to melanoma, metastatic carcinoma, CCS, and extrarenal rhabdoid tumor.
As a result, epithelioid MPNST is probably an underrecognized entity that most often arises in the superficial soft tissues of the extremities; it is associated with a nerve in approximately 60% of cases. Tumors are usually well circumscribed with a lobulated growth pattern and are composed of rounded cells with large, vesicular nuclei and prominent nucleoli. Labeling for S-100 protein is more frequently seen in epithelioid MPNST than in other forms of that tumor and are present in more than 90% of cases, usually with a strong and diffuse staining pattern.459 The majority of these tumors are also reactive for NSE and protein gene product 9.5 (PGP9.5), although these markers have a limited role, if any, in clinical practice; staining with CD56 and CD57 is less frequent.459,460 Despite their polygonal-cell appearance, cytokeratin and carcinoembryonic antigen (CEA) are lacking in epithelioid MPNST. Desmin, actin, UEAI, FLI1, and CD31 are consistently negative, allowing for exclusion of epithelioid LMS and angiosarcoma; melanosomal markers are also negative. Another reactant with potential utility in this differential diagnosis is podoplanin; Jokinen and colleagues461 reported expression of this marker in 75% of epithelioid MPNSTs but not in melanomas. Expression of SMARCB1 is lost in 50% of epithelioid MPNST, whereas metastatic melanomas are consistently positive; this marker may therefore be a useful diagnostic adjunct in a subset of cases.290
KEY DIAGNOSTIC POINTS Nerve Sheath Tumors • Schwannoma is diffusely S-100 protein positive. • Soft tissue perineurioma is positive for epithelial membrane antigen and shows variable expression of CD34 and claudin-1. • Malignant peripheral nerve sheath tumor shows focal positivity for S-100 protein and/or glial fibrillary acidic protein in approximately 50% of cases; SOX10 is a nuclear transcription factor expressed in a subset of cases.
Specific Soft Tissue and Bone Tumors
Adipocytic Tumors SPINDLE CELL LIPOMA AND PLEOMORPHIC LIPOMA
Spindle cell lipoma and pleomorphic lipoma are benign adipocytic tumors that arise on the neck and upper back and are composed of varying amounts of mature fat, bland spindle cells, ropey collagen bundles, and, in the case of pleomorphic lipoma, admixed hyperchromatic multinucleate giant cells. Although the diagnosis of both of these lipoma types is usually straightforward, cases with a minor (or absent) adipocytic component may be confused with other spindle cell neoplasms. The spindled and pleomorphic cells are strongly CD34 positive but are rarely S-100 protein positive.462,463 CD34 expression is also often found in some histologic mimics that include solitary fibrous tumor, neurofibroma, DFSP, and giant cell fibroblastoma. Lack of S-100 protein staining in the spindle cells is useful for distinguishing these lipoma variants from neurofibroma. Additionally, BCL2 reactivity may be found in spindle cell lipoma, similar to solitary fibrous tumor. Confident identification of spindle cell lipoma is usually possible with close attention to the histologic features and supportive immunoprofile of CD34 expression in the spindle cells. Desmin positivity in 20% of spindle cell lipomas was reported in one study,464 and loss of retinoblastoma protein (Rb) expression was recently reported to be a consistent finding in spindle cell and pleomorphic lipoma.287 WELL-DIFFERENTIATED AND DEDIFFERENTIATED LIPOSARCOMA
Atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLPS) is a locally aggressive neoplasm composed of adipocytes that show variation in cell size, nuclear atypia of adipocytes and stromal cells, and variable numbers of lipoblasts (Fig. 4-19). Because WDLPS shows no metastatic potential in the absence of dedifferentiation, tumors that occur at surgically amenable sites, where curative surgical excision is possible, are designated atypical lipomatous tumor. ALT/WDLPS accounts for the majority of liposarcomas. The most common anatomic sites of involvement are the extremities and retroperitoneum. Dedifferentiated liposarcoma (DDLPS) consists, in the majority of cases, of a nonlipogenic sarcoma that transitions from ALT/WDLPS (see Fig. 4-19). Dedifferentiation may occur in primary tumors or in recurrences. Recently, the definition of DDLPS as nonlipogenic has been somewhat revised with the recognition of cases that contain admixed lipoblasts within the spindle cell or pleomorphic sarcomatous component; such cases closely resemble pleomorphic liposarcoma in those areas.465,466 Along these lines, whenever a pleomorphic or histologically heterogeneous sarcoma is seen in retroperitoneal or intraabdominal locations, because DDLPS is overwhelmingly the most likely diagnosis, the tumor should be sampled extensively to identify a well-differentiated adipocytic component. Immunohistochemically, the most useful markers for confirming a diagnosis of WDLPS or DDLPS are MDM2 and CDK4, which show nuclear expression in both
115
adipocytes and spindle cells (see Fig. 4-19).284,285 Expression of both of these markers is found in more than 90% of cases. The extent and intensity of staining for these markers is greatest in DDLPS, reflecting a greater degree of MDM2 amplification.467 The molecular basis for the overexpression of these cell-cycle–related proteins in WDLPS and DDLPS is the presence of 12q13 to q15 amplicons in supernumerary ring and/or giant marker chromosomes with resultant constant amplification of MDM2 and HMGA2 and frequent coamplification of CDK4. Sometimes S-100 protein highlights lipoblasts in WDLPS. Additional immunostains are helpful in excluding tumors that may fall into the morphologic differential diagnosis with DDLPS, including sarcomatoid carcinoma, melanoma, and other spindle cell sarcomas. The high-mobility group A2 (HMGA2) protein is another intranuclear architectural transcription factor that is overexpressed in DDLPS, although benign lipomatous tumors are also often positive for this marker.468 DDLPS may express desmin regardless of the presence or absence of a component of heterologous rhabdomyosarcomatous differentiation.469 Keratin is typically negative in WDLPS/DDLPS. In addition, recent studies have suggested that nuclear p16 expression may also be used to support the diagnosis of ALT/WDLPS and DDLPS.470,471 PLEOMORPHIC LIPOSARCOMA
Pleomorphic liposarcoma (PLPS) is relatively rare and most often arises on the extremities of older adults. Less frequently, PLPS arises in the retroperitoneum or in young patients, usually in the setting of Li-Fraumeni syndrome. PLPS is a high-grade spindle cell or pleomorphic sarcoma that contains variable numbers of pleomorphic lipoblasts with hyperchromatic atypical nuclei. Those tumors with prominent epithelioid features may mimic poorly differentiated carcinomas, especially RCC and adrenal cortical carcinomas.472,473 The number of lipoblasts is highly variable in PLPS, and in many cases, areas indistinguishable from myxofibrosarcoma are present; in the absence of identifiable lipoblasts, a diagnosis of myxofibrosarcoma is often rendered. Therefore in limited tissue samples or small needle biopsies that show an otherwise unclassified pleomorphic sarcoma, additional sampling of resection specimens is needed to identify lipoblasts, the defining feature of this diagnosis. In general, IHC is mainly used to exclude potential mimics of PLPS: keratins for carcinoma, with the caveat that epithelioid PLPS may show focal cytokeratin positivity;472 MDM2 and CDK4 to exclude DDLPS; and SMA and desmin to exclude pleomorphic LMS. Limited S-100 protein expression is seen in lipoblasts in approximately 30% of PLPS.472 PLPS has a complex karyotype with nondistinctive structural and numerical chromosomal aberrations.474,475 MYXOID LIPOSARCOMA
Myxoid liposarcoma (MLPS) is characterized by the recurrent translocation t(12;16)(q13;p11) that involves DDIT3 on 12q13 and FUS on 16p11 and, less commonly, by the translocation t(12;22)(q13;q12), which
116
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
C
D
E
F
Figure 4-19 Well-differentiated liposarcoma, showing characteristic atypical stromal cells (A) with nuclear expression of MDM2 (B) and CDK4 (C). Dedifferentiated liposarcoma (D) shows a greater extent of staining for MDM2 (E) and CDK4 (F).
involves EWSR1 and DDIT3.476,477 Morphologically, MLPS consists of small, uniform, oval to round cells in an abundant myxoid stroma with prominent “crow’sfeet” vessels and variable numbers of univacuolated or bivacuolated small lipoblasts.478 MLPS is graded with a three-tier system of low, intermediate, and high grades
based on cellularity, with highly cellular tumors, formerly known as round cell liposarcoma, representing high-grade MLPS. Often MLPS expresses S-100 protein within the lipoblastic and spindle or round cell component. In addition, a subset of MLPS may also express desmin, muscle-specific actin, and α-SMA focally;
Specific Soft Tissue and Bone Tumors
CD34 is typically negative.468 Low-grade MLPS may mimic cellular myxoma, myxofibrosarcoma, or myxoid spindle cell lipoma. Expression of CD34 can help in the differential diagnosis with spindle cell lipoma, but otherwise morphologic evaluation is the gold standard, often along with genetic studies to evaluate DDIT3 gene rearrangements or fusion transcripts of MLPS. Highgrade MLPS may resemble extraskeletal myxoid chondrosarcoma, or even poorly differentiated carcinoma. MLPS is usually negative for MDM2 and CDK4, reflecting the fact that such tumors manifest different karyotypic abnormalities from well-differentiated and dedifferentiated liposarcomas.479,480 However, studies have shown upregulation of CDK4 and overexpression of MDM2 in a small subset of these tumors, which may be a potential diagnostic pitfall.481
Other Primary Neoplasms of Soft Tissue Some primary tumors of soft tissues do not fit neatly into one of the preceding categories, because they show either divergent differentiation or no specific line of differentiation morphologically or immunophenotypically. For this reason they are considered individually in this section. DESMOPLASTIC SMALL ROUND CELL TUMOR
Desmoplastic small round cell tumor (DSRCT) is an aggressive, clinicopathologically distinct tumor characterized by an EWSR1-WT1 chimeric transcript as a result of a (11;22)(p13;q12) translocation. DSRCT classically arises in the abdomen of young men and shows extensive fibrosis, within which discrete nests of small round tumor cells with minimal cytoplasm are seen (Fig. 4-20). Rarely, DSRCT occurs at extraabdominal sites, and it has an unusual polyphenotypic immunoprofile, with frequent staining for keratin (86%), EMA (93%), NSE (81%), vimentin (97%), and desmin (90%; see Fig. 4-20).482,483 CD99 reactivity is seen in only a small minority of cases; instead, WT1 immunoreactivity is frequently seen by using a polyclonal antibody directed against the C-terminus of the protein, in contrast to the widely used monoclonal antibodies that recognize the N-terminus of the protein, which are useful for diagnosing malignant mesothelioma and serous adenocarcinoma.482,484 Actin, myogenin, MYOD1, and chromogranin are generally absent in DSRCT.483 Molecular studies to identify EWSR1 rearrangement or the EWSR1-WT1 fusion transcript may be helpful to confirm the diagnosis, with the caveat that EWSR1 rearrangement alone cannot distinguish DSRCT from Ewing sarcoma. KEY DIAGNOSTIC POINTS Desmoplastic Small Round Cell Tumor • Typically positive for WT1 (polyclonal antibody directed against the C-terminus), keratin 8/18, EMA, NSE, and desmin. • CD99 expression is uncommon, and myogenin is negative.
117
OSSIFYING FIBROMYXOID TUMOR
Ossifying fibromyxoid tumor (OFMT) is a slowly growing mesenchymal lesion with a propensity to arise on the extremities, in the deep subcutis, or in skeletal muscles. Microscopically, this neoplasm is composed of lobulated nests of compact, cytologically bland round cells in a variably myxoid or densely hyalinized stroma.485 An incomplete shell of lamellar bone is a usual feature, but it may be absent in some cases (so-called nonossifying OFMT). Although the vast majority of cases follow a benign course, atypical histologic features such as high mitotic activity, high cellularity, and nuclear atypia may portend aggressive behavior. The tumor cells of OFMT stain strongly and diffusely for S-100 protein in as many as 94% of cases.88,130,131 Occasional reactivity may also be seen for keratin, CD57, NSE, synaptophysin, and GFAP.486-489 OFMT often also shows expression of desmin and, less frequently, α-SMA.130,131,486,490 EMA and melanocytic markers are consistently absent in OFMT. A recent study has identified near-diploid karyotypes with rearrangements of 6p21 in OFMT and hemizygous loss of chromosome 22 in malignant examples. PHF1 gene rearrangement has recently been identified as a recurrent finding in OFMT.491 Fluoresence in situ hybridization (FISH) analysis for rearrangement of the PHF1 locus may therefore be a helpful diagnostic tool to identify OFMT cases, particularly those with atypical morphologic features. SYNOVIAL SARCOMA
Synovial sarcoma is a malignant soft tissue tumor that shows epithelial and mesenchymal differentiation and has distinct clinical, genetic, and morphologic features. Although it was once thought that synovial sarcoma arose in association with synovium, it is now well known that this is not the case and that these tumors may arise at any anatomic location.492 Synovial sarcoma is characterized by the recurrent translocation t(X;18) (p11.2;q11.2), which results in SS18-SSX fusion genes, present in more than 90% of cases.493 Synovial sarcoma typically affects young adults and most often arises in deep soft tissues of the extremities. Morphologically, synovial sarcoma takes three main forms: 1) biphasic, 2) monophasic, and 3) poorly differentiated. Biphasic synovial sarcoma (BSS) consists of a fascicular spindle cell component and an epithelial component that usually shows glandular differentiation, whereas monophasic synovial sarcoma (MSS) lacks the epithelial component (Fig. 4-21). Poorly differentiated synovial sarcoma (PDSS) is diagnosed when areas with high cellularity and high mitotic activity predominate, and the tumor cells often have a rounded morphology, thereby mimicking other round cell tumors such as Ewing sarcoma. Classic BSS is usually not a diagnostic problem, whereas MSS and PDSS can simulate the appearances of several other soft tissue neoplasms that include MPNST, solitary fibrous tumor, fibrosarcomatous DFSP, and Ewing sarcoma. The glandular component of synovial sarcoma expresses cytokeratins, including AE1/AE3. EMA expression is typically observed in both BSS and MSS;
118
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
C
D
Figure 4-20 Desmoplastic small round cell tumor composed of nests of uniform tumor cells with minimal cytoplasm (A). It has a polyphenotypic immunoprofile, showing expression of desmin (B), dotlike cytokeratin (C), and neuron-specific enolase (D).
however, unlike its biphasic counterpart, MSS tends to be focally and inconsistently reactive for cytokeratins. In particular, MSS may show reactivity for simple keratins: CK7, CK8, CK18, and CK19.494,495 S-100 protein expression is found in approximately 30% of synovial sarcomas.494 CD99 is commonly observed in MSS,60,496,497 but expression of this marker is also shared by some other spindle cell neoplasms. Strong positivity for BCL2 protein has also been noted in the spindle cell component of synovial sarcoma.296,498 In one of these studies, in which FISH analysis confirmed the presence of t(X;18), 79% of MSS cases were positive for BCL2, whereas 20 LMSs, 4 MPNSTs, and 4 fibrosarcomas lacked this marker.296 Nevertheless, BCL2 reactivity is present in a variety of other soft tissue tumors, such as spindle cell lipoma, Kaposi sarcoma, solitary fibrous tumor, and GIST. Interestingly, all of these lesions also typically express CD34, which is consistently absent in MSS and may therefore be a useful discriminant. TLE1 is a transcription factor that was found to be upregulated in synovial sarcoma with gene-expression profiling studies. Nuclear labeling for TLE1, present in
approximately 80% of synovial sarcomas overall, supports the diagnosis of synovial sarcoma (see Fig. 4-21). In a study by Foo and colleagues,306 expression of TLE1 was most frequent in PDSS; they found reactivity for TLE1 in 78% of BSS, 79% of MSS, and 91% of PDSS. Expression of TLE1 is found in approximately 15% of MPNSTs and 8% to 40% of solitary fibrous tumors, but staining in those tumor types is usually only weak in intensity.304,306 Interestingly, as many as 70% of synovial sarcomas show expression of calretinin; distinction from mesothelioma is helped by lack of nuclear staining for WT1.499 E-cadherin and N-cadherin expression is seen to varying degrees and extents in BSS. PDSS with round cell features can easily be mistaken on morphologic grounds for other small round cell tumors, such as Ewing sarcoma and high-grade MPNST. Cytogenetic and molecular studies for t(X;18) can help confirm the diagnosis of synovial sarcoma. CD99 reactivity may further complicate the diagnostic interpretation if the pathologist is not aware of its frequent presence in PDSS.494 Analysis of cytokeratin (CK) subsets may contribute to differential diagnosis in this
Specific Soft Tissue and Bone Tumors
A
C
setting; for example, CK7 has been reported in as many as 50% of PDSS cases but is typically absent in Ewing sarcoma.500 TLE1 is positive in nearly all cases of PDSS, whereas in Ewing sarcoma, it is consistently negative.306 Recently, overexpression of the cancer testis antigen NY-ESO-1 was reported in 76% of synovial sarcomas in a strong and diffuse pattern.501 Expression in other spindle cell mesenchymal tumors was limited. Whether this has diagnostic implications remains to be seen, but it may identify a potential role for targeted therapy against NY-ESO-1. EPITHELIOID SARCOMA
Epithelioid sarcoma (EPS) arises most often on the distal extremities of young adults and has a tendency for locoregional spread and lymph node metastasis. EPS has a characteristic histologic growth pattern, characterized by coalescing nodules of uniform epithelioid tumor cells with central necrosis. Potential mimics of epithelioid sarcoma include necrobiotic granuloma, melanoma, or metastatic carcinoma.502,503 The occasional presence of cytoplasmic vacuoles in EPS may also raise the diagnostic possibility of EHE or angiosarcoma. Rare examples manifest unusual histologic findings, such as a chondroidlike matrix504 or a rhabdoid appearance.505
119
B
Figure 4-21 Monophasic synovial sarcoma—a uniform, fascicular, spindle cell neoplasm (A)—expresses epithelial membrane antigen (B) and transducin-like enhancer 1 (C).
The latter finding is more common in proximal-type EPS, which occurs in the proximal extremities, including the groin region, and consists of larger tumor cells with more notable nuclear atypia. A consistent immunophenotypic attribute of epithelioid sarcoma is intense perinuclear zone keratin reactivity as a result of perinuclear collections of intermediate filaments (Fig. 4-22). The degree of cytokeratin labeling may be heterogeneous in any given tumor, and p63 protein, CK5, and CK6 tend to be absent in EPS, whereas histologically similar carcinomas often express those proteins.505 CD34 positivity is seen in approximately 50% of EPS cases,59,168 and HHF-35 is detected in approximately 30% of cases.167,506 Desmin and S-100 protein are both virtually always negative in conventional EPS, but expression of desmin may be found in occasional cases of proximal-type EPS.168 Epithelioid sarcoma shares some immunohistologic attributes with synovial sarcoma in that both tumor types are reactive for cytokeratin and EMA. However, morphologically the two tumors are usually readily distinguishable, and if uncertainty exists, demonstration of TLE1 expression will support a diagnosis of synovial sarcoma.306 IHC studies are helpful in the diagnostic separation of EPS and isolated necrobiotic granulomas (e.g., deep
120
Immunohistology of Neoplasms of Soft Tissue and Bone
A
C granuloma annulare and cellular rheumatoid nodule). The histiocytic cells of granulomas show reactivity for CD68 and CD163, whereas the tumor cells of EPS do not; conversely, necrobiotic granulomas lack reactivity for EMA, cytokeratins, and CD34.507 Because of the potential for epithelioid vascular tumors to demonstrate aberrant keratin reactivity, their confusion with epithelioid sarcomas is a real possibility. Indeed, as mentioned earlier in this discussion, pseudomyogenic (epithelioid sarcoma–like) hemangioendothelioma in particular shows diffuse expression of AE1/ AE3.334,335 Reactivity for CD31 is strong evidence for true endothelial differentiation in this setting, as is labeling for ERG. Obviously, however, because EPS is commonly CD34 positive, similar to vascular neoplasms, this marker cannot be used to separate those two tumor groups. Although keratin expression is not uncommon in epithelioid vascular tumors, EMA is rarely detected. As mentioned above, nuclear expression of SMARCB1 is retained in epithelioid vascular tumors, whereas it is lost in at least 90% of EPS cases (see Fig. 4-22).341 Various chromosomal deletions and gains have been reported in EPS, as have structural rearrangements at 18q11 and 22q11, the latter of which is reflected in the consistent loss of SMARCB1 in these tumors.288 A recent study identified consistent deletions of the SMARCB1 locus in EPS.291
B
Figure 4-22 Epithelioid sarcoma is composed of bland epithelioid cells with pale eosinophilic cytoplasm (A). Tumor cells express cytokeratin (B) and show loss of SMARCB1 protein expression (C).
KEY DIAGNOSTIC POINTS Epithelioid Sarcoma/Synovial Sarcoma/ Angiosarcoma • Keratin expression may be detected in each entity. • ERG is a sensitive and specific marker for angiosarcoma. • Epithelioid sarcoma shows loss of SMARCB1 expression in almost all cases. • TLE1 is positive in synovial sarcoma.
CLEAR CELL SARCOMA OF TENDONS AND APONEUROSES
Clear cell sarcoma (CCS) was formerly thought to represent a primary soft tissue counterpart of cutaneous malignant melanoma. However, it is now known that CCS exhibits a consistent and characteristic (12;22) (q13;q12) chromosomal translocation that is not shared by melanocytic lesions of the skin.508,509 CCS typically involves tendons or aponeuroses of the extremities of young adults. This neoplasm is characterized by epithelioid and/or spindle-shaped tumor cells with clear or, more often, palely eosinophilic cytoplasm in a nested or fascicular growth pattern with a delicate fibrovascular
Specific Soft Tissue and Bone Tumors
A
C
stroma (Fig. 4-23).127 Melanin pigmentation is rarely observed on hematoxyline and eosin (H&E) stain. The IHC features of CCS largely mirror those of malignant melanoma. They include reactivity for S-100 protein, tyrosinase, melan A, HMB-45, and MITF (see Fig. 4-23)510 with the absence of EMA and myogenic, neural, and endothelial markers. A helpful clue to the diagnosis of CCS is the presence of HMB-45 positivity that is stronger and more diffuse than S-100 protein expression, which would be very unusual for melanoma. Keratin expression is also lacking in CCS. As stated, the (12;22)(q13;q12) translocation, yielding an EWSR1/ATF1 fusion, is characteristic of CCS.508,509 Its presence can be evaluated by in situ hybridization (ISH) methods and polymerase chain reaction (PCR)–based technologies, which provide a useful diagnostic adjunct to immunohistology and may sometimes be necessary to exclude a diagnosis of melanoma. CLEAR CELL SARCOMA–LIKE TUMOR OF THE GASTROINTESTINAL TRACT
This tumor type has pathologic features that are distinct from conventional CCS of tendons and aponeuroses.
121
B
Figure 4-23 The tumor cells of clear cell sarcoma are variably spindled or epithelioid and show a predominantly nested growth pattern (A). Expression of both S100 protein (B) and HMB-45 (C) is seen in the majority of cases.
These tumors arise most often in the small intestine and more rarely in the stomach or colon; they typically show a predominantly sheetlike growth pattern with a focally alveolar or pseudopapillary architecture, and approximately 50% of cases contain prominent osteoclast-like giant cells. The cells of this tumor type show strong and diffuse S-100 protein expression but are negative for HMB-45 and other melanocytic markers.511-513 The designation malignant gastrointestinal neuroectodermal tumor (GNET) has recently been proposed.514 Furthermore, an EWSR1-CREB1 fusion gene is also frequently found that corresponds to a (2;22)(q34;q12) translocation, although the (12;22) translocation typical of CCS of tendons and aponeuroses may alternatively be present.511 ALVEOLAR SOFT-PART SARCOMA
Alveolar soft-part sarcoma (ASPS) is a rare distinctive soft tissue tumor. ASPS typically occurs in young patients, shows a female predominance, and most often involves the lower extremities and head and neck sites. More than 50% of patients have metastatic disease at the time of presentation.515 The histologic appearance is that of nested proliferation of large epithelioid tumor cells with abundant eosinophilic or clear cytoplasm,
122
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B
Figure 4-24 Alveolar soft-part sarcoma composed of nests of dyshesive epithelioid cells with abundant clear and granular eosinophilic cytoplasm (A). Nuclear expression of the transcription factor TFE3 (B) is characteristic of this tumor type.
often with a granular quality. Rare periodic acid-Schiff (PAS)–positive diastase-resistant crystals are found in 70% of cases. Tumor nuclei are round and relatively uniform with fine chromatin, and dyshesion of tumor cells within the nests results in the characteristic alveolar appearance (Fig. 4-24); some cases show more solid growth, however, which raises the differential diagnosis of RCC and paraganglioma. ASPS shows an unbalanced translocation t(X;17)(p11.2q25), which results in fusion of TFE3, a transcription factor gene on Xp11, with alveolar soft-part sarcoma locus/alveolar soft-part sarcoma chromosomal locus (ASPL/ASPSCR1) on 17q25.516 A selective marker of the der (17) (X;17) translocation of ASPS is available; namely, nuclear staining for the TFE3 protein.517 Expression of TFE3 has been reported in up to 100% of ASPS (see Fig. 4-24).280 However, nuclear TFE3 expression may also be encountered in metastatic Xp11 translocation RCC and in a small subset of PEComas.518,519 Hence, caution is advised against diagnostic overreliance on TFE3. Controversy has existed over the line of cellular differentiation in ASPS ever since its seminal description in 1952. Despite attempts to prove a neuroectodermal or endocrine nature for this tumor, more recent studies have suggested a possible myogenic phenotype. Immunoreactivity for several muscle-associated proteins such as desmin, actins (both α-sarcomeric actin and α-SMA), myosin, Z-band protein, and the MM isozyme of creatine kinase in ASPS have been reported, but these findings have not been confirmed by other groups.520-522 In our experience, desmin and actins are almost always negative. Other studies have evaluated expression of MYOD1 and myogenin in ASPS,521,523,524 and most analyses have found a complete absence of nuclear staining for both skeletal muscle transcription factors.525,526 Granular cytoplasmic reactivity with the MYOD1 antibody 5.8A has been reported in the majority of cases of ASPS but is regarded as a reproducible nonspecific artifact. Cytokeratins, synaptophysin, chromogranin, melanocyte-specific markers, and neurofilament proteins are negative in ASPS, and S-100 protein shows at
most limited, weak expression.526-528 CD147, a chaperone protein to the monocarboxylate transporter 1 (MCT1) is expressed in the cytoplasm of tumor cells in ASPS but is not used in clinical practice.529 EXTRASKELETAL MYXOID CHONDROSARCOMA
Extraskeletal myxoid chondrosarcoma (EMCS), formerly known as chordoid sarcoma, most commonly arises in the soft tissues of the lower extremities of middle-aged adults. Although skeletal and extraskeletal myxoid chondrosarcomas share similar morphologic features, there are fundamental differences between these tumors at ultrastructural and molecular levels, which indicate that they represent distinct and separate entities. EMCS shows no evidence of cartilaginous differentiation but demonstrates a reciprocal (9;22) (q22;q12) chromosomal translocation that results in fusion of the EWSR1 and NR4A3 genes.530-532 Histologically, EMCS is composed of a network of uniform round to spindled tumor cells with hyperchromatic nuclei and moderate amounts of eosinophilic cytoplasm, which tends to stretch from one cell to another, imparting the delicate reticular growth pattern characteristic of this tumor type. Tumors with a solid growth pattern, more atypical cytomorphology, and high mitotic activity are considered high-grade tumors. Immunohistochemically, EMCS is at most only focally reactive for S-100 protein (<20% of cases) and CD57, whereas skeletal (true) chondrosarcomas of the myxoid type diffusely label for those determinants. EMA may occasionally be positive, but keratin is absent in EMCS.533 E-cadherin, N-cadherin, and GFAP are also negative in EMCS.66 Negativity for GFAP can help in distinguishing EMCS from myxopapillary ependymoma. More recently it has been found that a subset of EMCS shows loss of SMARCB1 protein expression, particularly those with rhabdoid features.292 An occasionally reported peculiarity in the immunophenotype of EMCS is a putative tendency to show occult neuroendocrine differentiation, represented by reactivity for
Specific Soft Tissue and Bone Tumors
synaptophysin or chromogranin,534-536 although we have not been able to confirm this observation. NEUROBLASTOMA
The vast majority of neuroblastomas arise in young children in the abdominal cavity, either within the adrenal gland or associated with the sympathetic chain. Neuroblastoma is a small round blue cell tumor. The tumor cells have minimal cytoplasm, a fine chromatin pattern, and often form rosettes. A lobular or nested growth pattern may be seen, and between the tumor cells, variable amounts of palely eosinophilic fibrillary material (neuropil) are present, which represents cell processes of neurites. Cytodifferentiation to mature ganglion cells may also be present (ganglioneuroblastoma). Tumor cells express NSE, synaptophysin, chromogranin, and neurofilament protein, as well as NB-84, expression of which is found in more than 95% of neuroblastomas.278 Neuroblastoma is typically negative for CD99, which is helpful in the differential diagnosis with Ewing sarcoma. The common genetic findings in neuroblastoma are deletions of chromosome 1 and doubleminute chromosomes, and, in approximately 25% of cases, amplification of MYCN (N-myc). Detection of MYCN amplification is a poor prognostic factor. FISH or chromogenic in situ hybridization (CISH) analysis is used in clinical practice to evaluate such amplifications.537 Recent studies have implicated ALK mutations in a subset of neuroblastomas; ALK protein expression can also be detected in many neuroblastoma cases by IHC, although expression does not correlate with ALK mutation status.267,538 MYOEPITHELIAL TUMORS
Soft tissue myoepithelioma and its malignant counterparts (myoepithelial carcinoma) are uncommon tumors that usually arise in the extremities, either in subcutaneous or in deep soft tissues.19,539 They characteristically exhibit a lobulated growth pattern, often contain myxoid stroma, and show a reticular, trabecular, or nested architecture. The constituent cells in myoepithelial tumors show the same range of morphologic features seen in their salivary gland counterparts (epithelioid, spindled, plasmacytoid/hyaline, or clear). Intratumoral heterogeneity in terms of both architecture and cytology is common. At one end of the morphologic spectrum is the lesion previously known as parachordoma, which is composed of large epithelioid cells with abundant eosinophilic to clear, vacuolated cytoplasm.18,540 The differential diagnosis for this group of lesions primarily centers on extraskeletal myxoid chondrosarcoma and the very rare peripheral chordoma (chordoma periphericum); however, chondroid lipoma and ossifying fibromyxoid tumor may be considered as well. Myoepithelial tumors have a distinct immunoprofile that differs from that of extraskeletal myxoid chondrosarcoma. Myoepithelial tumors are typically positive for CK8 and CK18, EMA, S-100 protein, and vimentin, and they may express GFAP in approximately 50% of cases.18,19,539,540 In addition, p63 expression is found in
123
approximately 40% of myoepithelial tumors, both benign and malignant forms, but expression of p63 is occasionally detected in a wide range of soft tissue neoplasms.541 In contrast, extraskeletal myxoid chondrosarcoma lacks keratin 18 and is infrequently positive for S-100 protein. Similar to myoepithelioma, OFMT usually shows reactivity for S-100 protein, but desmin expression, which is relatively common in the latter tumor type, is rare in myoepithelial tumors. OFMT is generally negative for EMA and keratin. On the other hand, myoepithelioma and peripheral chordoma show considerable immunophenotypic overlap in reference to keratin subtypes, EMA, vimentin, and S-100 protein, but lack of brachyury (T) expression in myoepithelial tumors distinguishes them from chordoma.542 SOFT TISSUE OSTEOSARCOMA
Osteosarcoma is rare outside the skeletal system. When it occurs in soft tissues, osteosarcoma has an aggressive clinical course. Like their intraosseous counterparts, soft tissue osteosarcomas have a variety of histologic patterns that include osteoblastic, fibroblastic, chondroblastic, small cell, and (very rarely) telangiectatic variants. In some cases, areas that mimic myxofibrosarcoma may be identified. Malignant osteoid deposition is the key to making the diagnosis, but osteoid deposition may be very limited and is easily overlooked. Soft tissue osteosarcomas are thus probably underrecognized. Osteocalcin has been reported to be a helpful marker to confirm an osteoblastic phenotype, although its utility in this regard is limited because of low sensitivity and specificity. SATB2, a nuclear matrix-associated transcription factor and epigenetic regulator, may be a useful marker of osteoblastic differentiation.543 Strong labeling for the osseous isozyme alkaline phosphatase has been used in the past to distinguish extraskeletal osteosarcoma from other pleomorphic sarcomas. The major drawback of this marker is that it must be assessed by using cryostat sections or imprint smears (paraffin sections are unsuitable for study); it is rarely used in practice. Extraskeletal osteosarcomas are reactive for vimentin, and their matrical elements often label for CD57; α-SMA may be seen focally, and a subset of tumors coexpress desmin,544 perhaps representing myofibroblastic differentiation. S-100 protein is usually observed only in areas with overtly cartilaginous differentiation.544 Epithelial markers are only exceptionally present, and if found, this should raise the possibility of metaplastic carcinoma with heterologous osteosarcomatous differentiation.544,545 GFAP and neurofilament proteins are absent. Other diagnostic possibilities to consider include heterologous osteosarcomatous differentiation in DDLPS or MPNST, and therefore a panel of immunostains to include cytokeratins, MDM2/ CDK4, and S-100 protein is advisable. DEEP “AGGRESSIVE” ANGIOMYXOMA
Deep angiomyxoma is a peculiar neoplasm of the pelvic and perineal soft tissues that predominantly affects women. It is composed of loosely arranged, bland,
124
Immunohistology of Neoplasms of Soft Tissue and Bone
spindled to stellate cells embedded in a myxoid matrix punctuated by numerous, often hyalinized, variably sized blood vessels. IHC analysis of deep angiomyxoma has revealed reactivity for actin and often for desmin but not for S-100 protein, suggesting a myogenic or myofibroblastic pattern of differentiation.379 Similar to angiomyofibroblastoma, estrogen and progesterone receptors are expressed in the tumor cells. Ultrastructural studies likewise support a fibroblastic/myofibroblastic phenotype in deep angiomyxoma.546 EXTRARENAL MALIGNANT RHABDOID TUMOR
Extrarenal malignant rhabdoid tumor (MRT) is virtually exclusive to infants and young children and is histologically indistinguishable from MRT of the kidney. This tumor may arise at any site, including visceral locations and deep soft tissue of the axial region.547 The characteristic appearance of this neoplasm is that of a cellular tumor composed of polygonal or oval cells with large eccentric nuclei, vesicular chromatin, prominent nucleoli, and hyaline paranuclear cytoplasmic eosinophilic inclusions. MRT has a complex immunophenotype: tumor cells show consistent reactivity for EMA and keratin50,548 and a tendency for perinuclear accentuation of these markers; some MRT may therefore be difficult to separate diagnostically from such entities as proximal-type epithelioid sarcoma without clinical correlation.549-551 Expression of SMARCB1, a protein encoded by a gene at chromosome 22q11.2 and involved in chromatin remodeling, is absent in MRT and proximal-type epithelioid sarcoma as well as in conventional epithelioid sarcoma.341,550 SMARCB1 (formerly INI1) gene mutations have been identified in as many as 75% of cases of MRT, including those of the kidney and CNS (atypical teratoid/rhabdoid tumor). Positivity for muscle-specific actin, CEA, α-SMA, CD99, synaptophysin, NSE, and S-100 protein has also been detected in selected cases.50,547 HMB-45, melan A, chromogranin, myoglobin, and CD34 are absent. A report by Izumi and colleagues552 suggested that dysadherin, a membrane glycoprotein involved in intercellular adhesion, is present in epithelioid sarcoma but not in MRT. In adults, tumors with similar morphologic features invariably represent rhabdoid change in a wide spectrum of other tumor types, therefore IHC is also extremely helpful in that setting. Poorly differentiated malignant neoplasms of various types may on occasion show rhabdoid cytomorphology, including melanomas, mesotheliomas, and carcinomas. Although the immunophenotype of MRT overlaps with those of other sarcomas and poorly differentiated carcinomas, loss of SMARCB1 protein expression is exceptionally rare in malignant neoplasms of adults with rhabdoid cytomorphology (other than epithelioid sarcoma). CARTILAGINOUS TUMORS OF BONE Chondroblastoma
Chondroblastoma is a relatively rare cartilaginous tumor of bone that typically arises in the epiphyses of the long
bones, usually in young patients. Tumors are composed of uniform round to oval cells (chondroblasts) with clear or eosinophilic cytoplasm, round nuclei, and welldefined cell borders. Nuclear grooves are frequently notable, and scattered osteoclast-type giant cells are also present. In addition to nodules of chondroid material, a network of pericellular calcification is a characteristic finding in this tumor, so-called chicken-wire calcification. Aneurysmal bone cyst–like changes may be present. Chondroblastoma is usually strongly reactive for NSE and S-100 protein.553,554 In particular, the proliferating stromal cells in chondroblastomas show strong S-100 protein positivity, which facilitates their distinction from giant cell tumor of bone in most cases.555 Aberrant cytokeratin expression has been reported in occasional cases of chondroblastoma.556,557 The mononuclear chondroblastic tumor cells are negative for CD68.554,558 CHONDROSARCOMA
Chondrosarcoma is a malignant tumor of bone that shows pure cartilaginous differentiation. Secondary changes that include myxoid features, ossification, and calcification may be present. As a group, chondrosarcoma includes primary tumors and secondary tumors that arise in a precursor lesion (typically osteochondroma or enchondroma) or a hereditary disorder (Ollier disease and Maffuci syndrome). Distinctive, uncommon subtypes of chondrosarcoma include clear cell, mesenchymal, and periosteal chondrosarcoma. Dedifferentiated chondrosarcoma consists of a well-differentiated cartilaginous component juxtaposed to a high-grade noncartilaginous sarcoma. In general, IHC plays a limited role in the diagnosis of chondrosarcoma, which relies on morphologic, clinical, and radiologic features. Identification of a well-differentiated cartilaginous component next to an otherwise unclassified high-grade sarcoma allows the diagnosis of dedifferentiated chondrosarcoma to be reached. The only consistent IHC finding in chondrosarcoma is S-100 protein positivity; however, in dedifferentiated chondrosarcoma, tumor cells are usually negative for S-100 protein. Heterologous rhabdomyoblastic differentiation may occur in dedifferentiated chondrosarcoma, in which desmin and myogenin expression are observed. MESENCHYMAL CHONDROSARCOMA
Mesenchymal chondrosarcoma (MCS) is a rare, aggressive, malignant cartilaginous neoplasm. Histologically, mesenchymal chondrosarcoma consists of a population of undifferentiated small round cells, which may resemble Ewing sarcoma, but also contains islands of welldifferentiated cartilage. Another salient feature of MCS is the presence of branching hemangiopericytoma-like vasculature. Mesenchymal chondrosarcoma arises in young patients and shows a wide anatomic distribution, frequently occurring in craniofacial bones, ribs, and ilium. MCS may also arise primarily in soft tissue. In the absence of characteristic neoplastic cartilage, additional IHC and/or molecular studies may be needed to exclude Ewing sarcoma. Although the cartilaginous component
Specific Soft Tissue and Bone Tumors
of mesenchymal chondrosarcoma is S-100 protein positive, the small-cell component expresses CD99, CD57, and NSE; therefore immunohistochemically, there may also be overlap with Ewing sarcoma.275,559,560 However, unlike Ewing sarcoma, MCS is nonreactive for synaptophysin and also typically does not express desmin, actin, cytokeratin, or EMA. In addition, MCS lacks EWSR1 gene rearrangements. However, a recent study has identified a novel HEY1-NCOA2 fusion in MCS, which appears to be a consistent finding.561 CLEAR CELL CHONDROSARCOMA
Clear cell chondrosarcoma (CCC) is an uncommon subtype of chondrosarcoma that most commonly arises in the epiphyses of long bones of men, and it carries a relatively good prognosis when compared with other chondrosarcoma subtypes.562 The histologic pattern of this neoplasm is that of clear polygonal cells arranged in lobules with prominent zones of metaplastic ossification and scattered areas that resemble chondroblastoma. The tumor cells of CCC are reactive for S-100 protein, CD57, type II collagen, and lysozyme. Immunohistology plays a limited role in diagnosis of this variant.
Fibrous Tumors of Bone CHONDROMYXOID FIBROMA
Chondromyxoid fibroma is an uncommon benign tumor that most often arises in long bones, particularly in the proximal tibia and distal femur, but any bone may be involved. The tumor has a relatively distinct morphologic appearance: it is composed of stellate or spindleshaped cells within a myxoid stroma in a lobular growth pattern, and additional findings may include hyaline cartilage deposition, aneurysmal bone cyst–like changes, calcifications, and degenerative changes. The lesional cells of chondromyxoid fibroma express S-100 protein in the majority of cases.563,564 Expression of SMA and CD34 has been reported to occur at the periphery of tumor lobules.564 DESMOPLASTIC FIBROMA OF BONE
Desmoplastic fibroma is composed of long fascicles of fibroblasts and myofibroblasts with minimal atypia in a collagenous or hyalinized stroma. However, despite morphologic similarities to desmoid fibromatosis, nuclear expression of β-catenin is rare and occurs in just one of 13 cases in one study.565 As expected given their appearances, expression of SMA or desmin is a more frequent finding. This tumor has a tendency for locally aggressive behavior.
Bone-Forming Tumors OSTEOSARCOMA
Osteosarcoma is the most common nonhematopoietic primary malignant bone tumor. Conventional osteosarcoma is most frequent in young patients and shows a
125
male predominance. By definition, conventional osteosarcoma is a high-grade malignant tumor in which the neoplastic cells produce osteoid. Morphologic variants of conventional osteosarcoma include osteoblastic, chondroblastic, and fibroblastic types. Clinicopathologically distinct subtypes include telangiectatic osteosarcoma, small cell osteosarcoma, low-grade central osteosarcoma, parosteal osteosarcoma, periosteal osteosarcoma, high-grade surface osteosarcoma, and secondary osteosarcoma. IHC studies of osteosarcoma have revealed few characteristic features and are used primarily to help exclude other lesions that fall within the differential diagnosis, particularly sarcomatoid carcinoma and synovial sarcoma. The bone matrix proteins osteocalcin and osteonectin have been reported to highlight osteoid deposition, but their efficacy in identifying osteosarcoma still needs further substantiation. Positive staining for osteonectin has been identified in the neoplastic cells of osteosarcoma and osteoblastoma, but expression within the mononuclear cells in giant cell tumors and chondroblastomas has also been observed.566 Overall, the reported specificity of immunoreactivity for osteonectin and osteocalcin is approximately 40% and 95%, respectively, for the diagnosis of a boneforming tumor.313,315 Those two markers may be useful as part of a panel of immunostains to corroborate a diagnosis of extraskeletal osteosarcoma, but identification of osteoid matrix associated with the constituent malignant cells remains the sine qua non of diagnosis. A recent promising marker for identification of osteoblastic differentiation is SATB2, a nuclear matrix protein that plays a role in osteoblast lineage commitment.543 It is important to note that occasionally osteosarcomas may show expression of cytokeratins, as well as α-SMA and desmin, which can lead to misdiagnosis.544 Cytoplasmic expression of CD99 is not uncommon, especially in the small cell subtype, in which the differential diagnosis may include Ewing sarcoma; identification of osteoid deposition essentially excludes Ewing sarcoma. Recent studies have shown that overexpression of MDM2 and CDK4 is a frequent finding in low-grade osteosarcomas and corresponds to amplification of these two genes. MDM2 and CDK4 amplification occurs in 67% of low-grade osteosarcomas and in only 12% of high-grade osteosarcomas.567 This finding has been shown to be of diagnostic utility, because in one study, all parosteal and central low-grade osteosarcomas expressed MDM2 and/or CDK4, usually diffusely with moderate or strong intensity, whereas expression of these markers in benign morphologic mimics of these tumors was extremely limited, with expression seen only in one case of bizarre parosteal osteochondromatous proliferation.568 Expression of MDM2 and CDK4 is limited to low-grade osteosarcomas and their dedifferentiated counterparts, whereas staining is generally negative in primary, conventional, high-grade osteosarcoma.569 Finding expression of these two markers in an otherwise nondistinctive high-grade sarcoma of bone may therefore suggest evolution from (dedifferentiation of) a low-grade osteosarcoma.
126
Immunohistology of Neoplasms of Soft Tissue and Bone
A
B Figure 4-25 Ewing sarcoma (A) has a distinctive pattern of diffuse strong membranous expression of CD99 (B).
Other Selected Bone Tumors EWING SARCOMA
Ewing sarcoma (ES) is a small round cell neoplasm of bone and soft tissue that is in part defined by the recurrent chromosomal translocation t(11;22)(q24;q12) that involves EWSR1, located on 22q12 and FLI1 on 11q24, which is present in 90% of cases, but it also involves less common translocation variants. The classic morphologic appearance of ES is one of uniform, small round cells with central round nuclei with fine chromatin and small amounts of eosinophilic to clear cytoplasm (Fig. 4-25). The tumor cells may form Homer-Wright rosettes, and occasional cases consist of larger tumor cells with prominent nucleoli. Very rarely, a minor spindle cell component may be seen. In general, however, spindle cell morphology essentially excludes the diagnosis. Over the past 15 years, it has become clear that ES and primitive neuroectodermal tumor (PNET) represent the same tumor type.570,571 As classically defined, ES was distinguished from PNET by an absence of pseudorosettes and the lack of ultrastructurally or immunohistochemically detectable neuroectodermal features. However, this diagnostic separation is now considered to be antiquated and has been abandoned. The EWSR1 and FLI1 genes flank the translocation breakpoint in ES/PNET. Reverse transcription (RT) PCR methods have allowed for detection of the chimeric mRNA transcripts produced by the fusion of those genes.572,573 EWSR1/FLI1 transcripts have not been detected in other small cell tumors. A minority of Ewing sarcomas will harbor a translocation that involves EWSR1 and an alternative partner, the most common of which is ERG, located on 21q12. The (21;22) (q22;q12) translocation is present in 5% to 10% of ES. In less than 1% of tumors, the EWSR1 fusion partners include ETV1, ETVF (formerly E1AF), FEV, and ZSG. The resulting translocations lead to constitutive expression of an abnormal transcription factor that is critical for ES tumorigenesis. The transcripts encode the amino end of EWSR1, the transcriptional activation domain, and the C-terminus (the DNA-binding domain) of the
corresponding fusion partner. FLI1 and ERG belong to the ETS family of transcription factors, which regulate several genes involved in cellular differentiation and growth.574,575 Rearrangements of EWSR1 with non–ETS-family genes—including NFATC2, POU5F1, SMARCA5, ZSG, and SP3—are also rarely identified.575-578 Detection of EWSR1 rearrangement by FISH is also diagnostically useful, but because of the nonspecificity of this finding, care must be taken in its interpretation. ES is characterized by diffuse membranous MIC2/ CD99 expression (see Fig. 4-25). Expression of CD99, a glycoprotein, can be detected by various monoclonal antibodies that include HBA71, 12E7, RFB-1329, and O13. It is present in virtually all cases of ES.579,580 Although it was initially thought to be specific for ES/ PNET, CD99 has since been identified in a variety of other tumors. In the small round blue cell group of pediatric neoplasms, lymphoblastic lymphoma and selected cases of alveolar RMS represent the principal CD99-positive alternatives to ES/PNET. Other tumors that may show CD99 expression include neuroendocrine carcinomas, mesenchymal chondrosarcoma, solitary fibrous tumors, and synovial sarcoma. However, strong, diffuse membranous reactivity for CD99 favors ES over other diagnostic considerations (the other CD99-positive tumors usually show cytoplasmic and more heterogeneous staining). Obviously, CD99 must be considered along with several other determinants in making a final diagnostic interpretation. Concomitant nuclear labeling for FLI1 is seen in approximately 70% of ES/PNET cases, but because of its low specificity, the use of this marker in ES is declining.581 FLI1 is not specific to rearrangement type, probably because of cross-reactivity with the highly homologous ETS DNAbinding domain present in the C-terminus of both ERG and FLI1. However, strong nuclear ERG immunoreactivity is relatively specific for ES with EWSR1-ERG rearrangement.192 Typical ES/PNET is nonreactive for chromogranin, GFAP, desmin, muscle-specific actin, myogenin, CD31, and CD45.582,583 However, in studies of tumors
Specific Soft Tissue and Bone Tumors
confirmed by molecular identification of the (11;22) translocation, immunoreactivity for cytokeratin was present in 20% to 30% of ES cases.584 Nonetheless, keratin usually shows relatively limited expression in these tumors when present. NB84, a marker developed for recognition of neuroblastoma, is also detected in roughly 20% of ES/PNETs.278 Recently, through gene-expression profiling, NKX2.2, a homeodomain-containing transcription factor that plays a critical role in neuroendocrine/glial differentiation, was shown to be differentially upregulated in Ewing sarcoma. The NKX2.2 gene is a target of EWSR1FLI1 fusion protein. A large IHC study demonstrated diffuse staining for NKX2.2 in 93% of 30 genetically confirmed ES cases examined, and staining was mostly moderate to strong in intensity. NKX2.2 was also positive in 11% of other tumor types, including all olfactory neuroblastomas examined and a subset of small cell carcinomas, synovial sarcomas, mesenchymal chondrosarcomas, and malignant melanomas. The sensitivity and specificity of NKX2.2 for Ewing sarcoma was 93% and 89%, respectively, although this study requires confirmation by other groups.585 KEY DIAGNOSTIC POINTS Ewing Sarcoma • Ewing sarcoma is typically positive for FLI1, CD99 (diffuse membranous), and NSE. • Keratin is positive in 20% to 30% of cases, usually with a focal distribution. • A differential panel should include muscle markers, leukocyte common antigen, and terminal deoxynucleotidyl transferase to exclude rhabdomyosarcoma and lymphoma, respectively.
CHORDOMA
Chordoma is a malignant tumor with notochordlike differentiation that principally occurs within the axial
A
127
skeleton, most commonly the sacral area, followed by occipital, cervical, and thoracolumbar sites; peripheral lesions are extremely rare.586,587 Chordoma is classically composed of cords and sheets of tumor cells within an abundant myxoid stroma and usually shows a multilobular growth pattern. Tumor cells have abundant pale, eosinophilic, microvacuolated cytoplasm and so-called physaliphorous cells (Fig. 4-26). The myxoid stroma of chordoma may mimic cartilage. The morphologic differential diagnosis of chordoma includes metastatic adenocarcinoma (especially RCC), chondrosarcoma, myxopapillary ependymoma, myoepithelial tumors, and EHE.136 Keratin expression is characteristic of chordoma. The subtypes of keratin proteins in this neoplasm potentially include cytokeratins 1, 5, 8, 10, 14 through 16, 18, and 19, and CK5 predominates.588 Chordoma also expresses EMA, and S-100 protein expression is found in approximately 60% to 80% of cases.136,137 Recently, expression of brachyury (T), a transcription factor involved in notochord development, was found to be a sensitive and specific marker of chordoma. Expression of brachyury in chordoma has been reported to occur in nearly 100% of cases,317 although a few reports of examples of brachyury-negative chordoma exist in the literature (see Fig. 4-26). Brachyury expression is not found in morphologic mimics of chordoma such as chondrosarcoma, myoepithelial tumors, and RCC.317,319 With regard to differential diagnosis, expression of EMA and keratins by chordoma, as well as the morphologic appearances, raises the differential diagnosis of metastatic RCC; brachyury and PAX8 are helpful markers for distinguishing between these two tumors, because chordomas rarely express PAX8, with only one case reported to show weak expression in a study by Sangoi and colleagues.319 Chondrosarcoma shares S-100 protein expression but is uniformly negative for epithelial markers and brachyury.137,317 Moreover, N-cadherin is virtually always expressed by chordoma but only rarely by chondrosarcoma. Conversely, Huse and
B
Figure 4-26 Tumor cells of chordoma are typically large with voluminous eosinophilic, microvacuolated, or clear cytoplasm (A). Brachyury, a transcription factor involved in notochord development, is expressed in virtually all cases of chordoma (B).
128
Immunohistology of Neoplasms of Soft Tissue and Bone
associates589 have shown that low-grade cartilaginous neoplasms of the skull base are podoplanin/D2-40positive, whereas chordoma is not. Some cases of EHE express cytokeratins, but they also exhibit reactivity for CD31 and ERG and lack S-100 protein expression. Finally, ependymomas are positive for GFAP,136 unlike the other differential diagnostic possibilities discussed here. Dedifferentiated chordoma is very rare and consists of a high-grade sarcomatous component that arises in association with chordoma.590 Occasional S-100 protein–positive cells may persist in the dedifferentiated component, but cytokeratin is usually negative. Divergent mesenchymal differentiation may also be encountered.591 In 1973, Heffelfinger and associates592 described a particular variant of chordoma with cartilaginous areas that simulated the appearance of chondrosarcoma.593 They designated these lesions as chondroid chordomas and suggested that they had a better prognosis than that of conventional chordoma. Despite morphologic differences, the IHC staining pattern of chondroid chordoma is basically identical to that seen in classical forms of this tumor.137,592-594 KEY DIAGNOSTIC POINTS Chordoma • Chordoma typically expresses cytokeratin, epithelial membrane antigen, S-100 protein, and brachyury. • Differential diagnosis includes metastatic renal cell carcinoma and chondrosarcoma, both of which are brachyury negative.
GIANT CELL TUMORS OF BONE AND SOFT TISSUE
Giant cell tumors of bone are benign but locally aggressive neoplasms that typically involve the epiphyses or proximal metaphyses of long tubular bones. The tumors are composed of sheets of ovoid mononuclear cells that represent the neoplastic component, with admixed osteoclast-like giant cells that often contain large numbers of nuclei. The osteoclast-like giant cells usually stain strongly for CD68595-597; however, mononuclear elements label either more weakly for that marker or are negative. The stromal cells may express α-SMA, but they lack CD45, S-100 protein, desmin, and lysozyme.595,596 Recent reports have suggested that p63 expression may distinguish these forms of giant cell tumor from some of their histologic mimics, in particular tenosynovial giant cell tumors (giant cell tumor of tendon sheath and diffuse-type giant cell tumor) and aneurysmal bone cyst.598,599 However, we have not been able to reproduce these findings in our laboratory. Morphologically similar lesions may also arise rarely as primary lesions in the subcutis or deep soft tissue.600,601 Tenosynovial giant cell tumors occur in two forms, localized and diffuse types; the latter was formerly also known as pigmented villonodular synovitis. The localized type is well circumscribed and most commonly arises in the fingers, called a giant cell tumor of tendon sheath.
The diffuse type is more commonly associated with larger joints and shows irregularly infiltrative margins. The constituent cell types are similar in both variants and comprise bland, mononuclear histiocytoid cells, a subset of which are somewhat larger, with eccentric nuclei and more abundant cytoplasm, sometimes containing fine hemosiderin pigment, with variable numbers of admixed multinucleate osteoclast-like giant cells and foam cells. The IHC profile of both types of tenosynovial giant cell tumor is similar. The small mononuclear cells, which are of monocyte/macrophage lineage, express CD68 and CD163; the larger mononuclear cells express desmin and muscle-specific actin in some cases. The multinucleated cells are also positive for CD68 and CD45.601-603 In addition, the larger mononuclear cells show diffuse and strong expression of the chaperone glycoprotein clusterin.604 LANGERHANS CELL HISTIOCYTOSIS
Langerhans cell histiocytosis (LCH) is a neoplastic proliferation of Langerhans cells that can arise in bone or soft tissues. This tumor type is rare and accounts for less than 1% of all bone tumors. Patients of any age may be affected, and although the anatomic distribution is wide, there is a predilection for skull bones and ribs. Recognition of Langerhans cells among the characteristic admixed inflammatory cell population, usually with a prominent population of eosinophils, may be difficult. The neoplastic cells have oval nuclei, often with grooves or indentations, and palely eosinophilic or clear cytoplasm. IHC is extremely useful in confirming the diagnosis of LCH. The tumor cells are typically positive for S-100 protein, CD1a, and the more recently discovered langerin, a highly sensitive marker of Langerhans cells.605 Recent studies have identified BRAF V600E mutations in approximately 50% of cases of LCH.606 ERDHEIM-CHESTER DISEASE
Erdheim-Chester is a rare disorder in which lipid-laden histiocytes infiltrate bone and viscera, with resultant fibrosis and osteosclerosis. The long bones are most commonly affected, and imaging studies demonstrate bilateral sclerosis and increased uptake by bone scan. Histologically, a diffuse infiltrate of foamy histiocytes is seen associated with an admixed inflammatory infiltrate of lymphocytes, plasma cells, and occasional Toutontype giant cells. Sclerosis of involved bone is characteristic. IHC can help confirm the histiocytic nature of the cells, which are positive for CD68, CD163, and lysozyme. S-100 protein is rarely positive, and CD1a is consistently negative, aiding in the differential diagnosis with LCH. Interestingly, a recent study reported BRAF mutations in some cases of Erdheim-Chester disease, similar to LCH.607 ADAMANTINOMA
Adamantinoma of long bones is a rare low-grade malignant neoplasm that typically arises in the metaphysis/diaphysis of the mid tibia in young adults.
Summary
A
129
B
Figure 4-27 Adamantinoma is composed of nests of epithelioid cells with a somewhat basaloid appearance (A) and shows diffuse expression of cytokeratin 5 (B).
Occasionally it forms part of a lesional continuum with a disorder known as osteofibrous dysplasia (Campanacci disease).608,609 Adamantinoma characteristically shows a biphasic appearance histologically, being composed of nests of compact epithelial cells dispersed within a cellular stroma (Fig. 4-27). The epithelial cells often have a basaloid or squamoid appearance. Adamantinoma is reactive for cytokeratin, and keratins 5, 14, and 19 predominate, reflecting a basal phenotype (see Fig. 4-27).610-615 Keratins 1, 13, and 17 are variably positive, and keratins 8 and 18 are negative. EMA is also typically positive in the epithelial component. A recent study has documented expression of podoplanin in most cases of adamantinoma.616 Because of its biphasic morphology and its epithelial immunophenotype, there has been speculation as to whether adamantinoma was the intraosseous counterpart of synovial sarcoma. However, these two neoplasms have completely dissimilar cytogenetic profiles and thus are now known to be distinct examples of mesenchymal tumors that demonstrate epithelial differentiation.613,614,617 From a pathologic perspective, difficulties can arise when considering the differential diagnosis of adamantinoma and metastatic carcinoma, particularly in biopsy specimens. To date, no systematic IHC comparison of those two groups of lesions has been done. Nevertheless, the most straightforward approach to this problem is to
review imaging studies of the lesion in question; adamantinoma has a distinctive radiographic appearance,609 which differs markedly from that of metastatic carcinoma. Additional lineage-specific immunostains may be helpful in evaluating possible metastatic lesions.
Summary Proper diagnosis of soft tissue and bone tumors requires a great deal of clinical information in addition to morphology, IHC, and frequent ISH probe examinations. IHC provides vital supplemental information to morphology to guide proper appropriate studies such as ISH probes.
Acknowledgment We are grateful to Dr. Mark Wick, who was the author of this chapter in previous editions and was responsible for much of the content in the sections on biology of antigens and antibodies. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Denk H, Krepler R, Artlieb U, et al: Proteins of intermediate filaments: An immunohistochemical and biochemical approach to the classification of soft tissue tumors. Am J Pathol. 110:193–208, 1983. 2. Lazarides E: Intermediate filaments: A chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem. 51:219–250, 1982. 3. Erlandson RA: Diagnostic transmission electron microscopy of tumors, New York, 1994, Lippincott, Williams & Wilkins, p 165. 4. Osborn M, Mandys V, Beddow E, et al: Cystic fibrohistiocytic tumours presenting in the lung: primary or metastatic disease? Histopathology. 43:556–562, 2003. 5. Anderton BH: Intermediate filaments: A family of homologous structures. J Muscle Res Cell Motil. 2:141–166, 1981. 6. Hermann H, Aebi U: Intermediate filaments and their associates: Multitalented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol. 12:79–90, 2000. 7. Mischke D: The complexity of gene families involved in epithelial differentiation: Keratin genes and the epidermal differentiation complex. Subcell Biochem. 31:71–104, 1998. 8. Saavedra-Matiz CA, Chapman NH, Wijsman EM, et al: Linkage of hereditary distal myopathy with desmin accumulation to 2q. Hum Hered. 50:166–170, 2000. 9. Ferrari S, Cannizzaro LA, Battini R, et al: The gene encoding human vimentin is located on the short arm of chromosome 10. Am J Hum Genet. 41:616–626, 1987. 10. Yoshime T, Maruno M, Kumura E, et al: Stochastic determination of the chromosomal region responsible for expression of human glial fibrillary acidic protein in astrocytic tumors. Neurosci Lett. 247:29–32, 1998. 11. Goldman R, Goldman AE, Green K, et al: Intermediate filaments: Possible functions as cytoskeletal connecting links between the nucleus and the cell surface. Ann NY Acad Sci. 455:1–17, 1985. 12. Osborn M: Summary: Intermediate filaments 1984. Ann NY Acad Sci. 455:669–681, 1985. 13. Vandenburgh HH: Cell shape and growth regulation in skeletal muscle: Exogenous versus endogenous factors. J Cell Physiol. 116:363–371, 1983. 14. Miettinen M: Keratin immunohistochemistry: Update of applications and pitfalls. Pathol Annu. 28(Pt. 2):113–143, 1993. 15. Moll R, Franke WW, Schiller DL, et al: The catalogue of human cytokeratins: Patterns of expression in normal epithelia, tumors, and cultured cells. Cell. 31:11–24, 1982. 16. Miettinen M, Limon J, Niezabitowski A, et al: Patterns of keratin polypeptides in 110 biphasic, monophasic, and poorlydifferentiated synovial sarcomas. Virchows Arch. 437:275–283, 2000. 17. O’Hara BJ, Paetau A, Miettinen M: Keratin subsets and monoclonal antibodies HBME-1 in chordoma: Immunohistochemical differential diagnosis between tumors simulating chordoma. Hum Pathol. 29:119–126, 1998. 18. Fisher C, Miettinen M: Parachordoma: A clinicopathologic and immunohistochemical study of four cases of an unusual soft tissue neoplasm. Ann Diagn Pathol. 1:3–10, 1997. 19. Hornick JL, Fletcher CD: Myoepithelial tumors of soft tissue: a clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters. Am J Surg Pathol. 27:1183–1196, 2003. 20. Swanson PE, Dehner LP, Sirgi KE, et al: Cytokeratin immunoreactivity in malignant tumors of bone and soft tissue. Appl Immunohistochem. 2:103–112, 1994. 21. Bahrami A, Gown AM, Baird GS, et al: Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol. 21:795–806, 2008. 22. Dahl D: Immunohistochemical differences between neurofilaments in perikarya, dendrites, and axons: Immunofluorescence study with antisera raised to neurofilament polypeptides (200 kD, 150 kD, and 70 kD) isolated by anion exchange chromatography. Exp Cell Res. 149:397–408, 1983. 23. Hickey WF, Lee V, Trojanowski JQ, et al: Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triplet protein subunits: Advantage
129.e1
over antisera and technical limitations. J Histochem Cytochem. 31:1126–1135, 1983. 24. Shaw G: Neurofilaments: Abundant but mysterious neuronal structures. Bioessays. 4:161–166, 1986. 25. Tapscott SJ, Bennett GS, Toyama Y, et al: Intermediate filament proteins in the developing chick spinal cord. Dev Biol. 86:40–54, 1981. 26. Tremblay GF, Lee VMY, Trojanowski JQ: Expression of vimentin, glial filament, and neurofilament proteins in-primitive childhood brain tumors: A comparative immunoblot and immunoperoxidase study. Acta Neuropathol. 68:239–244, 1985. 27. Lee VMY, Carden MJ, Trojanowski JQ: Novel monoclonal antibodies provide evidence for the in-situ existence of a nonphosphorylated form of the large neurofilament subunit. J Neurosci. 6:850–858, 1986. 28. Brown A: Contiguous phosphorylated and non-phosphorylated domains along axonal neurofilaments. J Cell Sci. 111:455–467, 1998. 29. Sternberger LA, Sternberger NH: Monoclonal antibodies distinguish phosphorylated and non-phosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci USA. 80:6126–6130, 1983. 30. Ulfig N, Nickel J, Bohl J: Monoclonal antibodies SMI311 and SMI312 as tools to investigate the maturation of nerve cells and axonal patterns in human fetal brain. Cell Tissue Res. 291:433– 443, 1998. 31. Trojanowski JQ, Lee VMY, Schlaepfer WW: An immunohistochemical study of central and peripheral nervous system tumors with monoclonal antibodies against neurofilaments and glial filaments. Hum Pathol. 15:248–257, 1984. 32. Trojanowski JQ, Lee VMY: Expression of neurofilament antigens by normal and neoplastic human adrenal chromaffin cells. N Engl J Med. 313:101–104, 1985. 33. Miettinen M, Lehto VP, Virtanen I: Immunofluorescence microscopic evaluation of the intermediate filament expression of the adrenal cortex and medulla and their tumors. Am J Pathol. 118:360–366, 1985. 34. Schmidt U, Muller U, Metz KA, et al: Cytokeratin and neurofilament protein staining in Merkel cell carcinoma of the small cell type and small cell carcinoma of the lung. Am J Dermatopathol. 20:346–351, 1998. 35. Shaw G, Weber K: The intermediate filament complement of the brain: A comparison between different mammalian species. Eur J Cell Biol. 33:95–104, 1984. 36. Osborn M, Altmannsberger M, Shaw G, et al: Various-sympathetic derived human tumors differ in neurofilament-expression: Use in diagnosis of neuroblastoma, ganglioneuroblastoma, and pheochromocytoma. Virchows Arch B Cell Pathol. 40:141–156, 1982. 37. Trojanowski JQ: Neurofilament and glial filament proteins. In Wick MR, Siegal GP, editors: Monoclonal Antibodies in Diagnostic Immunohistochemistry, New York, 1988, Marcel De, pp 115–146. 38. Dolman CL: Glial fibrillary acidic protein and cartilage. Acta Neuropathol. 79:101–103, 1989. 39. Yasuda T, Sobue G, Ito T, et al: Human peripheral nerve sheath neoplasms: Expression of Schwann cell-related markers and their relation to malignant transformation. Muscle Nerve. 14:812–819, 1991. 40. Lodding P, Kindblom LG, Angervall L, et al: A clinicopathologic study of 29 cases. Virchows Arch A. 416:237–248, 1990. 41. Giangaspero F, Fratamico FC, Ceccarelli C, et al: Malignant peripheral nerve sheath tumors and spindle cell sarcomas: An immunohistochemical analysis of multiple markers. Appl Pathol. 7:134–144, 1989. 42. Kawahara E, Oda Y, Ooi A, et al: Expression of glial fibrillary acidic protein (GFAP) in peripheral nerve sheath tumors: A comparative study of immunoreactivity of GFAP, vimentin, S100 protein, and neurofilament in 38 schwannomas and 18 neurofibromas. Am J Surg Pathol. 12:115–120, 1988. 43. Memoli VA, Brown EF, Gould VE: Glial fibrillary acidic protein (GFAP) immunoreactivity in peripheral nerve sheath tumors. Ultrastruct Pathol. 7:269–275, 1984. 44. Gleason BC, Fletcher CD: Myoepithelial carcinoma of soft tissue in children: an aggressive neoplasm analyzed in a series of 29 cases. Am J Surg Pathol. 31:1813–1824, 2007.
129.e2
Immunohistology of Neoplasms of Soft Tissue and Bone
45. Hornick JL, Fletcher CD: Cutaneous myoepithelioma: a clinicopathologic and immunohistochemical study of 14 cases. Hum Pathol. 35:14–24, 2004. 46. Geisler N, Plessmann U, Weber K: Amino acid sequence characterization of mammalian vimentin, the mesenchymal intermediate filament protein. FEBS Lett. 163:22–24, 1983. 47. Gereben B, Leuheiber K, Rausch WD, et al: Inverse hierarchy of vimentin epitope expression in primary cultures of chicken and rat astrocytes: A double immunofluorescence study. Neurobiology. 6:141–150, 1998. 48. Geisler N, Weber K: Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: Desmin and vimentin. Proc Natl Acad Sci USA. 78:4120–4123, 1981. 49. Battifora H: Assessment of antigen damage in immunohistochemistry: The vimentin internal control. Am J Clin Pathol. 96:669–671, 1991. 50. Fanburg-Smith JC, Hengge M, Hengge UR, et al: Extrarenal rhabdoid tumors of soft tissue: a clinicopathologic and immunohistochemical study of 18 cases. Ann Diagn Pathol. 2:351–362, 1998. 51. Swanson PE: Monoclonal antibodies to human milk fat globule proteins. In Wick MR, Siegal GP, editors: Monoclonal Antibodies in Diagnostic Immunohistochemistry, New York, 1988, Marcel De, pp 227–283. 52. Petersen OW, VanDeuers B: Characterization of epithelial membrane antigen expression in human mammary epithelium by ultrastructural immunoperoxidase cytochemistry. J Histochem Cytochem. 34:801–809, 1986. 53. Uematsu Y, Rojas-Corona RR, Llena JF, et al: Distribution of epithelial membrane antigen in normal and neoplastic human ependyma. Acta Neuropathol. 78:325–328, 1989. 54. Heyderman E, Steele K, Ormerod MG: A new antigen on the epithelial membrane: its immunoperoxidase localisation in normal and neoplastic tissue. J Clin Pathol. 32:35–39, 1979. 55. Sloane JP, Ormerod MG: Distribution of epithelial membrane antigen in normal and neoplastic tissues and its value in diagnostic tumor pathology. Cancer. 47:1786–1795, 1981. 56. Wick MR, Swanson PE, Manivel JC: Immunohistochemical analysis of soft tissue sarcomas: Comparisons with electron microscopy. Appl Pathol. 6:169–196, 1988. 57. Doyle LA, Fletcher CD: EMA positivity in epithelioid fibrous histiocytoma: a potential diagnostic pitfall. J Cutan Pathol. 38:697–703, 2011. 58. Hornick JL, Fletcher CD: Soft tissue perineurioma: clinicopathologic analysis of 81 cases including those with atypical histologic features. Am J Surg Pathol. 29:845–858, 2005. 59. Miettinen M, Fanburg-Smith JC, Virolainen M, et al: Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis. Hum Pathol. 30:934–942, 1999. 60. Folpe AL, Schmidt RA, Chapman D, et al: Poorly differentiated synovial sarcoma: immunohistochemical distinction from primitive neuroectodermal tumors and high-grade malignant peripheral nerve sheath tumors. Am J Surg Pathol. 22:673–682, 1998. 61. Fanburg-Smith JC, Miettinen M: Angiomatoid “malignant” fibrous histiocytoma: a clinicopathologic study of 158 cases and further exploration of the myoid phenotype. Hum Pathol. 30:1336–1343, 1999. 62. Guillou L, Benhattar J, Gengler C, et al: Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group. Am J Surg Pathol. 31:1387–1402, 2007. 63. Pinkus GS, Kurtin PJ: Epithelial membrane antigen–a diagnostic discriminant in surgical pathology: immunohistochemical profile in epithelial, mesenchymal, and hematopoietic neoplasms using paraffin sections and monoclonal antibodies. Hum Pathol. 16:929–940, 1985. 64. Franke WW, Moll R, Mueller H, et al: Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins. Proc Natl Acad Sci USA. 80:543–547, 1983.
65. Arnemann J, Spurr NK, Magee AI, et al: The human gene (DSG2) coding for HDGC, a second member of the desmoglein subfamily of the desmosomal cadherins, is, like DSG1 coding for desmoglein DG1, assigned to chromosome 18. Genomics. 13:484–486, 1992. 66. Laskin WB, Miettinen M: Epithelial-type and neural-type cadherin expression in malignant noncarcinomatous neoplasms with epithelioid features that involve the soft tissues. Arch Pathol Lab Med. 126:425–431, 2002. 67. Sata H, Hasegawa T, Abe Y, et al: Expression of E-cadherin in bone and soft tissue sarcomas: A possible role in epithelial differentiation. Hum Pathol. 30:1344–1349, 1999. 68. Truong LD, Rangdaeng S, Cagle P, et al: The diagnostic utility of desmin: A study of 584 cases and review of the literature. Am J Clin Pathol. 93:305–314, 1990. 69. Rangdaeng S, Truong LD: Comparative immunohistochemical staining for desmin and muscle specific actin: A study of 576 cases. Am J Clin Pathol. 96:32–45, 1991. 70. Small JV, Sobieszek A: Studies on the function and composition of the 10-nm (100 A) filaments of vertebrate smooth muscle. J Cell Sci. 23:243–268, 1977. 71. Lazarides E, Balzer DR Jr: Specificity of desmin to avian and mammalian muscle cells. Cell. 14:429–438, 1978. 72. Geisler N, Weber K: The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins. EMBO J. 1:1649–1656, 1982. 73. Ip W, Heuser JE: Subunit structure of desmin and vimentin protofilaments and how they assemble into intermediate filaments. Ann NY Acad Sci. 455:185–199, 1985. 74. Ngai J, Capetanaki YG, Lazarides E: Expression of the genes encoding for the intermediate filament proteins vimentin and desmin. Ann NY Acad Sci. 455:144–155, 1985. 75. Tokuyasu KT, Maher PA, Dutton AH, et al: Intermediate filaments in skeletal and cardiac muscle tissue in embryonic and adult chicken. Ann NY Acad Sci. 455:200–212, 1985. 76. Schmid E, Osborn M, Rungger-Brandle E, et al: Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta. Exp Cell Res. 137:329–340, 1982. 77. Richardson FL, Stromer MH, Huiatt TW, et al: Immunoelectron and immunofluorescence localization of desmin in mature avian muscles. Eur J Cell Biol. 26:91–101, 1981. 78. Daste G, Gioanni J, Lauque D, et al: GC12, marker of cells of mesodermal origin: Value and application to cytodiagnosis of serous effusions. Arch Anat Cytol Pathol. 45:185–191, 1997. 79. Pollock L, Rampling D, Greenwald SE, et al: Desmin expression in forms: Influence of the desmin clone and immunohistochemical method. J Clin Pathol. 48:535–538, 1995. 80. Chang TK, Li CY, Smithson WA: Immunocytochemical study of small round cell tumors in routinely processed specimens. Arch Pathol Lab Med. 113:1343–1348, 1989. 81. Schurch W, Skalli O, Seemayer TA, et al: Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. I Smooth Muscle Tumors. Am J Pathol. 128:91–103, 1987. 82. Volpicelli ER, Fletcher CD: Desmin and CD34 positivity in cellular fibrous histiocytoma: an immunohistochemical analysis of 100 cases. J Cutan Pathol. 39:747–752, 2012. 83. Fetsch JF, Laskin WB, Lefkowitz M, et al: Aggressive angiomyxoma: a clinicopathologic study of 29 female patients. Cancer. 78:79–90, 1996. 84. Granter SR, Nucci MR, Fletcher CD: Aggressive angiomyxoma: Reappraisal of its relationship to angiomyofibroblastoma in a series of 16 cases. Histopathology. 30:3–10, 1997. 85. Fletcher CDM, Tsang WTW, Fisher C, et al: Angiomyofibroblastoma of the vulva: A benign neoplasm distinct from aggressive angiomyxoma. Am J Surg Pathol. 16:373–382, 1992. 86. Liegl B, Hornick JL, Fletcher CD: Primary cutaneous PEComa: distinctive clear cell lesions of skin. Am J Surg Pathol. 32:608– 614, 2008. 87. Hornick JL, Fletcher CD: Sclerosing PEComa: clinicopathologic analysis of a distinctive variant with a predilection for the retroperitoneum. Am J Surg Pathol. 32:493–501, 2008. 88. Graham RP, Dry S, Li X, et al: Ossifying fibromyxoid tumor of soft parts: a clinicopathologic, proteomic, and genomic study. Am J Surg Pathol. 35:1615–1625, 2011.
References 89. Fletcher CD: Angiomatoid “malignant fibrous histiocytoma”: an immunohistochemical study indicative of myoid differentiation. Hum Pathol. 22:563–568, 1991. 90. Fanburg-Smith JC, Miettinen M: Angiomatoid “malignant” fibrous histiocytoma: a clinicopathologic study of 158 cases and further exploration of the myoid phenotype. Hum Pathol. 30:1336–1343, 1999. 91. Lae ME, Roche PC, Jin L, et al: Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol. 26:823–835, 2002. 92. Hurlimann J: Desmin and neural marker expression in mesothelial cells and mesotheliomas. Hum Pathol. 25:753–757, 1994. 93. Toccanier-Pelte MF, Skalli O, Kapanci Y, et al: Characterization of stromal cells with myoid features in lymph nodes and spleen in normal and pathologic conditions. Am J Pathol. 129:109–118, 1987. 94. Folpe AL, Patterson K, Gown AM: Antibodies to desmin identify the blastemal component of nephroblastoma. Mod Pathol. 10:895–900, 1997. 95. Mayall FG, Goddard H, Gibbs AR: Intermediate filament expression in mesotheliomas: leiomyoid mesotheliomas are not uncommon. Histopathology. 21:453–457, 1992. 96. Miettinen M: Antibody specificity to muscle actins in the diagnosis and classification of soft tissue tumors. Am J Pathol. 130:205–215, 1988. 97. Tsukada T, McNutt MA, Ross R, et al: HHF35, a muscle actinspecific monoclonal antibody. II Reactivity in Normal, Reactive, and Neoplastic Human Tissue. Am J Pathol. 127:389–402, 1987. 98. Jones H, Steart PV, DuBoulay CE, et al: Alpha-smooth muscle actin as a marker for soft tissue tumors: A comparison with desmin. J Pathol. 162:29–33, 1990. 99. Cintorino M, Vindigni C, DelVecchio MT, et al: Expression of actin isoforms and intermediate filament proteins in childhood orbital rhabdomyosarcomas. J Submicrosc Cytol Pathol. 21:409– 419, 1989. 100. Bussolati G, Papotti M, Foschini MP, et al: The interest of actin immunocytochemistry in diagnostic histopathology. Basic Appl Histochem. 31:165–176, 1987. 101. Skalli O, Gabbiani G, Babai F, et al: Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. II. Rhabdomyosarcomas. Am J Pathol. 130:515–531, 1988. 102. Hornick JL, Fletcher CD: Pseudomyogenic hemangioendothelioma: a distinctive, often multicentric tumor with indolent behavior. Am J Surg Pathol. 35:190–201, 2011. 103. Montgomery EA, Meis JM: Nodular fasciitis. Its morphologic spectrum and immunohistochemical profile. Am J Surg Pathol. 15:942–948, 1991. 104. Perez-Montiel MD, Plaza JA, Dominguez-Malagon H, et al: Differential expression of smooth muscle myosin, smooth muscle actin, h-caldesmon, and calponin in the diagnosis of myofibroblastic and smooth muscle lesions of skin and soft tissue. Am J Dermatopathol. 28:105–111, 2006. 105. Ogut O, Granzier H, Jin JP: Acidic and basic troponin-T isoforms in mature fast-twitch skeletal muscle and effect on contractility. Am J Physiol. 276:1162–C1170, 1999. 106. Lutz GJ, Lieber RL: Skeletal muscle myosin-II structure and function. Exerc Sport Sci Rev. 27:63–77, 1999. 107. Lefevre G: Troponins: Biological and clinical aspects. Ann Biol Clin. 58:39–48, 2000. 108. Atkinson RA, Joseph C, Dal Piaz F, et al: Binding of alpha-actinin to titin: Implications for Z-disk assembly. Biochemistry. 39:5255– 5264, 2000. 109. Dias P, Chen B, Dilday B, et al: Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass. Am J Pathol. 156:399–408, 2000. 110. Watanabe K, Kusakabe T, Hoshi N, et al: h-Caldesmon in leiomyosarcoma and tumors with smooth muscle-like differentiation: Its specific expression in the smooth muscle cell tumors. Hum Pathol. 30:392–396, 1999. 111. Hisaoka M, Wei-Qi S, Jian W, et al: Specific but variable expression of h-caldesmon in leiomyosarcomas: an immunohistochemical reassessment of a novel myogenic marker. Appl Immunohistochem Mol Morphol. 9:302–308, 2001.
129.e3
112. Miettinen MM, Sarlomo-Rikala M, Kovatich AJ, et al: Calponin and h-caldesmon in soft tissue tumors: consistent h-caldesmon immunoreactivity in gastrointestinal stromal tumors indicates traits of smooth muscle differentiation. Mod Pathol. 12:756–762, 1999. 113. Mentzel T, Dei Tos AP, Sapi Z, et al: Myopericytoma of skin and soft tissues: Clinicopathologic and immunohistochemical study of 54 cases. Am J Surg Pathol. 30:104–113, 2006. 114. Ceballos KM, Nielsen GP, Selig MK, et al: Is anti-h-caldesmon useful for distinguishing smooth muscle and myofibroblastic tumors? An immunohistochemical study. Am J Clin Pathol. 114:746–753, 2000. 115. Lazard D, Sastre X, Frid MG, et al: Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc Natl Acad Sci U S A. 90:999–1003, 1993. 116. Moczygemba C, Guidry J, Wittung-Stafshede P: Heme orientation affects holo-myoglobin folding and unfolding kinetics. FEBS Lett. 470:203–206, 2000. 117. Carter RL, McCarthy KP, Machin LG, et al: Expression of desmin and myoglobin in rhabdomyosarcomas and in developing skeletal muscle. Histopathology. 15:585–595, 1989. 118. Carter RL, Jameson CF, Philp ER, et al: Comparative phenotypes in rhabdomyosarcoma and developing skeletal muscle. Histopathology. 17:301–309, 1990. 119. Parham DM, Webber B, Holt H, et al: Immunohistochemical study of childhood rhabdomyosarcomas and related neoplasms: Results of an Intergroup Rhabdomyosarcoma Study project. Cancer. 67:3072–3080, 1991. 120. Seidal T, Kindblom LG, Angervall L: Myoglobin, desmin, and vimentin in ultrastructurally proven rhabdomyomas and rhabdomyosarcomas: An immunohistochemical study utilizing a series of monoclonal and polyclonal antibodies. Appl Pathol. 5:201–219, 1987. 121. Hosoi H, Sugimoto T, Hayashi Y, et al: Differential expression of myogenic regulatory genes, Myo-D1 and myogenin, in human rhabdomyosarcoma sublines. Int J Cancer. 50:977–983, 1992. 122. Newsholme SJ, Zimmerman DM: Immunohistochemical evaluation of chemically induced rhabdomyosarcomas in rats: Diagnostic utility of Myo-D1. Toxicol Pathol. 25:470–474, 1997. 123. Cui S, Hano H, Harada T, et al: Evaluation of new monoclonal anti-Myo-D1 and anti-myogenin antibodies for the diagnosis of rhabdomyosarcoma. Pathol Int. 49:62–68, 1999. 124. Shiro B, Siegal GP: The use of monoclonal antibodies to S100 protein in diagnostic immunohistochemistry. In Wick MR, Siegal GP, editors: Monoclonal Antibodies in Diagnostic Immunohistochemistry, New York, 1988, Marcel De, pp 455–503. 125. Fujii T, Machino K, Andoh H, et al: Calcium-dependent control of caldesmon-actin interaction by S100 protein. J Biochem. 107:133–137, 1990. 126. Weiss SW, Langloss JM, Enzinger FM: Value of S100 protein in the diagnosis of soft tissue tumors, with particular reference to benign and malignant Schwann cell tumors. Lab Invest. 49:299– 308, 1983. 127. Hisaoka M, Ishida T, Kuo TT, et al: Clear cell sarcoma of soft tissue: A clinicopathologic, immunohistochemical, and molecular analysis of 33 cases. Am J Surg Pathol. 32:452–460, 2008. 128. Wick MR, Swanson PE, Scheithauer BW, et al: Malignant peripheral nerve sheath tumor: An immunohistochemical study of 62 cases. Am J Clin Pathol. 87:425–433, 1987. 129. Daimaru Y, Hashimoto H, Enjoji M: Malignant peripheral nerve-sheath tumors (malignant schwannomas). An immunohistochemical study of 29 cases. Am J Surg Pathol. 9:434–444, 1985. 130. Folpe AL, Weiss SW: Ossifying fibromyxoid tumor of soft parts: a clinicopathologic study of 70 cases with emphasis on atypical and malignant variants. Am J Surg Pathol. 27:421–431, 2003. 131. Miettinen M, Finnell V, Fetsch JF: Ossifying fibromyxoid tumor of soft parts–a clinicopathologic and immunohistochemical study of 104 cases with long-term follow-up and a critical review of the literature. Am J Surg Pathol. 32:996–1005, 2008. 132. Oliveira AM, Sebo TJ, McGrory JE, et al: Extraskeletal myxoid chondrosarcoma: a clinicopathologic, immunohistochemical, and ploidy analysis of 23 cases. Mod Pathol. 13:900– 908, 2000.
129.e4
Immunohistology of Neoplasms of Soft Tissue and Bone
133. Guillou L, Wadden C, Kraus MD, et al: S-100 protein reactivity in synovial sarcomas–a potentially frequent diagnostic pitfall: Immunohistochemical Analysis of 100 cases. Applied Immunohistochemistry. 4:167–175, 1996. 134. Miettinen M, Kopczynski J, Makhlouf HR, et al: Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic, immunohistochemical, and molecular genetic study of 167 cases. Am J Surg Pathol. 27:625–641, 2003. 135. Miettinen M, Sobin LH, Sarlomo-Rikala M: Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with a reference to CD117 (KIT). Mod Pathol. 13:1134–1142, 2000. 136. Coffin CM, Swanson PE, Wick MR, et al: An immunohistochemical comparison of chordoma with renal cell carcinoma, colorectal adenocarcinoma, and myxopapillary ependymoma: a potential diagnostic dilemma in the diminutive biopsy. Mod Pathol. 6:531–538, 1993. 137. Wojno KJ, Hruban RH, Garin-Chesa P, et al: Chondroid chordomas and low-grade chondrosarcomas of the craniospinal axis. An immunohistochemical analysis of 17 cases. Am J Surg Pathol. 16:1144–1152, 1992. 138. Demicco EG, Rosenberg AE, Bjornsson J, et al: Primary RosaiDorfman disease of bone: a clinicopathologic study of 15 cases. Am J Surg Pathol. 34:1324–1333, 2010. 139. Pinkus GS, Lones MA, Matsumura F, et al: Langerhans cell histiocytosis immunohistochemical expression of fascin, a dendritic cell marker. Am J Clin Pathol. 118:335–343, 2002. 140. Hornick JL, Jaffe ES, Fletcher CD: Extranodal histiocytic sarcoma: clinicopathologic analysis of 14 cases of a rare epithelioid malignancy. Am J Surg Pathol. 28:1133–1144, 2004. 141. Takahashi K, Isobe T, Ohtsuki Y, et al: Immunohistochemical localization and distribution of S-100 proteins in the human lymphoreticular system. Am J Pathol. 116:497–503, 1984. 142. Hammer JA, O’Shannessy DJ, DeLeon M, et al: Immunoreactivity of PMP22, P0, and other 19 to 28 kD glycoproteins in peripheral nerve myelin of mammals and fish with HNK-1 and related antibodies. J Neurosci Res. 35:546–558, 1993. 143. Merkouri E, Matsas R: Monoclonal antibody BM89 recognizes a novel cell surface glycoprotein of the L2/HNK-1 family in the developing mammalian nervous system. Neuroscience. 50:53–68, 1992. 144. Lanier LL, Testi R, Bindl J, et al: Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J Exp Med. 169:2233–2238, 1989. 145. Abo T, Balch CM: A differentiation antigen of human NK and killer cells, identified by a monoclonal antibody (HNK1). J Immunol. 127:1024–1029, 1981. 146. Hirose T, Scheithauer BW, Sano T: Perineurial malignant peripheral nerve sheath tumor (MPNST): A clinicopathologic, immunohistochemical, and ultrastructural study of seven cases. Am J Surg Pathol. 22:1368–1378, 1998. 147. Amann G, Zoubek A, Salzer-Kuntschik M, et al: Relation of neurological marker expression and EWS gene fusion types in MIC2/CD99-positive tumors of the Ewing family. Hum Pathol. 30:1058–1064, 1999. 148. Miettinen M, Cupo W: Neural cell adhesion molecule distribution in soft tissue tumors. Hum Pathol. 24:62–66, 1993. 149. Hartel PH, Fanburg-Smith JC, Frazier AA, et al: Primary pulmonary and mediastinal synovial sarcoma: A clinicopathologic study of 60 cases and comparison with five prior series. Mod Pathol. 20:760–769, 2007. 150. Salzer JL, Holmes WP, Colman DR: The amino acid sequences of the myelin-associated glycoproteins: Homology to the immunoglobulin gene superfamily. J Cell Biol. 104:959–965, 1987. 151. Dixit SN, Mainardi CL, Beachey EH, et al: 7S domain constitutes the amino-terminal end of type IV collagen: An immunohistochemical study. Coll Rel Res. 3:263–273, 1983. 152. Griffin CA, Emanuel BS, Hansen JR, et al: Human collagen genes encoding basement membrane alpha-1 (IV) and alpha-2 (IV) chains map to the distal long arm of chromosome 13. Proc Natl Acad Sci USA. 84:512–516, 1987. 153. Terranova VP, Rohrbach DH, Martin GR: Role of laminin in the attachment of PAM212 (epithelial) cells to basement membrane collagen. Cell. 22:719–726, 1980.
154. Foidart JM, Bere EW Jr, Yaar M, et al: Distribution and immunoelectron microscopic localization of laminin, a non-collagenous basement membrane glycoprotein. Lab Invest. 42:336–342, 1980. 155. Nigar E, Dervan PA: Quantitative assessment of basement membranes in soft tissue tumors: Computerized image analysis of laminin and type IV collagen. J Pathol. 185:184–187, 1998. 156. Folpe AL, Billings SD, McKenney JK, et al: Expression of claudin-1, a recently described tight junction-associated protein, distinguishes soft tissue perineurioma from potential mimics. Am J Surg Pathol. 26:1620–1626, 2002. 157. Hornick JL, Fletcher CD: Soft tissue perineurioma: clinicopathologic analysis of 81 cases including those with atypical histologic features. Am J Surg Pathol. 29:845–858, 2005. 158. Thway K, Fisher C, Debiec-Rychter M, et al: Claudin-1 is expressed in perineurioma-like low-grade fibromyxoid sarcoma. Hum Pathol. 40:1586–1590, 2009. 159. Nonaka D, Chiriboga L, Rubin BP: Sox10: A pan-schwannian and melanocytic marker. Am J Surg Pathol. 32:1291–1298, 2008. 160. Karamchandani JR, Nielsen TO, van de Rijn M, et al: Sox10 and S100 in the Diagnosis of Soft-tissue Neoplasms. Appl Immunohistochem Mol Morphol. 20:445–450, 2012. 161. Fischer BE, Thomas KB, Schlokat U, et al: Triplet structure of human von Willebrand factor. Biochem J. 331:483–488, 1998. 162. Kaufman RJ, Pipe SW: Regulation of factor VIII expression and activity by von Willebrand factor. Thromb Haemost. 82:201–208, 1999. 163. Mukai K, Rosai J: Factor VIII-related antigen: An endothelial marker. In DeLellis RA, editor: Diagnostic Immunohistochemistry, New York, 1984, Ma, pp 243–261. 164. Sehested M, Hou-Jensen K: Factor VIII-related antigen as an endothelial cell marker in benign and malignant diseases. Virchows Arch A. 391:217–225, 1981. 165. Ramani P, Bradley NJ, Fletcher CDM: QBEND10, a new monoclonal antibody to endothelium: Assessment of its diagnostic utility in paraffin sections. Histopathology. 17:237–242, 1990. 166. Traweek ST, Kandalaft PL, Mehta P, et al: The human hematopoietic progenitor cell antigen (CD34) in vascular neoplasms. Am J Clin Pathol. 96:25–31, 1991. 167. Sirgi KE, Wick MR, Swanson PE: B72.3 and CD34 immunoreactivity in malignant epithelioid soft tissue tumors. Adjuncts in the recognition of endothelial neoplasms. Am J Surg Pathol. 17:179–185, 1993. 168. Guillou L, Wadden C, Coindre JM, et al: “Proximal-type” epithelioid sarcoma, a distinctive aggressive neoplasm showing rhabdoid features. Clinicopathologic, immunohistochemical, and ultrastructural study of a series. Am J Surg Pathol. 21:130– 146, 1997. 169. Miettinen M, Lindenmayer AE, Chanbal A: Endothelial cell markers CD31, CD34, and BNH9 antibody to H- and Y-antigens: Evaluation of their specificity and sensitivity in the diagnosis of vascular tumors and comparison with von Willebrand factor. Mod Pathol. 7:82–90, 1994. 170. Natkunam Y, Rouse RV, Zhu S, et al: Immunoblot analysis of CD34 expression in histologically diverse neoplasms. Am J Pathol. 156:21–27, 2000. 171. Rizeq MN, van de Rijn M, Hendrickson MR, et al: A comparative immunohistochemical study of uterine smooth muscle neoplasms with emphasis on the epithelioid variant. Hum Pathol. 25:671–677, 1994. 172. Goldblum JR: CD34 positivity in fibrosarcomas which arise in dermatofibrosarcoma protuberans. Arch Pathol Lab Med. 119:238–241, 1995. 173. Goldblum JR, Tuthill RJ: CD34 and factor-XIIIa immunoreactivity in dermatofibrosarcoma protuberans and dermatofibroma. Am J Dermatopathol. 19:147–153, 1997. 174. Metzelaar MJ, Korteweg J, Sizma JJ, et al: Biochemical characterization of PECAM-1 (CD31 antigen) on human platelets. Thromb Haemost. 66:700–707, 1991. 175. Parums DV, Cordell JL, Michlein K, et al: JC70: A new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol. 43:752–757, 1990.
References 176. Ohsawa M, Naka N, Tomita Y, et al: Use of immunohistochemical procedures in diagnosing angiosarcomas: Evaluation of 98 cases. Cancer. 75:2867–2874, 1995. 177. McKenney JK, Weiss SW, Folpe AL: CD31 expression in intratumoral macrophages: A potential diagnostic pitfall. Am J Surg Pathol. 25:1167–1173, 2001. 178. DeYoung BR, Wick MR, Fitzgibbon JF, et al: CD31: An immunospecific marker for endothelial differentiation in human neoplasms. Appl Immunohistochem. 1:97–100, 1993. 179. Sato Y: Role of ETS family transcription factors in vascular development and angiogenesis. Cell Struct Funct. 26:19–24, 2001. 180. Watson DK, Smyth FE, Thompson DM, et al: The ERGB/FLI-1 gene: Isolation and characterization of a new member of the family of human ETS transcription factors. Cell Growth Differ. 3:705–713, 1992. 181. Torchia EC, Boyd K, Rehg JE, et al: EWS/FLI-1 induces rapid onset of myeloid/erythroid leukemia in mice. Mol Cell Biol. 27:7918–7934, 2007. 182. Liu F, Walmsley M, Rodaway A, et al: FLI-1 acts at the top of the transcriptional network driving blood and endothelial development. Curr Biol. 18:1234–1240, 2008. 183. Landry JR, Konston S, Knezevic K, et al: FLI-1, EFL1, and ETS1 regulate the proximal promoter of the LMO2 gene in endothelial cells. Blood. 106:2680–2687, 2005. 184. Zwerner JP, Joo J, Warner KL, et al: The EWS/FLI1 oncogenic transcription factor deregulates GLI1. Oncogene. 27:3282–3291, 2008. 185. Matsunobu T, Tanaka K, Nakamura T, et al: The possible role of EWS-FLI1 in evasion of senescence in Ewing family tumors. Cancer Res. 66:803–811, 2006. 186. Rossi S, Orvieto E, Furlanetto A, et al: Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody. Mod Pathol. 17:547–552, 2004. 187. Folpe AL, Chand EM, Goldblum JR, et al: Expression of FLI-1, a nuclear transcription factor, distinguishes vascular neoplasms from potential mimics. Am J Surg Pathol. 25:1061–1066, 2001. 188. Mhawech-Fauceglia P, Herrmann FR, Bshara W, et al: Friend leukemia integration-1 expression in malignant and benign tumors: A multiple tumor tissue microarray analysis using polyclonal antibody. J Clin Pathol. 60:694–700, 2007. 189. McKay KM, Doyle LA, Lazar AJ, et al: Expression of ERG, an Ets family transcription factor, distinguishes cutaneous angiosarcoma from histological mimics. Histopathology. 61:989–991, 2012. 190. Miettinen M, Sarlomo-Rikala M, Wang ZF: Claudin-5 as an immunohistochemical marker for angiosarcoma and hemangioendotheliomas. Am J Surg Pathol. 35:1848–1856, 2011. 191. Miettinen M, Wang ZF, Paetau A, et al: ERG transcription factor as an immunohistochemical marker for vascular endothelial tumors and prostatic carcinoma. Am J Surg Pathol. 35:432–441, 2011. 192. Wang WL, Patel NR, Caragea M, et al: Expression of ERG, an Ets family transcription factor, identifies ERG-rearranged Ewing sarcoma. Mod Pathol. 25:1378–1383, 2012. 193. Pardridge WM, Boado RJ, Farrell CR: Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization. J Biol Chem. 265:18035–18040, 1990. 194. Ahrens WA, Ridenour RV, 3rd, Caron BL, et al: GLUT-1 expression in mesenchymal tumors: an immunohistochemical study of 247 soft tissue and bone neoplasms. Hum Pathol. 39:1519–1526, 2008. 195. Yamaguchi U, Hasegawa T, Hirose T, et al: Sclerosing perineurioma: a clinicopathological study of five cases and diagnostic utility of immunohistochemical staining for GLUT1. Virchows Arch. 443:159–163, 2003. 196. Le Huu AR, Jokinen CH, Rubin BP, et al: Expression of prox1, lymphatic endothelial nuclear transcription factor, in Kaposiform hemangioendothelioma and tufted angioma. Am J Surg Pathol. 34:1563–1573, 2010. 197. Miettinen M, Wang ZF: Prox1 transcription factor as a marker for vascular tumors-evaluation of 314 vascular endothelial and 1086 nonvascular tumors. Am J Surg Pathol. 36:351–359, 2012.
129.e5
198. Koda R, Zhao L, Yaoita E, et al: Novel expression of claudin-5 in glomerular podocytes. Cell Tissue Res. 343:637–648, 2011. 199. Soini Y: Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours. Histopathology. 46:551–560, 2005. 200. Miettinen M, Sarlomo-Rikala M, Wang ZF: Claudin-5 as an immunohistochemical marker for angiosarcoma and hemangioendotheliomas. Am J Surg Pathol. 35:1848–1856, 2011. 201. Kurosawa S, Galvin JB, Esmon NC, et al: Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem. 262:2206–2212, 1987. 202. Karmochkine M, Boffa MC: Thrombomodulin: Physiology and clinical applications (excluding systemic diseases). Rev Med Interne. 18:119–125, 1997. 203. Appleton MA, Attanoos RL, Jasani B: Thrombomodulin as a marker of vascular and lymphatic tumours. Histopathology. 29:153–157, 1996. 204. Zhang YM, Bachmann S, Hemmer C, et al: Vascular origin of Kaposi’s sarcoma: Expression of leukocytic adhesion molecule-1, thrombomodulin, and tissue factor. Am J Pathol. 144:51–59, 1994. 205. Ordonez NG: Value of thrombomodulin immunostaining in the diagnosis of mesothelioma. Histopathology. 31:25–30, 1997. 206. Holthofer H, Virtanen I, Kariniemi AL, et al: Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest. 47:60–66, 1982. 207. Kahn HJ, Bailey D, Marks A: Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi’s sarcoma and a subset of angiosarcomas. Mod Pathol. 15:434–440, 2002. 208. Breiteneder-Geleff S, Matsui K, Soleiman A, et al: Podoplanin, novel 43 kD membrane protein of glomerular epithelial cells, is downregulated in puromycin nephrosis. Am J Pathol. 151:1141– 1152, 1997. 209. Raica M, Cimpean AM, Ribatti D: The role of podoplanin in tumor progression and metastasis. Anticancer Res. 28:2997– 3006, 2008. 210. Breiteneder-Geleff S, Soleiman A, Horvat R, et al: Podoplanin-a specific marker for lymphatic endothelium expressed in angiosarcoma. Verh Dtsch Ges Pathol. 83:270–275, 1999. 211. Breiteneder-Geleff S, Soleiman A, Kowalski H, et al: Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: Podoplanin as a specific marker for lymphatic endothelium. Am J Pathol. 154:385–394, 1999. 212. Yu H, Pinkus GS, Hornick JL: Diffuse membranous immunoreactivity for podoplanin (D2-40) distinguishes primary and metastatic seminomas from other germ cell tumors and metastatic neoplasms. Am J Clin Pathol. 128:767–775, 2007. 213. Chu AY, Litzky LA, Pasha TL, et al: Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma. Mod Pathol. 18:105–110, 2005. 214. Yu H, Gibson JA, Pinkus GS, et al: Podoplanin (D2-40) is a -novel marker for follicular dendritic cell tumors. Am J Clin Pathol. 128:776–782, 2007. 215. Makinen T, Norrmen C, Petrova TV: Molecular mechanisms of lymphatic vascular development. Cell Mol Life Sci. 64:1915– 1929, 2007. 216. Fukunaga M: Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumors. Histopathology. 48:396– 402, 2005. 217. Nakatsuka S, Oji Y, Horiuchi T, et al: Immunohistochemical detection of WT1 protein in a variety of cancer cells. Mod Pathol. 19:804–814, 2006. 218. Lawley LP, Cerimele F, Weiss SW, et al: Expression of Wilms tumor 1 gene distinguishes vascular malformations from proliferative endothelial lesions. Arch Dermatol. 141:1297–1300, 2005. 219. Timar J, Mezzaros L, Orosz Z, et al: WT1 expression in angiogenic tumors of the skin. Histopathology. 47:67–73, 2005. 220. Iljin K, Karkkainen MJ, Lawrence EC, et al: VEGFR3 gene structure, regulatory region, and sequence polymorphisms. FASEB J. 15:1028–1036, 2001. 221. Folpe AL, Veikkola T, Valtola R, et al: Vascular endothelial growth factor receptor-3 (VEGFR3): A marker of vascular tumors with presumed lymphatic differentiation, including Kaposi’s sarcoma, kaposiform and Dabska-type hemangioendo-
129.e6
Immunohistology of Neoplasms of Soft Tissue and Bone
theliomas, and a subset of angiosarcomas. Mod Pathol. 13:180– 185, 2000. 222. Leader M, Collins M, Patel J, et al: Staining for factor VIIIrelated antigen and Ulex europaeus I (UEA-I) in 230 tumors: An assessment of their specificity for angiosarcoma and Kaposi’s sarcoma. Histopathology. 10:1153–1162, 1986. 223. DuBoulay CEH: Demonstration of alpha-1-antitrypsin and alpha-1-antichymotrypsin in fibrous histiocytomas using the immunoperoxidase technique. Am J Surg Pathol. 6:559–564, 1982. 224. Kindblom LG, Jacobsen GK, Jacobsen M: Immunohistochemical investigations of tumors of supposed fibroblastic-histiocytic origin. Hum Pathol. 13:834–840, 1982. 225. Pulford KAF, Rigney EM, Micklem KJ, et al: KP1: A new monoclonal antibody that detects a monocyte/macrophage-associated antigen in routinely processed tissue sections. J Clin Pathol. 42:414–421, 1989. 226. Lau SK, Chu PG, Weiss LM: CD163: a specific marker of macrophages in paraffin-embedded tissue samples. Am J Clin Pathol. 122:794–801, 2004. 227. Reid MB, Gray C, Fear JD, et al: Immunohistologic demonstration of factors XIIIa and XIIIs in reactive and neoplastic fibroblastic and fibrohistiocytic lesions. Histopathology. 10:1171–1178, 1986. 228. Thewes M, Engst R, Boeck K, et al: Expression of cathepsins in dermal fibrous tumors: An immunohistochemical study. Eur J Dermatol. 8:86–89, 1998. 229. Gloghini A, Rizzo A, Zanette I, et al: KP1/CD68 expression in malignant neoplasms including lymphomas, sarcomas, and carcinomas. Am J Clin Pathol. 103:425–431, 1995. 230. Pulford KA, Rigney EM, Micklem KJ, et al: KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections. J Clin Pathol. 42:414–421, 1989. 231. Cassidy M, Loftus B, Whelan A, et al: KP-1: not a specific marker. Staining of 137 sarcomas, 48 lymphomas, 28 carcinomas, 7 malignant melanomas and 8 cystosarcoma phyllodes. Virchows Arch. 424:635–640, 1994. 232. Dei Tos AP, Doglioni C, Laurino L, et al: KP1 (CD68) expression in benign neural tumours. Further evidence of its low specificity as a histiocytic/myeloid marker. Histopathology. 23:185–187, 1993. 233. Tsang WY, Chan JK: KP1 (CD68) staining of granular cell neoplasms: is KP1 a marker for lysosomes rather than the histiocytic lineage? Histopathology. 21:84–86, 1992. 234. Nguyen TT, Schwartz EJ, West RB, et al: Expression of CD163 (hemoglobin scavenger receptor) in normal tissues, lymphomas, carcinomas, and sarcomas is largely restricted to the monocyte/ macrophage lineage. Am J Surg Pathol. 29:617–624, 2005. 235. Burgdorf WH, Duray P, Rosai J: Immunohistochemical identification of lysozyme in cutaneous lesions of alleged histiocytic nature. Am J Clin Pathol. 75:162–167, 1981. 236. Hsi ED, Nickoloff BJ: Dermatofibroma and dermatofibrosarcoma protuberans: an immunohistochemical study reveals distinctive antigenic profiles. J Dermatol Sci. 11:1–9, 1996. 237. Goldblum JR, Tuthill RJ: CD34 and factor-XIIIa immunoreactivity in dermatofibrosarcoma protuberans and dermatofibroma. Am J Dermatopathol. 19:147–153, 1997. 238. Sachdev R, Sundram UN: Frequent positive staining with NKI/ C3 in normal and neoplastic tissues limits its usefulness in the diagnosis of cellular neurothekeoma. Am J Clin Pathol. 126:554– 563, 2006. 239. Clarkson KS, Sturdgess IC, Molyneux AJ: The usefulness of tyrosinase in the immunohistochemical assessment of melanocytic lesions: a comparison of the novel T311 antibody (antityrosinase) with S-100, HMB45, and A103 (anti-melan-A). J Clin Pathol. 54:196–200, 2001. 240. Miettinen M, Fernandez M, Franssila K, et al: Microphthalmia transcription factor in the immunohistochemical diagnosis of metastatic melanoma: comparison with four other melanoma markers. Am J Surg Pathol. 25:205–211, 2001. 241. Jungbluth AA, King R, Fisher DE, et al: Immunohistochemical and reverse transcription-polymerase chain reaction expression analysis of tyrosinase and microphthalmia-associated transcription factor in angiomyolipomas. Appl Immunohistochem Mol Morphol. 9:29–34, 2001.
242. Jungbluth AA, Busam KJ, Gerald WL, et al: A103: An antimelan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues. Am J Surg Pathol. 22:595–602, 1998. 243. Busam KJ, Chen YT, Old LJ, et al: Expression of melan-A (MART1) in benign melanocytic nevi and primary cutaneous malignant melanoma. Am J Surg Pathol. 22:976–982, 1998. 244. Busam KJ, Iversen K, Coplan KA, et al: Immunoreactivity for A103, an antibody to melan-A (Mart-1), in adrenocortical and other steroid tumors. Am J Surg Pathol. 22:57–63, 1998. 245. Busam KJ, Iversen K, Coplan KC, et al: Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma. Am J Surg Pathol. 25:197–204, 2001. 246. Nelson WJ, Nusse R: Convergence of Wnt, beta-catenin, and cadherin pathways. Science. 303:1483–1487, 2004. 247. Bertario L, Russo A, Sala P, et al: Genotype and phenotype factors as determinants of desmoid tumors in patients with familial adenomatous polyposis. Int J Cancer. 95:102–107, 2001. 248. Salas S, Chibon F, Noguchi T, et al: Molecular characterization by array comparative genomic hybridization and DNA sequencing of 194 desmoid tumors. Genes Chromosomes Cancer. 49:560–568, 2010. 249. Tejpar S, Nollet F, Li C, et al: Predominance of beta-catenin mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor). Oncogene. 18:6615–6620, 1999. 250. Lazar AJ, Tuvin D, Hajibashi S, et al: Specific mutations in the beta-catenin gene (CTNNB1) correlate with local recurrence in sporadic desmoid tumors. Am J Pathol. 173:1518–1527, 2008. 251. Huss S, Nehles J, Binot E, et al: beta-Catenin (CTNNB1) mutations and clinicopathological features of mesenteric desmoidtype fibromatosis. Histopathology. 62:294–304, 2012. 252. Carlson JW, Fletcher CD: Immunohistochemistry for betacatenin in the differential diagnosis of spindle cell lesions: analysis of a series and review of the literature. Histopathology. 51:509–514, 2007. 253. Bhattacharya B, Dilworth HP, Iacobuzio-Donahue C, et al: Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions. Am J Surg Pathol. 29:653–659, 2005. 254. Montgomery E, Folpe AL: The diagnostic value of beta-catenin immunohistochemistry. Adv Anat Pathol. 12:350–356, 2005. 255. Coffin CM, Hornick JL, Zhou H, et al: Gardner fibroma: a clinicopathologic and immunohistochemical analysis of 45 patients with 57 fibromas. Am J Surg Pathol. 31:410–416, 2007. 256. Shiota M, Fujimoto J, Semba T, et al: Hyperphosphorylation of a novel 80 kDa protein-tyrosine kinase similar to Ltk in a human Ki-lymphoma cell line, AMS3. Oncogene. 9:1567–1574, 1994. 257. Cessna MH, Zhou H, Sanger WG, et al: Expression of ALK1 and p80 in inflammatory myofibroblastic tumor and its mesenchymal mimics: a study of 135 cases. Mod Pathol. 15:931–938, 2002. 258. Sigel JE, Smith TA, Reith JD, et al: Immunohistochemical analysis of anaplastic lymphoma kinase expression in deep soft-tissue calcifying fibrous pseudotumor: evidence of a late sclerosing stage of inflammatory myofibroblastic tumor? Ann Diagn Pathol. 5:10–14, 2001. 259. Lawrence B, Perez-Atayde A, Hibbard MK, et al: TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors. Am J Pathol. 157:377–384, 2000. 260. Cook JR, Dehner LP, Collins MH, et al: Anaplastic lymphoma kinase (ALK) expression in the inflammatory myofibroblastic tumor: a comparative immunohistochemical study. Am J Surg Pathol. 25:1364–1371, 2001. 261. Patel AS, Murphy KM, Hawkins AL, et al: RANBP2 and CLTC are involved in ALK rearrangements in inflammatory myofibroblastic tumors. Cancer Genet Cytogenet. 176:107–114, 2007. 262. Bridge JA, Kanamori M, Ma Z, et al: Fusion of the ALK gene to the clathrin heavy chain gene, CLTC, in inflammatory myofibroblastic tumor. Am J Pathol. 159:411–415, 2001. 263. Marino-Enriquez A, Wang WL, Roy A, et al: Epithelioid inflammatory myofibroblastic sarcoma: An aggressive intra-abdominal variant of inflammatory myofibroblastic tumor with nuclear membrane or perinuclear ALK. Am J Surg Pathol. 35:135–144, 2011.
References 264. Debelenko LV, Raimondi SC, Daw N, et al: Renal cell carcinoma with novel VCL-ALK fusion: new representative of ALKassociated tumor spectrum. Mod Pathol. 24:430–442, 2011. 265. Marino-Enriquez A, Ou WB, Weldon CB, et al: ALK rearrangement in sickle cell trait-associated renal medullary carcinoma. Genes Chromosomes Cancer. 50:146–153, 2011. 266. Soda M, Choi YL, Enomoto M, et al: Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 448:561–566, 2007. 267. Berry T, Luther W, Bhatnagar N, et al: The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma. Cancer Cell. 22:117–130, 2012. 268. van Gaal JC, Flucke UE, Roeffen MH, et al: Anaplastic lymphoma kinase aberrations in rhabdomyosarcoma: clinical and prognostic implications. J Clin Oncol. 30:308–315, 2012. 269. Li XQ, Hisaoka M, Shi DR, et al: Expression of anaplastic lymphoma kinase in soft tissue tumors: An immunohistochemical and molecular study of 249 cases. Hum Pathol. 35:711–721, 2004. 270. Ozdemirli M, Fanburg-Smith JC, Hartmann DP, et al: Precursor B-lymphoblastic lymphoma presenting as a solitary bone tumor and mimicking Ewing’s sarcoma: A report of four cases and review of the literature. Am J Surg Pathol. 22:795–804, 1998. 271. Robertson PB, Neiman RS, Worapongpaiboon S, et al: O13 (CD99) positivity in hematologic proliferations correlates with TdT positivity. Mod Pathol. 10:277–282, 1997. 272. Soslow RA, Bhargava V, Warnke RA: MIC2, TdT, bcl-2, and CD34 expression in paraffin-embedded high-grade lymphoma/acute lymphoblastic leukemia distinguishes between distinct clinicopathologic entities. Hum Pathol. 28:1158–1165, 1997. 273. Weiss LM, Bindl JM, Picozzi VJ, et al: Lymphoblastic lymphoma: An immunophenotypic study of 26 cases with comparison to T-cell acute lymphoblastic leukemia. Blood. 67:474–478, 1986. 274. Renshaw AA: O13 (CD99) in spindle cell tumors: Reactivity with hemangiopericytoma, solitary fibrous tumor, synovial sarcoma, and meningioma, but rarely with sarcomatoid mesothelioma. Appl Immunohistochem. 3:250–256, 1995. 275. Granter SR, Renshaw AA, Fletcher CD, et al: CD99 reactivity in mesenchymal chondrosarcoma. Hum Pathol. 27:1273–1276, 1996. 276. Soslow RA, Wallace M, Goris J, et al: MIC2 gene expression in cutaneous neuroendocrine carcinoma (Merkel cell carcinoma). Appl Immunohistochem. 4:235–240, 1996. 277. Lumadue JA, Askin FB, Perlman EJ: MIC2 analysis of small cell carcinoma. Am J Clin Pathol. 102:692–694, 1994. 278. Miettinen M, Chatten J, Paetau A, et al: Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors. Am J Surg Pathol. 22:327–332, 1998. 279. Thomas JO, Nijjar J, Turley H, et al: NB84: a new monoclonal antibody for the recognition of neuroblastoma in routinely processed material. J Pathol. 163:69–75, 1991. 280. Argani P, Lal P, Hutchinson B, et al: Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions: a sensitive and specific immunohistochemical assay. Am J Surg Pathol. 27:750–761, 2003. 281. Argani P, Olgac S, Tickoo SK, et al: Xp11 translocation renal cell carcinoma in adults: expanded clinical, pathologic, and genetic spectrum. Am J Surg Pathol. 31:1149–1160, 2007. 282. Argani P, Aulmann S, Illei PB, et al: A distinctive subset of PEComas harbors TFE3 gene fusions. Am J Surg Pathol. 34:1395– 1406, 2010. 283. Malinowska I, Kwiatkowski DJ, Weiss S, et al: Perivascular epithelioid cell tumors (PEComas) harboring TFE3 gene rearrangements lack the TSC2 alterations characteristic of conventional PEComas: further evidence for a biological distinction. Am J Surg Pathol. 36:783–784, 2012. 284. Binh MB, Sastre-Garau X, Guillou L, et al: MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing welldifferentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol. 29:1340–1347, 2005. 285. Dei Tos AP, Doglioni C, Piccinin S, et al: Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours. J Pathol. 190:531–536, 2000.
129.e7
286. Binh MB, Garau XS, Guillou L, et al: Reproducibility of MDM2 and CDK4 staining in soft tissue tumors. Am J Clin Pathol. 125:693–697, 2006. 287. Chen BJ, Marino-Enriquez A, Fletcher CD, et al: Loss of retinoblastoma protein expression in spindle cell/pleomorphic lipomas and cytogenetically related tumors: an immunohistochemical study with diagnostic implications. Am J Surg Pathol. 36:1119– 1128, 2012. 288. Hollmann TJ, Hornick JL: INI1-deficient tumors: diagnostic features and molecular genetics. Am J Surg Pathol. 35:e47–63, 2011. 289. Bourdeaut F, Lequin D, Brugieres L, et al: Frequent hSNF5/INI1 germline mutations in patients with rhabdoid tumor. Clin Cancer Res. 17:31–38, 2011. 290. Hornick JL, Dal Cin P, Fletcher CD: Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma. Am J Surg Pathol. 33:542–550, 2009. 291. Sullivan LM, Folpe AL, Pawel BR, et al: Epithelioid sarcoma is associated with a high percentage of SMARCB1 deletions. Mod Pathol. 26:385–392, 2012. 292. Kohashi K, Oda Y, Yamamoto H, et al: SMARCB1/INI1 protein expression in round cell soft tissue sarcomas associated with chromosomal translocations involving EWS: a special reference to SMARCB1/INI1 negative variant extraskeletal myxoid chondrosarcoma. Am J Surg Pathol. 32:1168–1174, 2008. 293. Calderaro J, Moroch J, Pierron G, et al: SMARCB1/INI1 inactivation in renal medullary carcinoma. Histopathology. 61:428– 435, 2012. 294. Cheng JX, Tretiakova M, Gong C, et al: Renal medullary carcinoma: rhabdoid features and the absence of INI1 expression as markers of aggressive behavior. Mod Pathol. 21:647–652, 2008. 295. Boyd C, Smith MJ, Kluwe L, et al: Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis. Clin Genet. 74:358–366, 2008. 296. Suster S, Fisher C, Moran CA: Expression of bcl-2 oncoprotein in benign and malignant spindle cell tumors of soft tissue, skin, serosal surfaces, and gastrointestinal tract. Am J Surg Pathol. 22:863–872, 1998. 297. Hasegawa T, Matsuno Y, Shimoda T, et al: Frequent expression of bcl-2 protein in solitary fibrous tumors. Jpn J Clin Oncol. 28:86–91, 1998. 298. Krskova L, Kalinova M, Brizova H, et al: Molecular and immunohistochemical analyses of BCL2, KI-67, and cyclin D1 expression in synovial sarcoma. Cancer Genet Cytogenet. 193:1–8, 2009. 299. Chen G, Courey AJ: Groucho/TLE family proteins and transcriptional repression. Gene. 249:1–16, 2000. 300. Grbavec D, Lo R, Liu Y, et al: Transducin-like Enhancer of split 2, a mammalian homologue of Drosophila Groucho, acts as a transcriptional repressor, interacts with Hairy/Enhancer of split proteins, and is expressed during neuronal development. Eur J Biochem. 258:339–349, 1998. 301. Javed A, Guo B, Hiebert S, et al: Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription. J Cell Sci. 113(Pt 12):2221– 2231, 2000. 302. Baird K, Davis S, Antonescu CR, et al: Gene expression profiling of human sarcomas: insights into sarcoma biology. Cancer Res. 65:9226–9235, 2005. 303. Nielsen TO, West RB, Linn SC, et al: Molecular characterisation of soft tissue tumours: a gene expression study. Lancet. 359:1301– 1307, 2002. 304. Terry J, Saito T, Subramanian S, et al: TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies. Am J Surg Pathol. 31:240–246, 2007. 305. Su L, Sampaio AV, Jones KB, et al: Deconstruction of the SS18SSX fusion oncoprotein complex: insights into disease etiology and therapeutics. Cancer Cell. 21:333–347, 2012. 306. Foo WC, Cruise MW, Wick MR, et al: Immunohistochemical staining for TLE1 distinguishes synovial sarcoma from histologic mimics. Am J Clin Pathol. 135:839–844, 2011. 307. Jagdis A, Rubin BP, Tubbs RR, et al: Prospective evaluation of TLE1 as a diagnostic immunohistochemical marker in synovial sarcoma. Am J Surg Pathol. 33:1743–1751, 2009.
129.e8
Immunohistology of Neoplasms of Soft Tissue and Bone
308. Moller E, Hornick JL, Magnusson L, et al: FUS-CREB3L2/ L1-positive sarcomas show a specific gene expression profile with upregulation of CD24 and FOXL1. Clin Cancer Res. 17:2646–2656, 2011. 309. Doyle LA, Moller E, Dal Cin P, et al: MUC4 is a highly sensitive and specific marker for low-grade fibromyxoid sarcoma. Am J Surg Pathol. 35:733–741, 2011. 310. Doyle LA, Wang WL, Dal Cin P, et al: MUC4 Is a sensitive and extremely useful marker for sclerosing epithelioid fibrosarcoma: association with FUS gene rearrangement. Am J Surg Pathol. 36:1444–1451, 2012. 311. Garnero P, Grimaux M, Seguin P, et al: Characterization of immunoreactive forms of human osteocalcin generated in vivo and in vitro. J Bone Miner Res. 9:255–264, 1994. 312. Bradbeer JN, Virdi AS, Serre CM, et al: A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: Implications for the identification of cells of the osteoblastic lineage in vitro. J Bone Miner Res. 9:1221–1228, 1994. 313. Fanburg JC, Rosenberg AE, Weaver DL, et al: Osteocalcin and osteonectin immunoreactivity in the diagnosis of osteosarcoma. Am J Clin Pathol. 108:464–473, 1997. 314. Takada J, Ishii S, Ohta T, et al: Usefulness of a novel monoclonal antibody against human osteocalcin in immunohistochemical diagnosis. Virchows Arch A. 420:507–511, 1992. 315. Fanburg-Smith JC, Bratthauer GL, Miettinen M: Osteocalcin and osteonectin immunoreactivity in extraskeletal osteosarcoma: A study of 28 cases. Hum Pathol. 30:32–38, 1999. 316. Naylor SL, Helin-Davies D, Charoenworawat P, et al: The human osteonectin gene (OSN) has Taq I and Msp I polymorphisms. Nucleic Acids Res. 17:6753, 1989. 317. Vujovic S, Henderson S, Presneau N, et al: Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas. J Pathol. 209:157–165, 2006. 318. Tirabosco R, Mangham DC, Rosenberg AE, et al: Brachyury expression in extra-axial skeletal and soft tissue chordomas: a marker that distinguishes chordoma from mixed tumor/ myoepithelioma/parachordoma in soft tissue. Am J Surg Pathol. 32:572–580, 2008. 319. Sangoi AR, Karamchandani J, Lane B, et al: Specificity of brachyury in the distinction of chordoma from clear cell renal cell carcinoma and germ cell tumors: a study of 305 cases. Mod Pathol. 24:425–429, 2011. 320. Ozdemirli M, Fanburg-Smith JC, Hartmann DP, et al: Differentiating lymphoblastic lymphoma and Ewing’s sarcoma: Lymphocyte markers and gene rearrangement. Mod Pathol. 14:1175–1182, 2001. 321. Lucas DR, Bentley G, Dan ME, et al: Ewing sarcoma vs. Lymphoblastic Lymphoma: A Comparative Immunohistochemical Study. Am J Clin Pathol. 115:11–17, 2001. 322. Mathewson RC, Kjeldsberg CR, Perkins SL: Detection of terminal deoxynucleotidyl transferase (TdT) in non-hematopoietic small round cell tumors of children. Pediatr Pathol Lab Med. 17:835–844, 1997. 323. Ramnani D, Lindberg G, Gokaslan ST, et al: Signet-ring cell variant of small lymphocytic lymphoma with a prominent sinusoidal pattern. Ann Diagn Pathol. 3:220–226, 1999. 324. Tse CC, Chan JK, Yuen RW, et al: Malignant lymphoma with myxoid stroma: A new pattern in need of recognition. Histopathology. 18:31–35, 1991. 325. Tsang WY, Chan JK, Tang SK, et al: Large cell lymphoma with a fibrillary matrix. Histopathology. 29:80–82, 1992. 326. North PE, Waner M, Mizeracki A, et al: GLUT1: A newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol. 31:11–22, 2000. 327. Haber RS, Weiser KR, Pritsker A, et al: GLUT1 glucose transporter expression in benign and malignant thyroid nodules. Thyroid. 7:363–367, 1997. 328. Younes M, Lechago LV, Lechago J: Overexpression of the human erythrocyte glucose transporter occurs as a late event in human colorectal carcinogenesis and is associated with an increased incidence of lymph node metastases. Clin Cancer Res. 2:1151–1154, 1996. 329. Younes M, Brown RW, Stephenson M, et al: Overexpression of Glut1 and Glut3 in stage I nonsmall cell lung cancer is associated with poor survival. Cancer. 80:1046–1051, 1997.
330. Zukerberg LR, Nickoloff BJ, Weisee SW: Kaposiform hemangioendothelioma of infancy and childhood: An aggressive-neoplasm associated with Kasaback-Merritt syndrome and lymphangiomatosis. Am J Surg Pathol. 17:321–328, 1993. 331. Mentzel T, Mazzoleni G, Dei Tos AP, et al: Kaposiform hemangioendothelioma in adults. Clinicopathologic and immunohistochemical analysis of three cases. Am J Clin Pathol. 108:450–455, 1997. 332. Lyons LL, North PE, MacMoune-Lai F, et al: Kaposiform hemangioendothelioma: A study of 33 cases emphasizing its pathologic, immunophenotypic, and biologic uniqueness from juvenile hemangioma. Am J Surg Pathol. 28:559–568, 2004. 333. Arai E, Kuramochi A, Tsuchida T, et al: Usefulness of D2-40 immunohistochemistry for differentiation between kaposiform hemangioendothelioma and tufted angioma. J Cutan Pathol. 33:492, 2006. 334. Billings SD, Folpe AL, Weiss SW: Epithelioid sarcoma-like hemangioendothelioma. Am J Surg Pathol. 27:48–57, 2003. 335. Hornick JL, Fletcher CD: Pseudomyogenic hemangioendothelioma: a distinctive, often multicentric tumor with indolent behavior. Am J Surg Pathol. 35:190–201, 2011. 336. Fletcher CD, Hogendoorn P, Mertens F, et al: World Health Organization Classification of Tumors of Soft Tissue and Bone, Zurich Lyon, France, 2013, IARC. 337. Trombetta D, Magnusson L, von Steyern FV, et al: Translocation t(7;19)(q22;q13)-a recurrent chromosome aberration in pseudomyogenic hemangioendothelioma? Cancer Genet. 204:211– 215, 2011. 338. Errani C, Zhang L, Sung YS, et al: A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites. Genes Chromosomes Cancer. 50:644–653, 2011. 339. Tanas MR, Sboner A, Oliveira AM, et al: Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma. Sci Transl Med. 3:98ra82, 2011. 340. Mentzel T, Beham A, Calonje E, et al: Epithelioid hemangioendothelioma of skin and soft tissues: clinicopathologic and immunohistochemical study of 30 cases. Am J Surg Pathol. 21:363–374, 1997. 341. Hornick JL, Dal Cin P, Fletcher CD: Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma. Am J Surg Pathol. 33:542–550, 2009. 342. Miettinen M, Fetsch JF: Distribution of keratins in normal endothelial cells and a spectrum of vascular tumors: implications in tumor diagnosis. Hum Pathol. 31:1062–1067, 2000. 343. Al-Abbadi MA, Almasri NM, Al-Quran S, et al: Cytokeratin and epithelial membrane antigen expression in angiosarcomas: an immunohistochemical study of 33 cases. Arch Pathol Lab Med. 131:288–292, 2007. 344. Miettinen M, Sarlomo-Rikala M, Lasota J: KIT expression in angiosarcomas and fetal endothelial cells: lack of mutations of exon 11 and exon 17 of C-kit. Mod Pathol. 13:536–541, 2000. 345. Hornick JL, Fletcher CD: Immunohistochemical staining for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Am J Clin Pathol. 117:188–193, 2002. 346. Ueda T, Oji Y, Naka N, et al: Overexpression of the Wilms’ tumor gene WT1 in human bone and soft tissue sarcomas. Cancer Sci. 94:271–276, 2003. 347. Mentzel T, Schildhaus HU, Palmedo G, et al: Postradiation cutaneous angiosarcoma after treatment of breast carcinoma is characterized by MYC amplification in contrast to atypical vascular lesions after radiotherapy and control cases: clinicopathological, immunohistochemical and molecular analysis of 66 cases. Mod Pathol. 25:75–85, 2012. 348. Guo T, Zhang L, Chang NE, et al: Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions. Genes Chromosomes Cancer. 50:25–33, 2011. 349. Robin YM, Guillou L, Michels JJ, et al: Human herpesvirus 8 immunostaining: A sensitive and specific method for diagnosing Kaposi sarcoma in paraffin-embedded sections. Am J Clin Pathol. 121:330–334, 2004. 350. Cheuk W, Wong KO, Wong CS, et al: Immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish Kaposi sarcoma from its mimickers. Am J Clin Pathol. 121:335–342, 2004.
References 351. Weninger W, Partanen TA, Breiteneder-Geleff S, et al: Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi’s sarcoma tumor cells. Lab Invest. 79:243–251, 1999. 352. Hansen T, Katenkamp D: Rhabdomyoma of the head and neck: morphology and differential diagnosis. Virchows Arch. 447:849– 854, 2005. 353. Kapadia SB, Meis JM, Frisman DM, et al: Fetal rhabdomyoma of the head and neck: A clinicopathological and immunophenotypic study of 24 cases. Hum Pathol. 24:754–765, 1993. 354. Tanda F, Rocca PC, Bosincu L, et al: Rhabdomyoma of the tunica vaginalis of the testis: A histologic, immunohistochemical, and ultrastructural study. Mod Pathol. 10:608–611, 1997. 355. Newton WA, Jr, Soule EH, Hamoudi AB, et al: Histopathology of childhood sarcomas, Intergroup Rhabdomyosarcoma Studies I and II: clinicopathologic correlation. J Clin Oncol. 6:67–75, 1988. 356. Scrable H, Witte D, Shimada H, et al: Molecular differential pathology of rhabdomyosarcoma. Genes Chromosomes Cancer. 1:23–35, 1989. 357. Bridge JA, Liu J, Weibolt V, et al: Novel genomic imbalances in embryonal rhabdomyosarcoma revealed by comparative genomic hybridization and fluorescence in situ hybridization: an intergroup rhabdomyosarcoma study. Genes Chromosomes Cancer. 27:337–344, 2000. 358. Raney RB, Anderson JR, Barr FG, et al: Rhabdomyosarcoma and undifferentiated sarcoma in the first two decades of life: a selective review of intergroup rhabdomyosarcoma study group experience and rationale for Intergroup Rhabdomyosarcoma Study V. J Pediatr Hematol Oncol. 23:215–220, 2001. 359. Dias P, Parham DM, Shapiro DN, et al: Myogenic regulatory protein (MyoD1) expression in childhood solid tumors: Diagnostic utility in rhabdomyosarcoma. Am J Pathol. 13:1283–1291, 1990. 360. Wang NP, Marx J, McNutt MA, et al: Expression of myogenic regulatory proteins (myogenin and Myo-D1) in small blue round cell tumors of childhood. Am J Pathol. 147:1799–1810, 1995. 361. Coindre JM, deMascarel A, Trojani M, et al: Immunohistochemical study of rhabdomyosarcoma: Unexpected staining with S100 protein and cytokeratin. J Pathol. 155:127–132, 1988. 362. Coffin CM, Rulon J, Smith L, et al: Pathologic features of rhabdomyosarcoma before and after treatment: A clinicopathologic and immunohistochemical analysis. Mod Pathol. 10:1175–1187, 1997. 363. Barr FG, Galili N, Holick J, et al: Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat Genet. 3:113–117, 1993. 364. Barr FG, Nauta LE, Davis RJ, et al: In vivo amplification of the PAX3-FKHR and PAX7-FKHR fusion genes in alveolar rhabdomyosarcoma. Hum Mol Genet. 5:15–21, 1996. 365. Barr FG: Molecular genetics and pathogenesis of rhabdomyosarcoma. J Pediatr Hematol Oncol. 19:483–491, 1997. 366. Furlong MA, Mentzel T, Fanburg-Smith JC: Pleomorphic rhabdomyosarcoma in adults: a clinicopathologic study of 38 cases with emphasis on morphologic variants and recent skeletal muscle-specific markers. Mod Pathol. 14:595–603, 2001. 367. Gaffney EF, Dervan PA, Fletcher CD: Pleomorphic rhabdomyosarcoma in adulthood: Analysis of 11 cases with definition of diagnostic criteria. Am J Surg Pathol. 17:601–609, 1993. 368. Li G, Ogose A, Kawashima H, et al: Cytogenetic and real-time quantitative reverse-transcriptase polymerase chain reaction analyses in pleomorphic rhabdomyosarcoma. Cancer Genet Cytogenet. 192:1–9, 2009. 369. Mentzel T, Kuhnen C: Spindle cell rhabdomyosarcoma in adults: clinicopathological and immunohistochemical analysis of seven new cases. Virchows Arch. 449:554–560, 2006. 370. Mentzel T, Katenkamp D: Sclerosing, pseudovascular rhabdomyosarcoma in adults. Clinicopathological and immunohistochemical analysis of three cases. Virchows Arch. 436:305–311, 2000. 371. Chiles MC, Parham DM, Qualman SJ, et al: Sclerosing rhabdomyosarcomas in children and adolescents: a clinicopathologic review of 13 cases from the Intergroup Rhabdomyosarcoma Study Group and Children’s Oncology Group. Pediatr Dev Pathol. 8:141, 2005.
129.e9
372. Jo VY, Marino-Enriquez A, Fletcher CD: Epithelioid rhabdomyosarcoma: clinicopathologic analysis of 16 cases of a morphologically distinct variant of rhabdomyosarcoma. Am J Surg Pathol. 35:1523–1530, 2011. 373. Bridge JA, Meloni AM, Neff JR, et al: Deletion 5q in desmoid tumor and fluorescence in situ hybridization for chromosome 8 and/or 20 copy number. Cancer Genet Cytogenet. 92:150–151, 1996. 374. Rakheja D, Molberg KH, Roberts CA, et al: Immunohistochemical expression of beta-catenin in solitary fibrous tumors. Arch Pathol Lab Med. 129:776–779, 2005. 375. Barak S, Wang Z, Miettinen M: Immunoreactivity for calretinin and keratins in desmoid fibromatosis and other myofibroblastic tumors: a diagnostic pitfall. Am J Surg Pathol. 36:1404–1409, 2012. 376. Neilsen GP, O’Connell JX, Dickersin GR, et al: Collagenous fibroma (desmoplastic fibroblastoma): A report of seven cases. Mod Pathol. 9:781–785, 1996. 377. Eckert F, Schaich B: Tendon sheath fibroma: A case report with immunohistochemical studies. Hautarzt. 43:92–96, 1992. 378. Sciot R, Samson I, van der Berghe H, et al: Collagenous fibroma (desmoplastic fibroblastoma): Genetic link with fibroma of tendon sheath? Mod Pathol. 12:565–568, 1999. 379. Ockner DM, Sayadi H, Swanson PE, et al: Genital angiomyofibroblastoma. Comparison with aggressive angiomyxoma and other myxoid neoplasms of skin and soft tissue. Am J Clin Pathol. 107:36–44, 1997. 380. Neilsen GP, Rosenberg AE, Young RH, et al: Angiomyofibroblastoma of the vulva and vagina. Mod Pathol. 9:284–291, 1996. 381. Klemperer P, Rabin CB: Primary neoplasms of the pleura: A report of five cases. Arch Pathol. 11:385–412, 1931. 382. Guillou L, Gebhard S, Coindre JM: Lipomatous hemangiopericytoma: a fat-containing variant of solitary fibrous tumor? Clinicopathologic, immunohistochemical, and ultrastructural analysis of a series in favor of a unifying concept. Hum Pathol. 31:1108– 1115, 2000. 383. Hanau CA, Miettinen M: Solitary fibrous tumor: Histological and immunohistochemical spectrum of benign and malignant variants presenting at different sites. Hum Pathol. 26:440–449, 1995. 384. de Saint Aubain Somerhausen N, Rubin BP, Fletcher CD: Myxoid solitary fibrous tumor: A study of seven cases with emphasis on differential diagnosis. Mod Pathol. 12:463–471, 1999. 385. Bhargava R, Shia J, Hummer AJ, et al: Distinction of endometrial stromal sarcomas from “hemangiopericytomatous” tumors using a panel of immunohistochemical stains. Mod Pathol. 18:40–47, 2005. 386. Ng TL, Gown AM, Barry TS, et al: Nuclear beta-catenin in mesenchymal tumors. Mod Pathol. 18:68–74, 2005. 387. Fletcher CD, Dal Cin P, de Wever I, et al: Correlation between clinicopathological features and karyotype in spindle cell sarcomas. A report of 130 cases from the CHAMP study group. Am J Pathol. 154:1841–1847, 1999. 388. Bertucci F, Bouvier-Labit C, Finetti P, et al: Comprehensive genome characterization of solitary fibrous tumors using highresolution array-based comparative genomic hybridization. Genes Chromosomes Cancer. 52:156–164, 2012. 389. Hajdu M, Singer S, Maki RG, et al: IGF2 over-expression in solitary fibrous tumours is independent of anatomical location and is related to loss of imprinting. J Pathol. 221:300–307, 2010. 390. Evans HL: Low-grade fibromyxoid sarcoma. A report of 12 cases. Am J Surg Pathol. 17:595–600, 1993. 391. Goodlad JR, Mentzel T, Fletcher CD: Low grade fibromyxoid sarcoma: clinicopathological analysis of eleven new cases in support of a distinct entity. Histopathology. 26:229–237, 1995. 392. Evans HL: Low-grade fibromyxoid sarcoma: a clinicopathologic study of 33 cases with long-term follow-up. Am J Surg Pathol. 35:1450–1462, 2011. 393. Panagopoulos I, Storlazzi CT, Fletcher CD, et al: The chimeric FUS/CREB3l2 gene is specific for low-grade fibromyxoid sarcoma. Genes Chromosomes Cancer. 40:218–228, 2004. 394. Panagopoulos I, Moller E, Dahlen A, et al: Characterization of the native CREB3L2 transcription factor and the FUS/CREB3L2 chimera. Genes Chromosomes Cancer. 46:181–191, 2007.
129.e10
Immunohistology of Neoplasms of Soft Tissue and Bone
395. Mertens F, Fletcher CD, Antonescu CR, et al: Clinicopathologic and molecular genetic characterization of low-grade fibromyxoid sarcoma, and cloning of a novel FUS/CREB3L1 fusion gene. Lab Invest. 85:408–415, 2005. 396. Folpe AL, Lane KL, Paull G, et al: Low-grade fibromyxoid sarcoma and hyalinizing spindle cell tumor with giant rosettes: a clinicopathologic study of 73 cases supporting their identity and assessing the impact of high-grade areas. Am J Surg Pathol. 24:1353–1360, 2000. 397. Meis-Kindblom JM, Kindblom LG, Enzinger FM: Sclerosing epithelioid fibrosarcoma. A variant of fibrosarcoma simulating carcinoma. Am J Surg Pathol. 19:979–993, 1995. 398. Antonescu CR, Rosenblum MK, Pereira P, et al: Sclerosing epithelioid fibrosarcoma: a study of 16 cases and confirmation of a clinicopathologically distinct tumor. Am J Surg Pathol. 25:699– 709, 2001. 399. Rekhi B, Folpe AL, Deshmukh M, et al: Sclerosing epithelioid fibrosarcoma—A report of two cases with cytogenetic analysis of FUS gene rearrangement by FISH technique. Pathol Oncol Res. 2010. 400. Eyden BP, Manson C, Banerjee SS, et al: Sclerosing epithelioid fibrosarcoma: a study of five cases emphasizing diagnostic criteria. Histopathology. 33:354–360, 1998. 401. Shibahara H, Tamada S, Higashi M, et al: MUC4 is a novel prognostic factor of intrahepatic cholangiocarcinoma-mass forming type. Hepatology. 39:220–229, 2004. 402. Hanaoka J, Kontani K, Sawai S, et al: Analysis of MUC4 mucin expression in lung carcinoma cells and its immunogenicity. Cancer. 92:2148–2157, 2001. 403. Andrianifahanana M, Moniaux N, Schmied BM, et al: Mucin (MUC) gene expression in human pancreatic adenocarcinoma and chronic pancreatitis: a potential role of MUC4 as a tumor marker of diagnostic significance. Clin Cancer Res. 7:4033–4040, 2001. 404. Meis-Kindblom JM, Kindblom LG: Acral myxoinflammatory fibroblastic sarcoma: a low-grade tumor of the hands and feet. Am J Surg Pathol. 22:911–924, 1998. 405. Antonescu CR, Zhang L, Nielsen GP, et al: Consistent t(1;10) with rearrangements of TGFBR3 and MGEA5 in both myxoinflammatory fibroblastic sarcoma and hemosiderotic fibrolipomatous tumor. Genes Chromosomes Cancer. 50:757–764, 2011. 406. Fetsch JF, Laskin WB, Miettinen M: Superficial acral fibromyxoma: a clinicopathologic and immunohistochemical analysis of 37 cases of a distinctive soft tissue tumor with a predilection for the fingers and toes. Hum Pathol. 32:704–714, 2001. 407. Elco CP, Marino-Enriquez A, Abraham JA, et al: Hybrid myxoinflammatory fibroblastic sarcoma/hemosiderotic fibrolipomatous tumor: report of a case providing further evidence for a pathogenetic link. Am J Surg Pathol. 34:1723–1727, 2010. 408. Hallor KH, Sciot R, Staaf J, et al: Two genetic pathways, t(1;10) and amplification of 3p11-12, in myxoinflammatory fibroblastic sarcoma, haemosiderotic fibrolipomatous tumour, and morphologically similar lesions. J Pathol. 217:716–727, 2009. 409. Antonescu CR, Dal Cin P, Nafa K, et al: EWSR1-CREB1 is the predominant gene fusion in angiomatoid fibrous histiocytoma. Genes Chromosomes Cancer. 46:1051–1060, 2007. 410. Rossi S, Szuhai K, Ijszenga M, et al: EWSR1-CREB1 and EWSR1-ATF1 fusion genes in angiomatoid fibrous histiocytoma. Clin Cancer Res. 13:7322–7328, 2007. 411. Fletcher CDM, Unni KK, Mertens F, editors. World Health Organization Classification of Tumours Pathology and Genetics of Tumours of Soft Tissue and Bone, Lyon, 2002, IARC Press. 412. Gleason B, Hornick JL: Inflammatory myofibroblastic tumours: Where are we now? J Clin Pathol. 61:428–437, 2008. 413. Coffin CM, Hornick JL, Fletcher CD: Inflammatory myofibroblastic tumor: comparison of clinicopathologic, histologic, and immunohistochemical features including ALK expression in atypical and aggressive cases. Am J Surg Pathol. 31:509–520, 2007. 414. Coffin CM, Watterson J, Priest JR, et al: Extrapulmonary inflammatory myofibroblastic tumor (inflammatory pseudotumor). A clinicopathologic and immunohistochemical study of 84 cases. Am J Surg Pathol. 19:859–872, 1995. 415. Yamamoto H, Oda Y, Saito T, et al: p53 Mutation and MDM2 amplification in inflammatory myofibroblastic tumours. Histopathology. 42:431–439, 2003.
416. Griffin CA, Hawkins AL, Dvorak C, et al: Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors. Cancer Res. 59:2776–2780, 1999. 417. Morris SW, Kirstein MN, Valentine MB, et al: Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science. 263:1281–1284, 1994. 418. Coffin CM, Jaszcz W, O’Shea PA, et al: So-called congenitalinfantile fibrosarcoma: does it exist and what is it? Pediatr Pathol. 14:133–150, 1994. 419. Knezevich SR, McFadden DE, Tao W, et al: A novel ETV6NTRK3 gene fusion in congenital fibrosarcoma. Nat Genet. 18:184–187, 1998. 420. Nascimento AF, Bertoni F, Fletcher CD: Epithelioid variant of myxofibrosarcoma: expanding the clinicomorphologic spectrum of myxofibrosarcoma in a series of 17 cases. Am J Surg Pathol. 31:99–105, 2007. 421. Mentzel T, Calonje E, Wadden C, et al: Myxofibrosarcoma. Clinicopathologic analysis of 75 cases with emphasis on the lowgrade variant. Am J Surg Pathol. 20:391–405, 1996. 422. Mertens F, Fletcher CD, Dal Cin P, et al: Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: a report of the CHAMP Study Group. Chromosomes and MorPhology. Genes Chromosomes Cancer. 22:16–25, 1998. 423. Willems SM, Debiec-Rychter M, Szuhai K, et al: Local recurrence of myxofibrosarcoma is associated with increase in tumour grade and cytogenetic aberrations, suggesting a multistep tumour progression model. Mod Pathol. 19:407–416, 2006. 424. Billings SD, Folpe AL, Weiss SW: Do leiomyomas of deep soft tissue exist? An analysis of highly differentiated smooth muscle tumors of deep soft tissue supporting two distinct subtypes. Am J Surg Pathol. 25:1134–1142, 2001. 425. Kraft S, Fletcher CD: Atypical intradermal smooth muscle neoplasms: clinicopathologic analysis of 84 cases and a reappraisal of cutaneous “leiomyosarcoma”. Am J Surg Pathol. 35:599–607, 2011. 426. Azumi N, Ben-Ezra J, Battifora H: Immunophenotypic diagnosis of leiomyosarcomas and rhabdomyosarcomas with monoclonal antibodies to muscle-specific actin and desmin in formalin-fixed tissue. Mod Pathol. 1:469–474, 1988. 427. Iwata J, Fletcher CD: Immunohistochemical detection of cytokeratin and epithelial membrane antigen in leiomyosarcoma: a systematic study of 100 cases. Pathol Int. 50:7–14, 2000. 428. Miettinen M: Immunoreactivity for cytokeratin and epithelial membrane antigen in leiomyosarcoma. Arch Pathol Lab Med. 112:637–640, 1988. 429. Miettinen M, Sobin LH, Sarlomo-Rikala M: Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with a reference to CD117 (KIT). Mod Pathol. 13:1134–1142, 2000. 430. Winder SJ, Walsh MP: Calponin: Thin filament-linked regulation of smooth muscle contraction. Cell Signal. 5:677–686, 1993. 431. Kaddu S, Baham A, Cerroni L, et al: Cutaneous leiomyosarcoma. Am J Surg Pathol. 21:970–987, 1997. 432. Rao UN, Finkelstein SD, Jones MW: Comparative immunohistochemical and molecular analysis of uterine and extrauterine leiomyosarcomas. Mod Pathol. 1(2):1001–1009, 1999. 433. Oda Y, Miyajima K, Kawaguchi K, et al: Pleomorphic leiomyosarcoma: Clinicopathologic and immunohistochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous histiocytoma. Am J Surg Pathol. 25:1030–1038, 2001. 434. Chen E, O’Connell F, Fletcher CD: Dedifferentiated leiomyosarcoma: clinicopathological analysis of 18 cases. Histopathology. 59:1135–1143, 2011. 435. Nicolas MM, Tamboli P, Gomez JA, et al: Pleomorphic and dedifferentiated leiomyosarcoma: clinicopathologic and immunohistochemical study of 41 cases. Hum Pathol. 41:663–671, 2010. 436. Merchant W, Calonje E, Fletcher CD: Inflammatory leiomyosarcoma: a morphological subgroup within the heterogeneous family of so-called inflammatory malignant fibrous histiocytoma. Histopathology. 27:525–532, 1995. 437. Bowman F, Gultekin H, Dickman PS: Latent Epstein-Barr virus infection demonstrated in low-grade leiomyosarcomas of adults with acquired immunodeficiency syndrome, but not in adjacent
References Kaposi’s lesion or smooth muscle tumors in immunocompetent patients. Arch Pathol Lab Med. 121:834–838, 1997. 438. Jenson HB, Montalvo EA, McClain KL, et al: Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma. J Med Virol. 57:36–46, 1999. 439. Hornick JL, Fletcher CD: PEComa: what do we know so far? Histopathology. 48:75–82, 2006. 440. Folpe AL, Mentzel T, Lehr HA, et al: Perivascular epithelioid cell neoplasms of soft tissue and gynecologic origin: A clinicopathologic study of 26 cases and review of the literature. Am J Surg Pathol. 29:1558–1575, 2005. 441. Gray MH, Rosenberg AE, Dickersin GR, et al: Glial fibrillary acidic protein and keratin expression by benign and malignant nerve sheath tumors. Hum Pathol. 20:1089–1096, 1989. 442. Fine SW, McClain SA, Li M: Immunohistochemical staining for calretinin is useful for differentiating schwannomas from neurofibromas. Am J Clin Pathol. 122:552–559, 2004. 443. Carney JA: Psammomatous melanotic schwannoma: A distinctive, heritable tumor with special associations, including cardiac myxoma and the Cushing syndrome. Am J Surg Pathol. 14:206– 222, 1990. 444. Fanburg-Smith JC, Majidi M, Miettinen M: Keratin expression in schwannoma; a study of 115 retroperitoneal and 22 peripheral schwannomas. Mod Pathol. 19:115–121, 2006. 445. Hornick JL, Bundock EA, Fletcher CD: Hybrid schwannoma/ perineurioma: clinicopathologic analysis of 42 distinctive benign nerve sheath tumors. Am J Surg Pathol. 33:1554–1561, 2009. 446. Shimokama T, Watanabe T: Leiomyoma exhibiting a marked granular change: Granular cell leiomyoma versus granular cell schwannoma. Hum Pathol. 23:327–331, 1992. 447. Mazur MT, Shultz JJ, Myers JL: Granular cell tumor: Immunohistochemical analysis of 21 benign tumors and one malignant tumor. Arch Pathol Lab Med. 114:692–696, 1990. 448. Kurtin PJ, Bonin DM: Immunohistochemical demonstration of the lysosome-associated glycoprotein CD68 (KP-1) in granular cell tumors and schwannomas. Hum Pathol. 25:1172–1178, 1994. 449. Filie AC, Lage JM, Azumi N: Immunoreactivity of S100 protein, alpha-1-antitrypsin, and CD68 in adult and congenital granular cell tumors. Mod Pathol. 9:888–892, 1996. 450. Fine SW, Li M: Expression of calretinin and the alpha–subunit of inhibin in granular cell tumors. Am J Clin Pathol. 119:259– 264, 2003. 451. Lazar AJ, Fletcher CD: Primitive nonneural granular cell tumors of skin: clinicopathologic analysis of 13 cases. Am J Surg Pathol. 29:927–934, 2005. 452. LeBoit PE, Barr RJ, Burall S, et al: Primitive polypoid granular-cell tumor and other cutaneous granular-cell neoplasms of apparent nonneural origin. Am J Surg Pathol. 15:48–58, 1991. 453. Vered M, Dobriyan A, Buchner A: Congenital granular cell epulis presents an immunohistochemical profile that distinguishes it from the granular cell tumor of the adult. Virchows Arch. 454:303–310, 2009. 454. Kourea HP, Cordon-Cardo C, Dudas M, et al: Expression of p27(kip) and other cell cycle regulators in malignant peripheral nerve sheath tumors and neurofibromas: the emerging role of p27(kip) in malignant transformation of neurofibromas. Am J Pathol. 155:1885–1891, 1999. 455. Mertens F, Dal Cin P, De Wever I, et al: Cytogenetic characterization of peripheral nerve sheath tumours: a report of the CHAMP study group. J Pathol. 190:31–38, 2000. 456. Miettinen M: Modern Soft Tissue Pathology, New York, NY, 2010, Cambridge University Press. 457. King R, Busam K, Rosai J: Metastatic malignant melanoma resembling malignant peripheral nerve sheath tumor: Report of 16 cases. Am J Surg Pathol. 23:1499–1505, 1999. 458. Morphopoulos GD, Banerjee SS, Ali HH, et al: Malignant peripheral nerve sheath tumour with vascular differentiation: A report of four cases. Histopathology. 28:401–410, 1996. 459. Laskin WB, Weiss SW, Bratthauer GL: Epithelioid variant of malignant peripheral nerve sheath tumor (malignant epithelioid schwannoma). Am J Surg Pathol. 15:1136–1145, 1991. 460. Hoang MP, Sinkre P, Albores-Saavedra J: Expression of protein gene product 9. In epithelioid and conventional malignant
129.e11
peripheral nerve sheath tumors. Arch Pathol Lab Med. 125:1321– 1325, 2001. 461. Jokinen CH, Dadras SS, Goldblum JR, et al: Diagnostic implications of podoplanin expression in peripheral nerve sheathneoplasms. Am J Clin Pathol. 129:886–893, 2008. 462. Suster S, Fisher C: Immunoreactivity for the human hematopoietic progenitor cell antigen (CD34) in lipomatous tumors. Am J Surg Pathol. 21:195–200, 1997. 463. Templeton SF, Solomon AR Jr: Spindle cell lipoma is strongly CD34-positive: an immunohistochemical study. J Cutan Pathol. 23:546–550, 1996. 464. Tardio JC, Aramburu JA, Santonja C: Desmin expression in spindle cell lipomas: a potential diagnostic pitfall. Virchows Arch. 445:354–358, 2004. 465. Boland JM, Weiss SW, Oliveira AM, et al: Liposarcomas with mixed well-differentiated and pleomorphic features: a clinicopathologic study of 12 cases. Am J Surg Pathol. 34:837–843, 2010. 466. Marino-Enriquez A, Fletcher CD, Dal Cin P, et al: Dedifferentiated liposarcoma with “homologous” lipoblastic (pleomorphic liposarcoma-like) differentiation: clinicopathologic and molecular analysis of a series suggesting revised diagnostic criteria. Am J Surg Pathol. 34:1122–1131, 2010. 467. Sandberg AA: Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: Liposarcoma. Cancer Genet Cytogenet. 155:1–24, 2004. 468. Italiano A, Bianchini L, Keslair F, et al: HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas, whereas CDK4 belongs to a distinct inconsistent amplicon. Int J Cancer. 122:2233–2241, 2008. 469. Binh MB, Guillou L, Hostein I, et al: Dedifferentiated liposarcomas with divergent myosarcomatous differentiation developed in the internal trunk: a study of 27 cases and comparison to conventional dedifferentiated liposarcomas and leiomyosarcomas. Am J Surg Pathol. 31:1557–1566, 2007. 470. He M, Aisner S, Benevenia J, et al: P16 Immunohistochemistry as an alternative marker to distinguish atypical lipomatous tumor from deep-seated lipoma. Appl Immunohistochem Mol Morphol. 17:51–56, 2009. 471. Thway K, Flora R, Shah C, et al: Diagnostic utility of p16, CDK4, and MDM2 as an immunohistochemical panel in distinguishing well-differentiated and dedifferentiated liposarcomas from other adipocytic tumors. Am J Surg Pathol. 36:462–469, 2012. 472. Hornick JL, Bosenberg MW, Mentzel T, et al: Pleomorphic liposarcoma: clinicopathologic analysis of 57 cases. Am J Surg Pathol. 28:1257–1267, 2004. 473. Miettinen M, Enzinger FM: Epithelioid variant of pleomorphic liposarcoma: a study of 12 cases of a distinctive variant of highgrade liposarcoma. Mod Pathol. 12:722–728, 1999. 474. Fritz B, Schubert F, Wrobel G, et al: Microarray-based copy number and expression profiling in dedifferentiated and pleomorphic liposarcoma. Cancer Res. 62:2993–2998, 2002. 475. Rieker RJ, Joos S, Bartsch C, et al: Distinct chromosomal imbalances in pleomorphic and in high-grade dedifferentiated liposarcomas. Int J Cancer. 99:68–73, 2002. 476. Tallini G, Akerman M, Dal Cin P, et al: Combined morphologic and karyotypic study of 28 myxoid liposarcomas. Implications for a revised morphologic typing, (a report from the CHAMP Group). Am J Surg Pathol. 20:1047–1055, 1996. 477. Dal Cin P, Sciot R, Panagopoulos I, et al: Additional evidence of a variant translocation t(12;22) with EWS/CHOP fusion in myxoid liposarcoma: clinicopathological features. J Pathol. 182:437–441, 1997. 478. Kilpatrick SE, Doyon J, Choong PF, et al: The clinicopathologic spectrum of myxoid and round cell liposarcoma. A study of 95 cases. Cancer. 77:1450–1458, 1996. 479. De Vreeze RS, de Jong D, Tielen IH, et al: Primary retroperitoneal myxoid/round cell liposarcoma is a nonexisting disease: An immunohistochemical and molecular biological analysis. Mod Pathol. 22:223–231, 2009. 480. Oda Y, Yamamoto H, Takahiira T, et al: Frequent alteration of p16 (INK4a/p14 (ARF) and p53 pathways in the round cell component of myxoid/round cell liposarcoma: p53 gene alterations and reduced p14 (ARF) expression both correlate with poor prognosis. J Pathol. 207:410–421, 2005.
129.e12
Immunohistology of Neoplasms of Soft Tissue and Bone
481. Dei Tos AP, Piccinin S, Doglioni C, et al: Molecular aberrations of the G1-S checkpoint in myxoid and round cell liposarcoma. Am J Pathol. 151:1531–1539, 1997. 482. Gerald WL, Ladanyi M, de Alava E, et al: Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22) (p13]2): Desmoplastic small round-cell tumor and its variants. J Clin Oncol. 16:3028–3036, 1998. 483. Ordonez NG: Desmoplastic small round cell tumor: II: an ultrastructural and immunohistochemical study with emphasis on new immunohistochemical markers. Am J Surg Pathol. 22:1314– 1327, 1998. 484. Ordi J, de Alava E, Torne A, et al: Intraabdominal desmoplastic small round-cell tumor with EWS/ERG fusion transcripts. Am J Surg Pathol. 22:1026–1032, 1998. 485. Enzinger FM, Weiss SW, Liang CY: Ossifying fibromyxoid tumor of soft parts. A clinicopathological analysis of 59 cases. Am J Surg Pathol. 13:817–827, 1989. 486. Matsumoto K, Yamamoto T, Min W, et al: Ossifying fibromyxoid tumor of soft parts: Clinicopathologic, immunohistochemical and ultrastructural study of four cases. Pathol Int. 49:742–746, 1999. 487. Fukunaga M, Ushigome S, Ishikawa E: Ossifying subcutaneous tumor with myofibroblastic differentiation: A variant of ossifying fibromyxoid tumor of soft parts? Pathol Int. 44:727–734, 1994. 488. Miettinen M, Finnell V, Fetsch JF: Ossifying fibromyxoid tumor of soft parts–a clinicopathologic and immunohistochemical study of 104 cases with long-term follow-up and a critical review of the literature. Am J Surg Pathol. 32:996–1005, 2008. 489. Miettinen M: Ossifying fibromyxoid tumor of soft parts: Additional observations of a distinctive soft tissue tumor. Am J Clin Pathol. 95:142–149, 1991. 490. Ekfors TO, Kulju T, Aaltonen M, et al: Ossifying fibromyxoid tumour of soft parts: Report of four cases including one mediastinal and one infantile. APMIS. 106:1124–1130, 1998. 491. Gebre-Medhin S, Nord KH, Moller E, et al: Recurrent rearrangement of the PHF1 gene in ossifying fibromyxoid tumors. Am J Pathol. 181:1069–1077, 2012. 492. Miettinen M, Virtanen I: Synovial sarcoma–a misnomer. Am J Pathol. 117:18–25, 1984. 493. Clark J, Rocques PJ, Crew AJ, et al: Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat Genet. 7:502–508, 1994. 494. Pelmus M, Guillou L, Hostein I, et al: Monophasic fibrous and poorly differentiated synovial sarcoma: immunohistochemical reassessment of 60 t(X;18)(SYT-SSX)-positive cases. Am J Surg Pathol. 26:1434–1440, 2002. 495. Lopes JM, Bjerkehagen B, Holm R, et al: Immunohistochemical profile of synovial sarcoma with emphasis on the epithelial-type differentiation: A study of 49 primary tumours, recurrences, and metastases. Pathol Res Pract. 190:168–177, 1994. 496. Machen SK, Fisher C, Gautam RS, et al: Utility of cytokeratin subsets for distinguishing poorly differentiated synovial sarcoma from peripheral primitive neuroectodermal tumour. Histopathology. 33:501–507, 1998. 497. Ordonez NG, Mahfouz SM, MacKay B: Synovial sarcoma: An immunohistochemical and ultrastructural study. Hum Pathol. 21:733–749, 1990. 498. Nicholson AG, Goldstraw P, Fisher C: Synovial sarcoma of the pleura and its differentiation from other primary pleural tumours: A clinicopathological and immunohistochemical review of three cases. Histopathology. 33:508–513, 1998. 499. Miettinen M, Limon J, Niezabitowski A, et al: Calretinin and other mesothelioma markers in synovial sarcoma: analysis of antigenic similarities and differences with malignant mesothelioma. Am J Surg Pathol. 25:610–617, 2001. 500. Masui F, Matsuno Y, Yokoyama R, et al: Synovial sarcoma, histologically mimicking primitive neuroectodermal tumor/ Ewing’s sarcoma at distant sites. Jpn J Clin Oncol. 29:438–441, 1999. 501. Lai JP, Robbins PF, Raffeld M, et al: NY-ESO-1 expression in synovial sarcoma and other mesenchymal tumors: significance for NY-ESO-1-based targeted therapy and differential diagnosis. Mod Pathol. 25:854–858, 2012.
502. Enzinger FM: Epitheloid sarcoma. A sarcoma simulating a granuloma or a carcinoma. Cancer. 26:1029–1041, 1970. 503. Chase DR, Enzinger FM: Epithelioid sarcoma. Diagnosis, prognostic indicators, and treatment. Am J Surg Pathol. 9:241–263, 1985. 504. Chetty R, Slavin JL: Epithelioid sarcoma with extensive chondroid differentiation. Histopathology. 24:400–401, 1994. 505. Laskin WB, Miettinen M: Epithelioid sarcoma: new insights based on an extended immunohistochemical analysis. Arch Pathol Lab Med. 127:1161–1168, 2003. 506. Miettinen M, Fanburg-Smith JC, Virolainen M, et al: Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis. Hum Pathol. 30:934–942, 1999. 507. Wick MR, Manivel JC: Epithelioid sarcoma and isolated necrobiotic granuloma: A comparative immunocytochemical study. J Cutan Pathol. 13:253–260, 1986. 508. Hiraga H, Nojima T, Abe S, et al: Establishment of a new continuous clear cell sarcoma cell line: Morphological and cytogenetic characterization and detection of chimaeric EWS/ATF-1 transcripts. Virchows Arch. 431:45–51, 1997. 509. Stenman G, Kindblom LG, Angervall L: Reciprocal translocation t(12;22)(q13]3) in clear-cell sarcoma of tendons and aponeuroses. Genes Chromosomes Cancer. 4:122–127, 1992. 510. Meis-Kindblom JM: Clear cell sarcoma of tendons and aponeuroses: A historical perspective and tribute to the man behind the entity. Adv Anat Pathol. 13:286–292, 2006. 511. Antonescu CR, Nafa K, Segal NH, et al: EWS-CREB1: a recurrent variant fusion in clear cell sarcoma–association with gastrointestinal location and absence of melanocytic differentiation. Clin Cancer Res. 12:5356–5362, 2006. 512. Zambrano E, Reyes-Mugica M, Franchi A, et al: An osteoclastrich tumor of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: reports of 6 cases of a GIST simulator. Int J Surg Pathol. 11:75–81, 2003. 513. Stockman DL, Miettinen M, Suster S, et al: Malignant gastrointestinal neuroectodermal tumor: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of 16 cases with a reappraisal of clear cell sarcoma-like tumors of the gastrointestinal tract. Am J Surg Pathol. 36:857–868, 2012. 514. Reference deleted in proofs. 515. Ogose A, Yazawa Y, Ueda T, et al: Alveolar soft part sarcoma in Japan: multi-institutional study of 57 patients from the Japanese Musculoskeletal Oncology Group. Oncology. 65:7–13, 2003. 516. Ladanyi M, Lui MY, Antonescu CR, et al: The der(17)t(X;17) (p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25. Oncogene. 20:48–57, 2001. 517. Pang LJ, Chang B, Zou H, et al: Alveolar soft part sarcoma: A biomarker diagnostic strategy using TFE3 immunoassay and ASPL-TFE3 fusion transcripts in paraffin-embedded tumor tissues. Diagn Mol Pathol. 17:245–252, 2008. 518. Wu A, Kunji LP, Cheng L, et al: Renal cell carcinoma in children and young adults: Analysis of clinicopathological, immunohistochemical, and molecular characteristics with an emphasis on the spectrum of Xp11 translocation-associated and unusual clear cell subtypes. Histopathology. 53:533–544, 2008. 519. Cho HY, Chung DH, Khurana H, et al: The role of TFE3 in PEComa. Histopathology. 53:236–249, 2008. 520. Foschini MP, Ceccarelli C, Eusebi V, et al: Alveolar soft-part sarcoma: Immunological evidence of rhabdomyoblastic differentiation. Histopathology. 13:101–108, 1988. 521. Foschini MP, Eusein V: Alveolar soft part sarcoma: A new type of rhabdomyosarcoma? Semin Diagn Pathol. 4:58–68, 1994. 522. Hirose T, Kudo E, Hasaegawa T, et al: Cytoskeletal properties of alveolar soft part sarcoma. Hum Pathol. 21:204–211, 1990. 523. Menesce LP, Eyden BP, Edmondson D, et al: Immunophenotype and ultrastructure of alveolar soft part sarcoma. J Submicrosc Cytol Pathol. 2593:377–387, 1993. 524. Rosai J, Dias P, Parham DM, et al: MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature. Am J Surg Pathol. 15:974–981, 1991. 525. Nakano H, Tateishi A, Imamura T, et al: RT-PCR suggests human skeletal muscle origin of alveolar soft part sarcoma. Oncology. 58:319–323, 2000.
References 526. Ordonez NG, Mackay B: Alveolar soft-part sarcoma: A review of the pathology and histogenesis. Ultrastruct Pathol. 22:275– 292, 1998. 527. Miettinen M, Ekfors T: Alveolar soft part sarcoma: Immunohistochemical evidence for muscle cell differentiation. Am J Clin Pathol. 93:32–38, 1990. 528. Auerbach HE, Brooks JJ: Alveolar soft part sarcoma. A clinicopathologic and immunohistochemical study. Cancer. 60:66–73, 1987. 529. Ladanyi M, Antonescu CR, Drobnjak M, et al: The precrystalline cytoplasmic granules of alveolar soft part sarcoma contain monocarboxylate transporter 1 and CD147. Am J Pathol. 160:1215–1221, 2002. 530. Kilpatrick SE, Inwards CY, Fletcher CD, et al: Myxoid chondrosarcoma (chordoid sarcoma) of bone: A report of two cases and review of the literature. Cancer. 79:1903–1910, 1997. 531. Antonescu CR, Argani P, Erlandson RA, et al: Skeletal and extraskeletal myxoid chondrosarcoma: A comparative clinicopathologic, ultrastructural, and molecular study. Cancer. 83:1504–1521, 1998. 532. Orndal C, Carlen B, Akerman M, et al: Chromosomal abnormality t(9;22)(q22;q12) in an extraskeletal myxoid chondrosarcoma characterized by fine needle aspiration cytology, electron microscopy, immunohistochemistry and DNA flow cytometry. Cytopathology. 2:261–270, 1991. 533. Aigner T, Oliveira AM, Nascimento AG: Extraskeletal myxoid chondrosarcomas do not show a chondrocytic phenotype. Mod Pathol. 17:214–221, 2004. 534. Domanski HA, Carlen B, Mertens F, et al: Extraskeletal myxoid chondrosarcoma with neuroendocrine differentiation: A case report with fine-needle aspiration biopsy, histopathology, electron microscopy, and cytogenetics. Ultrastruct Pathol. 27:363– 368, 2003. 535. Goh YW, Spagnolo DV, Platten M, et al: Extraskeletal myxoid chondrosarcoma: A light microscopic, immunohistochemical, ultrastructural, and immunoultrastructural study indicating neuroendocrine differentiation. Histopathology. 39:514–524, 2001. 536. Algros MP, Collonge-Rame MA, Bedgejian I, et al: Neuroectodermal differentiation of extraskeletal myxoid chondrosarcoma: A classical feature? Ann Pathol. 23:244–248, 2003. 537. Schmidt ML, Lal A, Seeger RC, et al: Favorable prognosis for patients 12 to 18 months of age with stage 4 nonamplified MYCN neuroblastoma: a Children’s Cancer Group Study. J Clin Oncol. 23:6474–6480, 2005. 538. Duijkers FA, Gaal J, Meijerink JP, et al: High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome. Am J Pathol. 180:1223–1231, 2012. 539. Gleason B, Hornick JL: Myoepithelial tumours of skin and soft tissue: An update. Diagn Histopathol. 14:552–562, 2008. 540. Folpe AL, Agoff SN, Willis J, et al: Parachordoma is immunohistochemically and cytogenetically distinct from axial chordoma and extraskeletal myxoid chondrosarcoma. Am J Surg Pathol. 23:1059–1067, 1999. 541. Jo VY, Fletcher CD: P63 Immunohistochemical staining is limited in soft tissue tumors. Am J Clin Pathol. 136:762–766, 2011. 542. Tirabosco R, Mangham DC, Rosenberg AE, et al: Brachyury expression in extra-axial skeletal and soft tissue chordomas: A marker that distinguishes chordoma from mixed tumor/ myoepithelioma/parachordoma in soft tissue. Am J Surg Pathol. 32:572–580, 2008. 543. Conner JR, Hornick JL: SATB2 is a novel marker of osteoblastic differentiation in bone and soft tissue tumours. Histopathology. 63:36–49, 2013. 544. Lidang Jensen LM, Schumacher B, Jensen MO, et al: Extraskeletal osteosarcomas: A clinicopathologic study of 25 cases. Am J Surg Pathol. 22:588–594, 1998. 545. Hasegawa T, Hirose T, Hizawa K, et al: Immunophenotypic heterogeneity in osteosarcomas. Hum Pathol. 22:583–590, 1991. 546. Begin LR, Clement PB, Kirk ME, et al: Aggressive angiomyxoma of pelvic soft parts: A clinicopathologic study of nine cases. Hum Pathol. 16:621–628, 1985. 547. Kodet R, Newton WA, Jr, Sachs N, et al: Rhabdoid tumors of soft tissues: a clinicopathologic study of 26 cases enrolled on the
129.e13
Intergroup Rhabdomyosarcoma Study. Hum Pathol. 22:674– 684, 1991. 548. Perrone T, Swanson PE, Twiggs L, et al: Malignant rhabdoid tumor of the vulva: Is distinction from epithelioid sarcoma possible? A pathologic and immunohistochemical study. Am J Surg Pathol. 13:848–858, 1989. 549. Shiratsuchi H, Oshiro Y, Saito T, et al: Cytokeratin subunits of inclusion bodies in rhabdoid cells: Immunohistochemical and clinicopathological study of malignant rhabdoid tumor and epithelioid sarcoma. Int J Surg Pathol. 9:37–48, 2001. 550. Oda Y, Tsuneyoshi M: Extrarenal rhabdoid tumors of soft tissue: Clinicopathological and molecular genetic review and distinction from other soft-tissue sarcomas with rhabdoid features. Pathol Int. 56:287–295, 2006. 551. Argenta PA, Thomas S, Chura JC: Proximal-type epithelioid sarcoma vs. malignant rhabdoid tumor of the vulva: a case report, review of the literature, and argument for consolidation. Gynecol Oncol. 107:130–135, 2007. 552. Izumi T, Oda Y, Hasegawa T, et al: Prognostic significance of dysadherin expression in epithelioid sarcoma and its diagnostic utility in distinguishing epithelioid sarcoma from malignant rhabdoid tumor. Mod Pathol. 19:820–831, 2006. 553. Nakamura Y, Becker LE, Marks A: S-100 protein in tumors of cartilage and bone: An immunohistochemical study. Cancer. 52:1820–1824, 1983. 554. Posl M, Amling M, Ritzel H, et al: Morphologic characteristics of chondroblastoma: A retrospective study of 56 cases of the Hamburg bone tumor register. Pathologe. 17:26–34, 1996. 555. Monda L, Wick MR: S-100 protein immunostaining in the differential diagnosis of chondroblastoma. Hum Pathol. 16:287– 293, 1985. 556. Semmelink HJ, Pruszczynski M, Wiersma-van Tilburg A, et al: Cytokeratin expression in chondroblastomas. Histopathology. 16:257–263, 1990. 557. Edel G, Ueda Y, Nakanishi J, et al: Chondroblastoma of bone: A clinical, radiological, light and immunohistochemical study. Virchows Arch A Pathol Anat Histopathol. 421:355–366, 1992. 558. Brecher ME, Simon MA: Chondroblastoma: An immunohistochemical study. Hum Pathol. 19:1043–1047, 1988. 559. Swanson PE, Lillemoe TJ, Manivel JC, et al: Mesenchymal chondrosarcoma: An immunohistochemical study. Arch Pathol Lab Med. 114:943–948, 1990. 560. Rushing EJ, Armonda RA, Ansari Q, et al: Mesenchymal chondrosarcoma: A clinicopathologic and flow cytometric study of 13 cases presenting in the central nervous system. Cancer. 77:1884–1891, 1996. 561. Wang L, Motoi T, Khanin R, et al: Identification of a novel, recurrent HEY1-NCOA2 fusion in mesenchymal chondrosarcoma based on a genome-wide screen of exon-level expression data. Genes Chromosomes Cancer. 51:127–139, 2012. 562. Swanson PE: Clear cell tumors of bone. Semin Diagn Pathol. 14:281–291, 1997. 563. Bleiweiss IJ, Klein MJ: Chondromyxoid fibroma: report of six cases with immunohistochemical studies. Mod Pathol. 3:664– 666, 1990. 564. Nielsen GP, Keel SB, Dickersin GR, et al: Chondromyxoid fibroma: a tumor showing myofibroblastic, myochondroblastic, and chondrocytic differentiation. Mod Pathol. 12:514–517, 1999. 565. Hauben EI, Jundt G, Cleton-Jansen AM, et al: Desmoplastic fibroma of bone: an immunohistochemical study including betacatenin expression and mutational analysis for beta-catenin. Hum Pathol. 36:1025–1030, 2005. 566. Serra M, Morini MC, Scotlandi K, et al: Evaluation of osteonectin as a diagnostic marker of osteogenic bone tumors. Hum Pathol. 23:1326–1331, 1992. 567. Mejia-Guerrero S, Quejada M, Gokgoz N, et al: Characterization of the 12q15 MDM2 and 12q13-14 CDK4 amplicons and clinical correlations in osteosarcoma. Genes Chromosomes Cancer. 49:518–525, 2010. 568. Yoshida A, Ushiku T, Motoi T, et al: Immunohistochemical analysis of MDM2 and CDK4 distinguishes low-grade osteosarcoma from benign mimics. Mod Pathol. 23:1279–1288, 2010. 569. Yoshida A, Ushiku T, Motoi T, et al: MDM2 and CDK4 immunohistochemical coexpression in high-grade osteosarcoma:
129.e14
Immunohistology of Neoplasms of Soft Tissue and Bone
correlation with a dedifferentiated subtype. Am J Surg Pathol. 36:423–431, 2012. 570. Navarro S, Cavazzana AO, Llombart-Bosch A, et al: Comparison of Ewing’s sarcoma of bone and peripheral neuroepithelioma: An immunocytochemical and ultrastructural analysis of two primitive neuroectodermal neoplasms. Arch Pathol Lab Med. 118:608–615, 1994. 571. Parham DM, Hijazi Y, Steinberg SM, et al: Neuroectodermal differentiation in Ewing’s sarcoma family of tumors does not predict tumor behavior. Hum Pathol. 30:911–918, 1999. 572. Lee CS, Southey MC, Waters K, et al: EWS/FLI-1 fusion transcript detection and MIC2 immunohistochemical staining in the detection of Ewing’s sarcoma. Pediatr Pathol Lab Med. 16:379– 392, 1996. 573. Scotlandi K, Serra M, Manara MC, et al: Immunostaining of the p30/32MIC2 antigen and molecular detection of EWS rearrangements for the diagnosis of Ewing’s sarcoma and peripheral neuroectodermal tumor. Hum Pathol. 27:408–416, 1996. 574. Arvand A, Denny CT: Biology of EWS/ETS fusions in Ewing’s family tumors. Oncogene. 20:5747–5754, 2001. 575. Sankar S, Lessnick SL: Promiscuous partnerships in Ewing’s sarcoma. Cancer Genet. 204:351–365, 2011. 576. Wang L, Bhargava R, Zheng T, et al: Undifferentiated small round cell sarcomas with rare EWS gene fusions: identification of a novel EWS-SP3 fusion and of additional cases with the EWS-ETV1 and EWS-FEV fusions. J Mol Diagn. 9:498–509, 2007. 577. Ng TL, O’Sullivan MJ, Pallen CJ, et al: Ewing sarcoma with novel translocation t(2;16) producing an in-frame fusion of FUS and FEV. J Mol Diagn. 9:459–463, 2007. 578. Szuhai K, Ijszenga M, de Jong D, et al: The NFATc2 gene is involved in a novel cloned translocation in a Ewing sarcoma variant that couples its function in immunology to oncology. Clin Cancer Res. 15:2259–2268, 2009. 579. Ambros IM, Ambros PF, Strehl S, et al: MIC2 is a specific marker for Ewing’s sarcoma and peripheral primitive neuroectodermal tumors: Evidence for a common histogenesis of Ewing’s sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer. 67:1886–1893, 1991. 580. Fellinger EJ, Garin-Chesa P, Triche TJ, et al: Immunohistochemical analysis of Ewing’s sarcoma cell surface antigen p30/32MIC2. Am J Pathol. 139:317–325, 1991. 581. Folpe AL, Hill CE, Parham DM, et al: Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on CD99-positive mimics of Ewing’s sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol. 24:1657–1662, 2000. 582. Halliday BE, Slagel DD, Elsheikh TE, et al: Diagnostic utility of MIC-2 immunocytochemical staining in the differential diagnosis of small blue cell tumors. Diagn Cytopathol. 19:410–416, 1998. 583. Miettinen M, Lehto VP, Virtanen J: Histogenesis of Ewing’s sarcoma: An evaluation of intermediate filaments and endothelial cell markers. Virchows Arch Cell Pathol. 41:277, 1988. 584. Gu M, Antonescu CR, Guiter G, et al: Cytokeratin immunoreactivity in Ewing’s sarcoma: Prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol. 24:410–416, 1999. 585. Yoshida A, Sekine S, Tsuta K, et al: NKX2.2 is a useful immunohistochemical marker for Ewing sarcoma. Am J Surg Pathol. 36:993–999, 2012. 586. Miettinen M, Gannon FH, Lackman R: Chordomalike soft tissue sarcoma in the leg: A light and electron microscopic and immunohistochemical study. Ultrastruct Pathol. 16:577–586, 1992. 587. Hu Y, Gao Y, Zhang X: A clinicopathological and immunohistochemical study of 34 cases of chordoma. Chung Hua Ping Li Hsueh Tsa Chih. 25:142–144, 1996. 588. Naka T, Iwamoto Y, Shinohara N, et al: Cytokeratin sub-typing in chordomas and the fetal notochord: An immunohistochemical analysis of aberrant expression. Mod Pathol. 10:545–551, 1997. 589. Huse JT, Pasha TL, Zhang PJ: D2-40 functions as an effective chondroid marker distinguishing true chondroid tumors from chordoma. Acta Neuropathol. 113:87–94, 2007.
590. Meis JM, Raymond AK, Evans HL, et al: “Dedifferentiated” chordoma: A clinicopathologic and immunohistochemical study of three cases. Am J Surg Pathol. 11:516–525, 1987. 591. Bisceglia M, D’Angelo VA, Guglielmi G, et al: Dedifferentiated chordoma of the thoracic spine with rhabdomyosarcomatous differentiation: Report of a case and review of the literature. Ann Diagn Pathol. 11:262–272, 2007. 592. Heffelfinger MJ, Dahlin DC, MacCarty CS, et al: Chordomas and cartilaginous tumors at the skull base. Cancer. 32:410–420, 1973. 593. Rosenberg AE, Brown GA, Bhan AK, et al: Chondroid chordoma—a variant of chordoma: A morphologic and immunohistochemical study. Am J Clin Pathol. 101:36–41, 1994. 594. Ishida T, Dorfman HD: Chondroid chordoma versus low-grade chondrosarcoma of the base of the skull: Can immunohistochemistry resolve the controversy? J Neurooncol. 18:199–206, 1994. 595. Masui F, Ushigome S, Fujii K: Giant cell tumor of bone: An -immunohistochemical comparative study. Pathol Int. 48:355– 361, 1998. 596. Watanabe K, Tajino T, Kusakabe T, et al: Giant cell tumor of bone: Frequent actin immunoreactivity in stromal tumor cells. Pathol Int. 47:680–684, 1997. 597. Fornasier VL, Protzner K, Zhang I, et al: The prognostic significance of histomorphometry and immunohistochemistry in giant cell tumors of bone. Hum Pathol. 27:754–760, 1996. 598. Lee CH, Espinosa I, Jensen KC, et al: Gene expression profiling identifies p63 as a diagnostic marker for giant cell tumor of the bone. Mod Pathol. 21:531–539, 2008. 599. Dickson BC, Li SQ, Wunder JS, et al: Giant cell tumor of bone express p63. Mod Pathol. 21:369–375, 2008. 600. Oliveira AM, Dei Tos AP, Fletcher CD, et al: Primary giant cell tumor of soft tissues: A study of 22 cases. Am J Surg Pathol. 24:248–256, 2000. 601. O’Connell JX, Wehrli BM, Nielsen GP, et al: Giant cell tumors of soft tissue: A clinicopathologic study of 18 benign and malignant tumors. Am J Surg Pathol. 24:386–395, 2000. 602. Folpe AL, Weiss SW, Fletcher CDM, et al: Tenosynovial giant cell tumors: evidence for a desmin-positive dendritic cell subpopulation. Mod Pathol. 11:939–944, 1998. 603. Somerhausen NS, Fletcher CD: Diffuse-type giant cell tumor: clinicopathologic and immunohistochemical analysis of 50 cases with extraarticular disease. Am J Surg Pathol. 24:479–492, 2000. 604. Boland JM, Folpe AL, Hornick JL, et al: Clusterin is expressed in normal synoviocytes and in tenosynovial giant cell tumors of localized and diffuse types: diagnostic and histogenetic implications. Am J Surg Pathol. 33:1225–1229, 2009. 605. Sholl LM, Hornick JL, Pinkus JL, et al: Immunohistochemical analysis of langerin in langerhans cell histiocytosis and pulmonary inflammatory and infectious diseases. Am J Surg Pathol. 31:947–952, 2007. 606. Sahm F, Capper D, Preusser M, et al: BRAFV600E mutant protein is expressed in cells of variable maturation in Langerhans cell histiocytosis. Blood. 120:e28–34, 2012. 607. Haroche J, Charlotte F, Arnaud L, et al: High prevalence of BRAF V600E mutations in Erdheim-Chester disease but not in other non-Langerhans cell histiocytoses. Blood. 120:2700–2703, 2012. 608. Kahn LB: Adamantinoma, osteofibrous dysplasia, and differentiated adamantinoma. Skeletal Radiol. 32:245–258, 2003. 609. Hazelbag HM, Hogendoorn PC: Adamantinoma of the long bones: An anatomicoclinical review of its relationship to osteofibrous dysplasia. Ann Pathol. 21:499–511, 2001. 610. Kuruvilla G, Steiner GC: Osteofibrous dysplasia-like adamantinoma of bone: A report of five cases with immunohistochemical and ultrastructural studies. Hum Pathol. 29:809–814, 1998. 611. Jundt G, Remberger K, Roessner A, et al: Adamantinoma of long bones: A histopathological and immunohistochemical study of 23 cases. Pathol Res Pract. 191:112–120, 1995. 612. Benassi MS, Campanacci L, Gamberi G, et al: Cytokeratin expression and distribution in adamantinoma of the long bones and osteofibrous dysplasia of tibia and fibula: An immunohistochemical study correlated to histogenesis. Histopathology. 25:71– 76, 1994.
References 613. Ishida T, Iijima T, Kikuchi F, et al: A clinicopathological and immunohistochemical study of osteofibrous dysplasia, differentiated adamantinoma, and adamantinoma of long bones. Skeletal Radiol. 21:493–502, 1992. 614. Knapp RH, Wick MR, Scheithauer BW, et al: Adamantinoma of bone: An electron microscopic and immunohistochemical study. Virchows Arch A. 398:75–86, 1982. 615. Hazelbag HM, Fleuren GJ, vd Broek LJ, et al: Adamantinoma of the long bones: keratin subclass immunoreactivity pattern with
129.e15
reference to its histogenesis. Am J Surg Pathol. 17:1225–1233, 1993. 616. Kashima TG, Dongre A, Flanagan AM, et al: Podoplanin expression in adamantinoma of long bones and osteofibrous dysplasia. Virchows Arch. 459:41–46, 2011. 617. Gleason BC, Liegl-Atzwanger B, Kozakewich HP, et al: Osteofibrous dysplasia and adamantinoma in children and adolescents: A clinicopathologic reappraisal. Am J Surg Pathol. 32:363–376, 2008.
C H A P T E R 5
IMMUNOHISTOLOGY LYMPHOMA
OF
HODGKIN
PARUL BHARGAVA, MARSHALL E. KADIN
Overview 130 Biology of Antigens and Antibodies 130 Antibody Specifications 137 Diagnostic Immunohistochemistry 139 Molecular Anatomic Pathology 144 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications 144 Theranostic Applications 145 Summary 145
Overview Hodgkin lymphoma (HL) is widely accepted to be a malignant clonal proliferation of B lymphocytes—or, rarely, T lymphocytes—surrounded by variable numbers of inflammatory cells and fibrosis. Morphologically, HLs share the following characteristics: • The neoplastic cells, designated Hodgkin/ReedSternberg cells (H/RSCs) are large mononuclear (Hodgkin cells), binucleate (classic), or multinucleate cells with huge eosinophilic nucleoli with surrounding halos (i.e., inclusion-like nucleoli), called classic H/RSCs and variants; less frequently, they are large and polylobated with delicate nuclear outlines and inconspicuous nucleoli, known as lymphocytic and histiocytic cells (L&H cells) or popcorn cells. • H/RSCs are commonly of B-cell lineage with a surrounding rosette of T cells. • The H/RSCs are a minor population (1 in 10 to 1 in 1000 cells), with most of the tumor mass made up of nonneoplastic background inflammatory cells and fibrosis. The two major histologic types are known as classic Hodgkin lymphoma (CHL), which represents nearly 95% of HL, and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), which makes up 5% of HL (Figs. 5-1 and 5-2).1-3 Classic Hodgkin lymphoma is 130
divided into four subtypes, based on the composition of the inflammatory background and cytomorphology of H/RSCs: 1) nodular sclerosis (NS) type (~70%), 2) mixed cellularity (MC) type (~20% to 25%), 3) lymphocyte-rich (LR) type (~5%), and 4) lymphocyte depleted (LD) type (<1%). NLPHL is a B-cell neoplasm derived from germinal center B cells that are continually undergoing somatic mutations of immunoglobulin genes.3-5 These cells have an intact B-cell phenotype and B-cell gene expression. CHL belongs to the group of germinal center/post germinal center B-cell lymphomas in which the H/ RSCs have undergone extensive somatic mutations of immunoglobulin genes.6 The H/RSCs in CHL have downregulated B-cell antigens and gene expression. Accordingly, these two major subtypes of HL express distinctive antigen profiles that can be used to distinguish NLPHL from CHL, particularly NLPHL from the morphologically similar lymphocyte-rich variant of CHL. The German Hodgkin Study Group showed the utility of an immunohistochemical (IHC) approach to improve the accuracy of identifying NLPHL. IHC disproved the solely morphologic diagnosis of NLPHL by an expert panel in 25 of 104 cases, whereas 13 cases originally not confirmed as NLPHL showed an NLPHLlike immunophenotypic pattern with a significantly better survival than CHL.7
Biology of Antigens and Antibodies Principal Antibodies (CD45, CD20, BSAP/PAX5, CD30, CD15) The H/RSCs in NLPHL, also known as popcorn cells or L&H cells because of their distinctive morphology, generally express leukocyte common antigen (LCA) or CD45. CD45 is either absent or is weakly expressed in a subset of H/RSCs in CHL (Fig. 5-3).8,9 CD20 is expressed by L&H cells in all cases of NLPHL in keeping with their B-cell origin (Fig. 5-4). H/RSCs of only a small subset of CHL cases express B-cell antigen CD20.10-13 The reported proportion of CHL cases (mainly NS and MC subtypes) with CD20
Biology of Antigens and Antibodies
A
B
C
D
131
E Figure 5-1 Classic Hodgkin lymphoma. A, Lymphocyte rich. B, Nodular sclerosis. C, Lacunar cells in nodular sclerosis. D, Mixed cellularity. E, Lymphocyte depletion.
expression is highly variable, ranging from 5% to nearly 60% in paraffin immunohistochemistry (IHC).14-16 In a given case of CHL, only a minor proportion of H/RSCs express CD20 antigen, and intensity of staining is typically weak and variable. Limited studies to evaluate CD20 in the lymphocyte-rich CHL subtype have shown that 30% to 40% are CD20 immunoreactive.17,18 B-cell–specific activator protein (BSAP) is a transcription factor expressed in nonneoplastic B cells and B-cell–derived lymphomas. It is encoded by the PAX5 gene and influences several B-cell functions such as B-cell antigen expression, immunoglobulin (Ig) expression, and Ig class switch. BSAP/PAX5 is expressed by
most H/RSCs in CHL19 as well as in L&H cells of NLPHL.20 Notably, the intensity of BSAP/PAX5 expression is weaker in H/RSCs of CHL compared with surrounding nonneoplastic B-cells. Unlike other commonly used B-cell markers (e.g., CD20, CD79), which are often unreactive in H/RSCs of CHL, BSAP/PAX5 immunoreactivity in most CHL cases is useful in supporting their B-cell origin. BSAP/PAX5 is generally not expressed in normal and most malignant T cells, and thus PAX5/BSAP immunoreactivity favors CHL over T/null-cell anaplastic large cell lymphomas (ALCLs).19 In all cases of CHL (Fig. 5-5) and in a minor proportion of NLPHL, the tumor cells express CD30, usually in
132
Immunohistology of Hodgkin Lymphoma
A
B
Figure 5-2 Nodular lymphocyte-predominant Hodgkin lymphoma. A, Low magnification shows nodular pattern. B, Popcorn variants of Hodgkin/Reed-Sternberg cells.
a membranous-and-Golgi pattern. CD30 is a member of the tumor necrosis factor (TNF) superfamily.21-23 Activation of CD30 signaling by native CD30L or Epstein-Barr virus latent membrane protein 1 (EBV-LMP1) results in activation of nuclear factor kappa B (NF-κB) cell transcription factor, which has an antiapoptotic effect, promotes cell proliferation, and causes upregulation of cytokine production by H/RSCs.24,25 Notably, nonneoplastic “transformed” cells—that is, immunoblasts that express CD30—are often seen in NLPHL. H/RSCs in CHL, but not in NLPHL, express CD15 detected by antibody LeuM1 in 60% to 85% of cases, with an average of 68% (Fig. 5-6).14,26-28 The expression
Figure 5-3 Absence of CD45 leukocyte common antigen on Hodgkin/Reed-Sternberg cells in classic Hodgkin lymphoma.
pattern may be Golgi and membranous or Golgi only. The antigenic determinant for LeuM1 is a trisaccharide, 3-fucosyl-N-acetyllactosamine formed by the 1-3 fucosylation of a type 2 blood group backbone chain (Gal1-4GlcNAc); the carbohydrate backbone is identical to that of Lewis X, also known as X-hapten.29
B-Cell Receptor (CD79a), Transcription Factors (Oct-2, BOB.1, PU.1, JunB), and Immunoglobulins (J-Chain, IgD) CD79 is a dimeric transmembrane protein, which, along with surface immunoglobulin, is part of the B-cell receptor complex.30 It is a pan–B-cell marker expressed from the pre–B-cell stage to the plasma cell stage of differentiation.31 Like CD20, CD79 is generally expressed in all cases of NLPHL. However, H/RSCs of CHL are generally unreactive for CD79; in a minor subset of cases (0% to 20%), a small proportion of the neoplastic cells may be positive.13,32-34 Global loss of B-cell–specific gene expression is a distinctive feature of H/RSCs in CHL.35 The loss may be due to aberrant expression of ID2, a suppressor of B-cell–specific gene expression in HL.36 Oct-2 is a transcription factor that, along with its coactivator BOB.1/OBF.1, binds to Ig gene octomer sites, thus inducing Ig synthesis.37 Normal germinal center B cells demonstrate strong staining for Oct-2 and BOB.1. Because they are germinal-center derived, L&H cells in NLPHL are consistently immunoreactive for both Oct-2 and BOB.1.20 Conversely, the H/RSCs in
Biology of Antigens and Antibodies
A
133
B
Figure 5-4 B-cell antigen expression in nodular lymphocyte-predominant Hodgkin lymphoma. A, Large Hodgkin/Reed-Sternberg cells (H/ RSCs) in the center are surrounded by a nodule of smaller L26-positive (CD20+) B lymphocytes. B, CD20-positive H/RSCs in lymphocytepredominant Hodgkin lymphoma.
CHL do not express both (80%) or express only one (20%) of these two proteins.38,39 H/RSCs often do not express Ig, which is thought to be due to crippling mutations within Ig genes; absence of transcriptional activators, such as Oct-2/BOB.1, may represent novel mechanisms for Ig dysregulation.38 Although most T-cell lymphomas are Oct-2 negative, variable staining has been demonstrated in some peripheral T-cell lymphomas not otherwise specified (NOS) as well as a subset (~50%) of anaplastic lymphoma kinase (ALK)–positive ALCLs.40 Transcription factor PU.1 is not expressed in H/RSCs of CHL and is associated with defective Ig transcription.41 Conversely, H/RSCs of NLPHL express PU.1.42 JunB and c-Jun are part of the activator protein 1 (AP-1) family of transcription factors. AP-1 proteins are stimulated in a rapid and transient fashion by a number of extracellular signals that trigger growth factor pathways and/or stress signals (e.g., ultraviolet [UV] radiation). They promote mitogen-induced cell-cycle progression and regulate apoptosis. Recently, it has been
demonstrated that H/RSCs in CHL constitutively overexpress AP-1 proteins, including c-Jun and JunB (Fig. 5-7). Conversely, malignant cells in NLPHL had been shown to express neither c-Jun nor JunB.43 However, in our experience,17 JunB is expressed in a minor subset of NLPHL cases. Additionally, JunB antibody stains scattered lymphocytes, particularly in areas of progressively transformed germinal centers. Most of the other Band T-cell NHLs tested did not express or only weakly expressed JunB and/or c-Jun, with the exception of t(2;5)-positive ALCLs, which showed strong expression.43,44 J-chain is a 15-kD acidic protein synthesized by B cells and plasma cells that secrete polymeric Igs. J-chain expression has thus been observed in most H/RSCs in NLPHL but not in H/RSCs of CHL with dysregulated Ig genes.38,45,46 IgD expression in H/RSCs has been reported in a subset (27%) of NLPHL cases with an extrafollicular distribution of L&H cells and a relatively T-cell–rich background.47 In contrast, IgD expression is rarely seen in T-cell–rich B-cell lymphoma (TCRBCL).
Figure 5-5 Hodgkin/Reed-Sternberg cells in classic Hodgkin lymphoma stained for CD30 with antibody Ber-H2.
Figure 5-6 Hodgkin/Reed-Sternberg cells in classic Hodgkin lymphoma express CD15 detected by antibody LeuM1.
134
Immunohistology of Hodgkin Lymphoma
Figure 5-7 JunB expression in classic Hodgkin lymphoma.
Some studies demonstrated immunoreactivity for IgD in a minor subset of H/RSCs in CHL48; however, others were negative.49
Germinal Center/Post–Germinal Center Markers (BCL-6, IRF4, PRDM1, CD138) H/RSCs in NLPHL are germinal center derived and thus express BCL-6, a transcription factor of germinal center B cells, but they do not express CD10. H/RSCs in NLPHL do not express CD138/syndecan-1, a proteoglycan associated with post germinal center B cells.50 H/RSCs in NLPHL often are surrounded by a population of activated helper-inducer memory T cells (CD4+, CD57+, CD45R+, CD45+), which are normally confined to the light zone of germinal centers of secondary follicles (Fig. 5-8).4 These surrounding T cells also express other markers, such as c-MAF, PD-1/CD279, BCL-6, IRF4 (formerly MUM1), and CD134.
Figure 5-9 Hodgkin/Reed-Sternberg cells in classic Hodgkin lymphoma express CD40.
Conversely, H/RSCs in CHL are heterogeneous with respect to expression of BCL-6.51-53 IRF4 is strongly immunoreactive in H/RSCs in CHL.54,55 PRDM1 (formerly BLIMP-1), a master regulator of plasma cell differentiation, is expressed in a small proportion of H/RSCs in a minority of CHL cases,55 whereas CD138 is generally negative, which reflects their mixed germinal center or post germinal center origin.
Tumor Necrosis Factor Superfamily (CD40, CD95) In addition to CD30, H/RSCs in CHL express CD40,56 an antigen characteristic of germinal center B cells, activation of which inhibits apoptosis (Fig. 5-9).57 CD95 (Fas) is another member of the TNF superfamily that is expressed by germinal center B cells. When bound by its ligand (Fas-L), CD95 induces apoptosis in B cells that have low-affinity immunoglobulins. CD95 is expressed by H/RSCs in a majority of CHL.58 However, it is not specific for H/RSCs, because nonneoplastic B cells, B-cell non-Hodgkin lymphomas, and T-cell lymphomas may express CD95.
Epithelial Membrane Antigen Epithelial membrane antigen (EMA) is expressed in some cases of NLPHL (Fig. 5-10), but it is not expressed in CHL. However, EMA is commonly expressed by tumor cells in ALCL, which may be useful as a distinguishing feature between ALCL and CHL.
T-Cell and Cytotoxic Markers (CD2, CD3, CD4, CD5, CD8, CD57, Granzyme B, Perforin, TIA-1) Figure 5-8 Leu7-positive (CD57+) T lymphocytes surround Hodgkin/Reed-Sternberg cells in nodular lymphocyte-predominant Hodgkin lymphoma.
In 5% to 20% of CHL cases, H/RSCs appear to have variable expression of T-cell antigens (CD2, CD3, CD4, CD5, CD8) and antigens associated with cytotoxic molecules (granzyme B, perforin, and TIA-1; Fig. 5-11).59-65
Biology of Antigens and Antibodies
A
135
B
Figure 5-10 Expression of epithelial membrane antigen in nodular lymphocyte-predominant Hodgkin lymphoma. A, Low magnification of nodule. B, High magnification of individual Hodgkin/Reed-Sternberg cells.
In positive cases, a mean fraction of 40% of H/RSCs (from 20% to 100%) expressed the analyzed T-cell markers in one study.66 However, aberrant T-cell antigen expression was also detected in some CHLs with immunoglobulin gene rearrangements, presumably of B-cell derivation.67 A T-cell derivation was proven for H/RSCs by polymerase chain reaction (PCR) amplification of T-cell receptor genes from single-picked H/RSCs in approximately 1% to 2% of CHL cases.67-69 However, a recent retrospective study suggests that at least some of the cases previously classified as CHL of T-cell derivation (CD30+, CD15+, T-markers+) may represent nodal involvement by cutaneous T-cell lymphomas rather than true HL.70 Although the neoplastic H/RSCs in NLPHL are of B-cell derivation, a majority of H/RSCs in NLPHL have a surrounding ring of CD3-positive T cells, which to a variable extent coexpress CD57 (see Fig. 5-8). The surrounding inflammatory cells within the expanded nodules of NLPHL are B-cell rich with admixed CD4and CD57-positive T cells and scant CD8-positive cells, features that can be used to distinguish NLPHL from T-cell/histiocyte-rich large B-cell lymphoma.
A
Epstein-Barr Virus Epstein-Barr virus (EBV) is associated with the etiology of HL, and EBV-LMP1 and/or EBV-encoded small RNAs known as EBERs can be detected in approximately 50% of cases of CHL (Fig. 5-12).71 H/RSCs frequently express EBV-LMP1, a transforming protein that can confer a growth advantage on H/RSCs.72,73 The frequency of EBV detection in HL is much higher in the mixed cellularity and lymphocyte depletion types than in the nodular sclerosis type.73-75 EBV is frequently detected in cases of CHL in immunocompromised patients, such as those infected with human immunodeficiency virus (HIV) and those with posttransplant lymphoproliferative disorders.76 EBV is also detected at higher frequency in HL patients in developing countries.74,75
Dendritic or Antigen-Presenting Cell Markers (Fascin, CLIP-170/Restin) A 55-kD actin-bundling protein localized predominantly in dendritic cells in nonneoplastic tissues, fascin
B
Figure 5-11 Classic Hodgkin lymphoma with T-cell phenotype. A, Hodgkin/Reed-Sternberg cells stained for UCHL1 (CD45RO). B, Expression of cytotoxic molecule TIA-1 by Hodgkin/Reed-Sternberg cells and smaller, surrounding, tumor-infiltrating lymphocytes.
136
Immunohistology of Hodgkin Lymphoma
A
B
Figure 5-12 Detection of Epstein-Barr virus (EBV) in classic Hodgkin lymphoma. A, Expression of latent membrane protein 1. B, In situ hybridization study for EBV-encoded small RNAs (EBER).
is a sensitive marker expressed in all H/RSCs in CHL.77 The staining profile for fascin raises the possibility of a dendritic cell derivation, particularly an interdigitating reticulum cell, for the neoplastic cells of HL, notably in nodular sclerosis (Fig. 5-13). However, because fascin expression can be induced by EBV infection of B cells, the possibility of viral induction of fascin in lymphoid or other cell types must also be considered.77 Because 100% of CHL cases tested thus far have demonstrated fascin immunoreactivity, it has a high negative predictive value (i.e., absence of fascin expression is against a diagnosis of CHL); however, expression of fascin is not specific for CHL, and fascin expression has also been described in a majority of cases of ALCL (50% to 70%),78,79 which makes it less useful for this differential diagnosis. H/RSCs of CHL strongly express CLIP-170/restin, which colocalizes with membranes of intermediate macropinocytic vesicles and assists in the trafficking of macropinosomes to the cytoskeleton, a crucial step in antigen presentation. The strong expression of CLIP-170/restin in H/RSCs, dendritic cells, and activated B cells underscores their functional similarities and supports a function-based concept of H/RSCs as “professional” antigen-presenting cells.80
A
CD74 CD74 functions as a major histocompatibility complex (MHC) class II chaperone and is normally expressed by a variety of cell types including B cells, activated T cells, macrophages, activated endothelial cells, and epithelial cells. Although expressed by the H/RSCs of CHL, CD74 is not specific for this lymphoma; expression has been reported in a variety of non-HLs and in nonlymphoid epithelial malignancies.
Other Biologic Markers in Hodgkin Lymphoma It has been shown that expression of translation initiation factors eIF4E and eIF2alpha is increased in neoplastic cells of HL but not in surrounding lymphocytes.81 An increase in eIF4E expression may lead to constitutively high expression of NF-κB. H/RSCs have high expression of c-FLIP, which may protect them from apoptosis.82 Tissue inhibitor of metalloproteinases (TIMPs) are proteins with proteinase inhibition and cytokine properties. TIMP-1 is active primarily in B cells and B-cell lymphomas, whereas TIMP-2 is restricted to T cells. HL-derived cell lines express TIMP-1 and show low
B
Figure 5-13 Fascin expression in nodular sclerosis Hodgkin lymphoma. A, Low magnification of nodule. B, Staining of individual Hodgkin/ Reed-Sternberg cells at high magnification.
Antibody Specifications
expression of TIMP-2. TIMP-1 protein can be detected in frozen tissues of CHL lymph nodes, where it produces primarily a diffuse background staining and colocalization with CD30 in few H/RSCs.83 Galectin-1 is an immunoregulatory glycan-binding protein expressed by H/RSCs. H/RSC Gal-1 may contribute to the development and maintenance of an immunosuppressive Th2/Treg-skewed microenvironment in CHL and may provide the molecular basis of selective Gal-1 expression by H/RSCs.84 The programmed death 1 (PD-1) and programmed death ligand (PD-L) signaling system is involved in the functional impairment of T cells, such as in chronic viral infection or tumor immune evasion. PD-L expression is upregulated in H/RSCs in tissues and HL lines, as well as in some T-cell lymphomas, but not in B-cell lymphomas. PD-1/CD279 expression is elevated markedly in tumor-infiltrating T cells of HL and in peripheral blood T cells of HL patients.85
Cytokines and Chemokines in Hodgkin Lymphoma Clinical and histologic features of Hodgkin lymphoma have been associated with cytokine and chemokine production by tumor cells.86,87 The majority of CHL cases are characterized by expression of tumor necrosis factor receptor (TNFR) family members and their ligands and an unbalanced production of Th2 cytokines and chemokines.88 Chemokine receptor CCR7 is a lymphocyte homing receptor expressed in B, T, and activated dendritic cells, and it has been implicated in regulation of lymphocyte migration to secondary lymphoid organs. The promoter region of CCR7 has binding sites for both AP-1 and NF-κB. In line with c-Jun/JunB overexpression seen in CHL but not in NLPHL, CCR7 is overexpressed in most H/RSCs of CHL (Fig. 5-14).89 H/RSCs in CHL manifest variable expressions of CD25 (Tac, p55), the alpha chain of the receptor for interleukin 2 (IL-2).90,91 CD25 is not expressed by H/RSCs in NLPHL. Aggregation of TRAF adapter proteins TRAF2 and TRAF5 in H/RSCs is required for CD30 signaling and for activation of NF-κB.92 TGF-β and basic fibroblast growth factor produced by HL cells are associated with the pathogenesis of nodular sclerosis.93,94 IL-13 and IL-13 receptors are frequently expressed in H/RSCs and contribute to the production of TGF-β1–mediated fibrosis.95,96 H/RSCs secrete IL-5, which stimulates production of eosinophils and eosinophilia.97 H/RSCs also express eotaxin, which is a chemoattractant for eosinophils.98 Both neoplastic and reactive IL-10–producing cells are significantly more common in EBV-positive HL cases. IL-10 is an immunosuppressive cytokine that can help H/RSCs to escape local immune surveillance.99 Members of a family of transcription factors responsible for signal transducers and activators of transcription (STATs), the proteins STAT3, STAT5, and STAT6 are frequently constitutively activated in H/RSCs and can be demonstrated by IHC. STAT5 appears to be activated by IL-21.100 STAT6 mediates signaling by IL-13, and antibody-mediated neutralization of IL-13 causes
137
Figure 5-14 Hodgkin/Reed-Sternberg cells in classic Hodgkin lymphoma express CCR7.
significant decreases in levels of HL cell proliferation and phosphorylated STAT6 in HL cell lines.101 Table 5-1 summarizes the expression pattern of some of the newly recognized biologic markers in HL and in other entities in the differential diagnosis of HL.
Antibody Specifications The antibodies most commonly used for diagnosing HL are Ber-H2 (CD30), LeuM1 (CD15), LCA (CD45), L26 (CD20), CD75 (LN1), CD74 (LN2), PAX5, CD3, UCHL1 (CD45RO), ALK, fascin, and EBV-LMP1. EMA and CD57 can be used to recognize NLPHL. Monoclonal antibody LN1 reacts with H/RSCs in approximately one third of HL cases, most frequently in cases of NLPHL (>75% of cases).12 Monoclonal antibody LN2, which recognizes the MHC class II– associated invariant chain, reacts with H/RSCs in approximately two thirds of HL cases.12 All the antibodies mentioned previously can be used in formalin-fixed TABLE 5-1 New Biologic Markers of Hodgkin/ Reed-Sternberg Cells Antigen
CHL
NLPHL
TCRBCL
ALCL
NF-κB
+
U
U*
−
JunB/c-Jun
+
S
U
+
CCR7
+
−
U
U
Oct-2/BOB.1
S
+
+
S
J-chain
−
S
S
−
BSAP/PAX5
+ (weak)
+
+
R
†
*± In diffuse large B-cell lymphoma; not directly studied in T-cell–rich B-cell lymphoma (TCRBCL). † Both (80%); one (20%). ALCL, anaplastic large cell lymphoma; CHL, classic Hodgkin lymphoma; NF-κB, nuclear factor kappa B; NLPHL, nodular lymphocyte–predominant Hodgkin lymphoma; +, nearly all cases positive; S, sometimes positive; R, rare (<5%); −, negative; U, expression unknown.
Immunohistology of Hodgkin Lymphoma
paraffin-embedded (FFPE) tissues. Additional antibodies useful in the diagnosis of difficult cases are BNH.9102 and CBF.78 (Fig. 5-15 and Table 5-2).103 For antigen retrieval, we have replaced the microwave oven with a steamer that heats the sections to 95° to 98° C. The slides are immersed in Coplin jars that contain citrate buffer (pH 6, 0.01 mol/L) and are then heated in the steamer for 20 minutes. Afterward, slides are cooled at room temperature for 30 minutes, rinsed in double-distilled water, and then transferred to phosphate-buffered saline, pH 7.4.
100%
CHL NLPHL ALCL, Alk ALCL, Alk
90% Cases expressing antigen
138
80% 70% 60% 50% 40% 30% 20%
Immunostaining Pitfalls
10%
CD15 ANTIGEN
When the clinician relies on the demonstration of CD15 to make a diagnosis of HL, problems can arise, because more than 30% of CHL cases will not express CD15 detected by LeuM1 antibody. A comparative study by Ree and coworkers,104 which we confirmed in our laboratory, showed that anti–Lewis-X (BG-7; Signet Laboratories, Dedham, MA) is superior to LeuM1 for staining H/ RSCs and yield 87% versus 68.5% for LeuM1 (Fig. 5-16). It is also important to identify the cells that express CD15, because granulocytes express high levels of CD15 and are often present in various tumors other than HL.26
0%
CD30
CD20
CD45
EMA
EBV
Alk-1
Figure 5-15 Frequency of antigens in classic Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, and anaplastic large cell lymphoma. ALCL, Anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; CHL, classic Hodgkin lymphoma; EBV, Epstein-Barr virus; EMA, epithelial membrane antigen; NLPHL, nodular lymphocyte–predominant Hodgkin lymphoma.
TABLE 5-2 Antibodies for Detection of Hodgkin Lymphoma–Associated Antigens Antibody
Clone
Manufacturer
Dilution
Antigen Retrieval Method
CD30
Ber-H2
Dako
1 : 25
Steamer/citrate buffer pH 6, 20 min at 95° to 98° C
CD15
LeuM1
Becton-Dickinson
1 : 25
Same as CD30
CD45 (LCA)
2B11
Dako
1 : 200
Same as CD30
CD20
L26
Dako
1 : 100
Same as CD30
CD45RO
UCHL1
Dako
Pepsin digest 10 min at 37° C
CD3
UCHT1
Dako
Steamer/citrate buffer pH 6, 20 min at 95° to 98° C
CD40
MAB89
Immunotech
1 : 40
Same as CD3
ALK
ALK1
Dako
1 : 25
Same as CD3
Fascin
55K-2
Dako
1 : 75
Same as CD3
Lewis-X type 2 chain (BG-7)
P12
Signet
1 : 40
Steamer/citrate buffer pH 8, 20 min at 95° to 98° C
EBV-LMP1
CS1-4
Dako
1 : 50
Steamer/citrate buffer pH 6, 20 min at 95°C to 98° C
BCL-6
PG-B6p
Dako
1 : 10
Same as EBV-LMP1
CD57
Leu7
Dako
1 : 10
Same as EBV-LMP1
EMA
E29
Dako
1 : 50
Pepsin digest, 12 min at 37° C
CDw75
LN1
ICN Biomedicals
Undiluted
Steamer/citrate buffer pH 6, 20 min at 95° to 98° C
CD74
LN2
ICN Biomedicals
Undiluted
Same as CDw75
NF-κB
P65C
Zymed Lab
1 : 200
Same as CDw75
CCR7
CCR7.6B3
eBioscience
1 : 200
Steam for 30 min in 1 mmol/L EDTA pH 8.089
JunB
SC8051
Dako
1 : 75
HIER steamer/target retrieval solution 30 min
EBV-LMP, Epstein-Barr virus latent membrane protein; EDTA, ethylenediamine tetraacetic acid; EMA, epithelial membrane antigen; HIER, heat-induced epitope retrieval; NF-κB, nuclear factor kappa B.
Diagnostic Immunohistochemistry
A
139
B
Figure 5-16 Comparative staining of Hodgkin/Reed-Sternberg cells for CD15 antigenic determinant. With anti–Lewis-X (A) and anti-LeuM1 antibodies (B) in a case of lymphocyte depletion classic Hodgkin lymphoma shows increased sensitivity with anti–Lewis-X antibody.
CD30 ANTIGEN
The use of monoclonal antibody Ber-H2 with antigen retrieval methods has enabled sensitive detection of CD30 in FFPE tissues. However, some hematopathologists prefer to use B5-fixed tissues, which affords excellent cytomorphology of lymphoid tissues. B5 is a mercuric chloride–containing fixative that requires removal of mercury before immunostaining. This procedure is usually accomplished with Lugol’s solution followed by sodium thiosulfate. A study by Facchetti and coworkers105 showed that omitting the Lugol’s treatment is optimal for detection of CD30, even without wet heating with microwave or proteolytic predigestion of sections. It is also important to pay attention to the cytomorphology of the immunoreactive cells, aided by a good counterstain. In NLPHL, although several CD30 immunoreactive cells may be encountered, on closer review, these represent medium-sized reactive immunoblasts, whereas the larger, polylobulated neoplastic L&H H/RSCs are typically unreactive. CD45 (LEUKOCYTE COMMON ANTIGEN)
It is often difficult to discern CD45 expression in tumor cells owing to strong immunoreactivity of surrounding cells in CHL. Areas without adjacent cells should be sought to determine whether the tumor cells are CD45 immunoreactive.
have been shown to express PAX5; in one report, 1/23 (4%) ALK-positive ALCLs and 3/36 (8%) ALK-negative ALCLs expressed PAX5 attributed to amplifications of the PAX5 locus.106
Diagnostic Immunohistochemistry Although CHL and NLPHL have distinctive morphologic and immunophenotypic characteristics, it is important to note that an inconsistency of immunophenotype of H/RSCs has been reported in simultaneous and consecutive specimens in paraffin sections from the same patients. Chu and coworkers107 found that the immunophenotype of H/RSCs was identical in simultaneous biopsies in only 11 of 39 patients (28%) and remained constant in consecutive biopsies in only 4 of 21 patients (19%). Major differences were related to cell lineage– specific antigens, whereas minor differences involved mainly CD15 and CD74 antigens. In most cases of CHL and NLPHL, an accurate diagnosis can be rendered with an adequate size biopsy, proper fixation, thorough morphologic review, and appropriate antibody selection. However, several caveats and differential diagnostic considerations may cause confusion with HL. One of the major differential diagnoses includes non-HL subtypes with an abundance of nonneoplastic reactive background inflammatory cells. We will now discuss these subtypes and certain nonlymphoid malignancies and tumorlike nonneoplastic look-alikes.
CD20 AND T-CELL ANTIGENS
With CD20 and T-cell antigen staining in CHL, similar difficulties can arise when interpreting the staining of tumor cells versus surrounding cells. In this regard, PAX5 stain is most useful in establishing B-cell lineage of H/RSCs; the nuclear immunoreactivity in neoplastic cells, surrounding rim of small nonimmunoreactive T-cells, and the dim staining intensity of H/RSCs in CHL compared with surrounding nonneoplastic cells are all useful in interpretation of this stain. Of note, although PAX5 expression generally correlates with a B-cell lineage, rare cases of T-cell lineage lymphomas
Non-Hodgkin Lymphomas ANAPLASTIC LARGE CELL LYMPHOMA
CD30 is displayed on the tumor cells of ALCL, which is a non-HL of T-cell derivation with a different natural history than HL.21,108-111 The histologic features of ALCL, particularly cohesive tumor cell growth pattern and lymph node sinus infiltration by tumor cells, were once thought to be distinguishing characteristics; however, experience has shown otherwise (Fig. 5-17). Rare cases of cell-rich HL—particularly those classified
140
Immunohistology of Hodgkin Lymphoma
as nodular sclerosis type II in the British National Investigation,112 or the syncytial variant113—and some cases of lymphocyte-depletion HL can be confused with ALCL (see Fig. 5-1, E, and Fig. 5-17, A and B).114 In these cases, a panel of antibodies is used to make the distinction (Table 5-3; see also Table 5-1). Expression of ALK protein and establishing a T-cell lineage are most useful in establishing a diagnosis of ALCL. The monoclonal antibody ALK-1 is directed against ALK tyrosine kinase, which is most often activated by the translocation t(2;5)(p23; q35) and less frequently by other chromosomal rearrangements in ALCL (Fig. 5-18).115,116 ALK is rarely, if ever, expressed in the malignant cells
of HL.114 Demonstration of a B-cell phenotype (as with subset CD20 expression or BSAP/PAX5) in H/RSCs in CHL is also useful in excluding ALCL of T-cell derivation. However, rarely, PAX5 may also be expressed in ALCL, including in ALK-negative cases, which may be confused with HL (see above). LYMPHOEPITHELIOID CELL VARIANT OF PERIPHERAL T-CELL LYMPHOMA (LENNERT LYMPHOMA)
Lennert lymphoma is another T-cell lymphoma that can resemble CHL because of the presence of Hodgkin/
A
B
C
D
E
F
Figure 5-17 Cell-rich classic Hodgkin lymphoma with interfollicular and intrasinus distribution of tumor cells. A, Low magnification of interfollicular pattern. B, Low magnification of sinus infiltration. C, High magnification of Hodgkin/Reed-Sternberg cells (H/RSCs) within sinus of lymph node. D, Expression of CD30 by H/RSCs. E, Expression of fascin by H/RSCs. F, Expression of CD40 by H/RSCs.
Diagnostic Immunohistochemistry
141
TABLE 5-3 Antibody Panel for Differential Diagnosis of Hodgkin Lymphoma Classic Hodgkin Lymphoma
Nodular Lymphocyte– Predominant Hodgkin Lymphoma
Anaplastic Large Cell Lymphoma
Primary Mediastinal Large B-Cell Lymphoma
T-Cell–Rich B-Cell Lymphoma
CD30
+
S
+
S
−
CD15
+
−
R
−
−
CD20
S
+
−
+
+
CD3
−
−
+
−
−
CD40
+
+
−
+
+
CD45
−
+
S
+
+
EBV-LMP1
S
−
−
−
−
ALK
−
−
+
−
−
Fascin
+
−
+
+*
−*
PAX5
+
+
R
+
+
Antibody
*Limited cases studied. ALK, Anaplastic lymphoma receptor tyrosine kinase; EBV-LMP1, Epstein-Barr virus latent membrane protein; +, Nearly all cases positive; S, sometimes positive; R, rare (<5%); −, negative.
Reed-Sternberg (H/RS)–like cells, eosinophils, and plasma cells. Small clusters of epithelioid histiocytes resembling granulomas are a distinctive feature. In Lennert lymphoma, the H/RS-like cells express a CD4postive T-cell phenotype.117
PERIPHERAL T-CELL LYMPHOMA NOT OTHERWISE SPECIFIED
A recently described rare variant of NLPHL with cytomorphologically atypical T cells, seen most often in young patients, may morphologically resemble
A
B
C
D
Figure 5-18 Hodgkin-like anaplastic large cell lymphoma. With nodular pattern (A) and lacunar cells (B). C, CD30 staining of tumor cells within sinus. D, Expression of p80NPM/anaplastic lymphoma receptor tyrosine kinase by tumor cells in lacunar spaces.
142
Immunohistology of Hodgkin Lymphoma
peripheral T-cell lymphoma (PTCL) and PTCL not otherwise specified (PTCL-NOS) and may raise the possibility of concurrent nodal involvement by PTCLNOS.118 Although the reported proliferation index in the T-cell rich areas was relatively high (20% to 50%), they did not display any loss of pan–T-cell antigens and lacked clonal rearrangements of T-cell receptor genes in tested cases. They had variable CD4 to CD8 ratios and variably expressed germinal center markers, follicularhelper markers, cytotoxic markers, or T-regulatory markers. In contrast, PTCL-NOS may display loss of pan–T-cell antigens, are generally CD4 predominant, and have clonally rearranged T-cell receptor genes. ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA
Angioimmunoblastic T-cell lymphoma (AILT) is a nodal peripheral T-cell lymphoma derived from follicular T-helper cells. Although EBV-infected transformed B immunoblasts are often seen in AILT, in some cases, large cells that resemble Reed-Sternberg cells morphologically, as well as immunophenotypically (CD30+, CD15+, LMP1+), are present.119 In addition to recognition of morphologic features of AILT, helpful IHC stains include CD21 to demonstrate expanded CD21-positive follicular dendritic meshwork and coexpression of germinal center markers (CD10, BCL-6, PD1/CD279, CXCL13) in neoplastic T-helper cells of AILT. NODAL INVOLVEMENT BY CUTANEOUS CD30-POSTIVE LYMPHOMAS
Primary cutaneous lymphomas such as lymphomatoid papulomatosis (LyP), cutaneous anaplastic large cell lymphoma (C-ALCL), and transformed mycosis fungoides may have CD30-positive cells that cytomorphologically resemble H/RSCs. Lymph nodes that drain these primary cutaneous lymphomas can be secondarily involved by these cells, which can closely mimic CHL. Clinical history of skin lesions, presence of T-cell markers on CD30-immunoreactive large cells, and absence of PAX5 favor nodal involvement by cutaneous lymphoma.70
Figure 5-19 Hodgkin/Reed-Sternberg–like cells in primary mediastinal B-cell large cell lymphoma.
such as growth factor receptor–bound protein 2 (Grb2), have been shown to be expressed in all PMBCL but only in a minority of CHL cases.122 T-CELL–RICH B-CELL LYMPHOMA
T-cell–rich B-cell lymphoma was recognized as a non-HL that usually occurs in patients older than 50 years with advanced (stage III or IV) disease. Response to chemotherapy regimens commonly used to treat HL is poor, therefore it is important to distinguish TCRBCL from HL, particularly NLPHL or lymphocyte-rich classic Hodgkin lymphoma (LRCHL) (Fig. 5-20).121,123 The tumor cells in TCRBCL appear to be negative for CD30 and CD15 and for vimentin, all of which are expressed in H/RSCs in CHL. Furthermore, the reactive inflammatory infiltrate that is rich in TIA-1– positive lymphocytes in TCRBCL and CHL is rarely encountered in NLPHL, whereas CD57-positive lymphocytes characteristic of NLPHL are infrequent in TCRBCL.124 Unlike TCRLBCL, NLPHL generally has at least one nodular area, best highlighted by CD21 immunostaining (CD23 is often unreactive); the background cells in the nodules of NLPHL are B-cell rich in contrast to the T-cell–rich background of TCRBCL.
PRIMARY MEDIASTINAL B-CELL LYMPHOMA
Primary mediastinal B-cell lymphoma (PMBCL) can be confused with CHL because it presents as a mass in the anterior mediastinum of young adults and often contains H/RS-like cells in a background of collagen sclerosis (Fig. 5-19).120 Also, in one study, H/RS-like cells expressed CD30 in 35 of 51 cases (69%).121 Although CD30 is present in a majority of cases of PMBCL, it is irregularly expressed and is generally weak in intensity. Strong, uniform expression of B-cell markers such as CD20 and CD79a favor PMBCL over CHL. CD15 is typically absent in PMBCL but may occasionally be expressed. Absence of EBV (EBERs and LMP1) and lack of inflammatory background, particularly eosinophils (characteristic of HL), are other discriminating characteristics. CD10 may be expressed in a minor proportion of PMBCL cases. Recently, some newer markers,
Figure 5-20 Hodgkin/Reed-Sternberg–like cells in T-cell–rich B-cell lymphoma.
Diagnostic Immunohistochemistry
143
Nonlymphoid Tumors
Figure 5-21 Hodgkin-like posttransplant lymphoproliferative disorder.
HODGKIN-LIKE POSTTRANSPLANT LYMPHOPROLIFERATIVE DISORDER (HL-PTLD)
Hodgkin-like posttransplant lymphoproliferative disorder (HL-PTLD) has cells that resemble H/RSCs (Fig. 5-21) and has been reported in allograft recipients, post methotrexate therapy patients,125 and HIV-infected patients. CHL has also been reported in each of these conditions, and differential diagnosis is based on morphologic and immunophenotypic features.126 The histopathologic features often show a mixed population of small- to intermediate-sized lymphocytes admixed with histiocytes, plasma cells, and rare eosinophils and neutrophils in addition to scattered, large, pleomorphic mononuclear and binucleated cells without sclerosis and absence of nodularity that resembles mixed cellularity or lymphocyte-depletion HL.127 Whereas H/RSCs in CHL characteristically express CD30 and CD15, HL-PTLD cells often have an activated B-cell phenotype (i.e., CD20+, CD30+, CD45+, but CD15−). The atypical cells in HL-PTLD have been reported to express fascin, with a weak expression of Bcl-2; CD45 is variably expressed. Virtually all cases of HL-PTLD are EBV positive; EBV may be absent in up to 50% of CHL.
CD30, the most consistent marker of H/RSCs, is readily detected in FFPE tissues.21,132 However, tumor cells in some nonlymphoid malignancies—including embryonic carcinoma, melanoma, and pancreatic cancer—can also express CD30.132,133 Because sinus infiltration of lymph nodes is characteristic of CD30-positive ALCLs, there is potential for confusion of ALCLs with the few metastatic carcinomas that express CD30 antigen.21 Moreover, because malignant melanoma can express CD30, there is the possibility of mistaking an anaplastic melanoma for a primary CD30-positive ALCL.133 CD15 expressed on H/RSCs is also associated with carcinomas.134 Fortunately, it is not common to encounter a carcinoma that could be clinically or histologically mistaken for HL. However, the cohesive growth pattern of tumor cells in the syncytial variant of nodular sclerosis HL might rarely be mistaken for metastatic carcinoma expressing CD15.
Pseudoneoplastic Look-Alikes INFECTIOUS MONONUCLEOSIS
H/RS-like cells in infectious mononucleosis are similar in most respects to their morphologic counterparts in HL with respect to expression of EBERs, EBV-LMP1, and CD30 and to low expression of CD45.135 However, the H/RS-like cells in infectious mononucleosis are CD15 negative,136 have a post–germinal center phenotype (CD10, BCL-6−, IRF4), are polyclonal for cytoplasmic kappa and lambda light chains, and coexpress BOB.1 and Oct-2.137 CYTOMEGALOVIRUS LYMPHADENITIS
Lymph nodes infected with cytomegalovirus contain H/ RS-like cells that are caused by viral inclusions and are readily distinguished from HL by the absence of CD15 and CD30 (Fig. 5-22).
CLASSIC HODGKIN LYMPHOMA IN CHRONIC LYMPHOCYTIC LEUKEMIA AND HODGKIN-LIKE CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA
CHL transformation is a rare form of Richter’s transformation, which can occur in patients with B-chronic lymphocytic leukemia (B-CLL). H/RSCs are seen in a polymorphous background of inflammatory cells and are morphologically and immunophenotypically indistinguishable from CHL. Such transformations have been variously reported to be clonally distinct128-130 or clonally related130,131 to malignant cells in B-CLL. Separately, H/RS-like cells may be seen singly or clustered in a background of B-CLL. Although Reed-Sternberg– like cells in CLL are typically CD30 immunoreactive with variable expression of CD20, CD15, and LMP1, the background is composed of monomorphous B-CLL cells, thus these cases are not thought to represent Richter’s transformation.
Figure 5-22 Hodgkin/Reed-Sternberg–like cells in a lymph node infected by cytomegalovirus.
144
Immunohistology of Hodgkin Lymphoma
Figure 5-23 Granulomas in a lymph node involved by Hodgkin lymphoma.
INTERFOLLICULAR LYMPHADENITIS
Lymphadenitis that mimics Hodgkin disease has been described as a benign lymphadenopathy that can mimic interfollicular HL.138,139 Cervical lymph nodes are affected most often. There is no progression to lymphoma. The lymph nodes show follicular hyperplasia with a mottled interfollicular zone with epithelioid histiocytes, lymphocytes, eosinophils, and immunoblasts. Some immunoblasts with prominent nucleoli resemble H/RSCs. However, their nucleoli are typically smaller and basophilic, in contrast to the eosinophilic nucleoli of H/RSCs. IHC distinguishes this disorder from interfollicular HL because the H/RS-like cells display B- or T-cell antigens and lack CD15.139,140 GRANULOMATOUS LYMPHADENITIS
The presence of noncaseating granulomas is a wellknown histologic feature associated with several nonhematopoietic and hematopoietic malignancies, including HL (Fig 5-23). In HL, approximately 15% of patients have granulomas, which may be present in nodal and extranodal sites uninvolved by HL.141 The presence of granulomas alone, in the absence of diagnostic H/RSCs, should not be interpreted as evidence of HL. Conversely, granulomatous reaction may be present in a site involved by HL and on occasion may be extremely florid, necessitating a thorough morphologic review to detect small foci of HL. H/RSCs in such areas have the classic immunophenotype (CD30+, CD15+, LCA−, CD20−) as opposed to reactive immunoblasts (LCA+, CD20+, CD30+, CD15−).
Molecular Anatomic Pathology In a majority of cases, the diagnosis of CHL and NLPHL is based on morphology and immunophenotyping, and molecular testing is rarely utilized. PCR studies for immunoglobulin heavy chain gene (IgH) rearrangements have been used to distinguish TCRBCL from HL,
with the former demonstrating clonal bands more frequently than the latter.142 Although demonstration of clonal IgH bands in CHL generally required special cell enrichment techniques and/or single-cell microdissection, recent data suggest that detectable rearrangements without microdissection can be seen in a higher number of CHL cases with newer multiplex primers, making IgH analysis less useful in this context.143 T-cell receptor (TCR) gamma rearrangements have been described in the rare T-cell–derived CHL as well as a subset of B-cell derived CHL.67 In one case of CHL, identical rearrangements of the TCR alpha chain were found in the HL lymph node and coexistent cutaneous T-cell lymphoma.144 In another case studied by singlecell PCR, identical TCR beta-chain rearrangements were found in H/RSCs in a lymph node of mixed cellularity HL and in skin lesions with CD30- and CD15positive H/RSCs.37 Because nodal involvement by cutaneous T-cell lymphomas can closely mimic HL,70 cases with a T-cell immunophenotype and/or T-cell receptor gene rearrangements must be carefully assessed to evaluate this possibility.
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications The exact genomic alteration that drives lymphomagenesis in CHL and NLPHL is not completely understood. Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) is present in numerous cell types and is normally only transiently activated by stress, immune, and inflammatory signals.2 It has been shown that NF-κB is constitutively activated in cultured H/RSCs,24 primarily owing to mutations and/or increased turnover of its natural inhibitor I-kappa B (IκB).88 Constitutive NF-κB results in overexpressed antiapoptotic genes that allow H/RSCs to escape apoptosis despite losing ability to produce Ig. Overexpression of NF-κB can be demonstrated immunohistochemically in CHL; however, it is not specific to this tumor type and can be seen in a variety of other malignancies, including mediastinal B-cell lymphoma.145,146 Gene-expression profiling has demonstrated similarities between CHL and mediastinal large B-cell lymphomas (e.g., downregulated B-cell receptor signaling, increased JAK/STAT signaling, and IL-13 signaling)146 and between cell-rich CHL and ALK-negative ALCL (a “hyepractivated gene-expression program” with suppression of respective lineage markers).147 At a transcriptional level, HL is more closely related to ALK-positive ALCL than to the B-cell non-HL or B-cell samples investigated, although it is usually a B-cell derived lymphoma. The newly identified genes that discriminate HL and ALCL may be pathobiologically important and may serve as possible therapeutic targets.148 A tissue microarray study with IHC and in situ hybridization (ISH) to observe cell-cycle and apoptosisregulating genes has shown multiple alterations in cellcycle checkpoints and major tumor suppressor pathways,
Summary
some of which are linked to survival as well as to EBV positivity.149
Theranostic Applications Although newer therapeutic agents such as monoclonal antibodies are being evaluated, most are in the early experimental stages. Nonetheless, accurate documentation of H/RSC expression of potential therapeutic targets would likely be beneficial when evaluating such biopsies.
CD20 and Monoclonal Antibody (Rituximab) Therapy CD20 expression by a majority of L&H cells in NLPHL has been used as a basis for targeted monoclonal therapy.150 Rituximab therapy is also being tried in CHL, not only targeting the minor CD20-expressing H/ RSCs but also infiltrating background B-lymphocytes.151
CD40 CD40 is widely expressed on H/RSCs, and its ligand, CD40L, is expressed by many T cells that surround H/RSCs. In B cells, CD40 regulates progression from immunoglobulin isotype switch to cytokine secretion and ultimately terminates in Fas-mediated apoptosis to terminate the immune response. CD40 signal transduction pathways result in activation of NF-κB; this in turn activates transcription of IL-2, IL-6, IL-8, TNF, and granulocyte macrophage colony-stimulating factor (GM-CSF), which affects the proliferation and activation of many components of the immune system. CD40 activation of NF-κB is mediated by proteolysis of TRAF3, and a protease inhibitor has been used to block this pathway.152
CD30 Anti-CD30 monoclonal antibodies have been used in preclinical murine xenograft models of localized HL to demonstrate dose-dependent reduction in tumor mass. A significant increase also was seen in survival of mice bearing disseminated HL treated with anti-CD30.153 Antitumor activity of anti-CD30 has been enhanced by conjugation with monomethyl auristatin E, which induces G2/M growth arrest and cell death.154 Trials with monoclonal anti-CD30 antibodies as well as biospecific molecules are also being conducted.155,156 The anti-CD30 antibody drug conjugate brentuximab vedotin was recently reported to induce objective responses with manageable toxicity in 75% of patients with relapsed or refractory HL after autologous stem cell transplantation.157
Anti–Epstein-Barr Virus Therapy In almost 40% of HL patients, H/RSCs express EBVassociated antigens. EBV-specific cytotoxic T lymphocytes that express the anti-CD30 artificial chimeric
145
T-cell receptor have been used for immunotherapy of HL. Adoptive transfer of EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have shown that these cells persist in patients with HL to produce complete tumor responses.158 Treatment failure occurs if a subpopulation of malignant cells lacks or loses expression of EBV antigens. To overcome this limitation, investigators have prepared EBV-CTLs that retain antitumor activity conferred by their native receptor, while expressing a chimeric antigen receptor specific for CD30.159
Interleukin-2 Receptor Interleukin-2 receptor (CD25) has been used as a target for immunotherapy protocols to treat HL.160 A pitfall of these protocols is that difficulty is sometimes encountered in demonstrating CD25 expression by H/RSCs, against which the therapy is directed. Indeed, CD25 is often expressed on activated tumor-infiltrating lymphocytes (TILs) in HL, and it is important to distinguish them from H/RSCs. We found this possible in most cases, when a biotinylated tyramine enhancement step161 was applied to FFPE tissues.91
Chemokine Receptor 4 (CCR4) CCR4 is a chemokine receptor expressed on H/RSCs in 24% of patients with HL. A chimeric anti-CCR4 antibody—KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity—is being developed as a novel treatment for patients with CCR4-positive HL.162
Galectin Galectin 1 (Gal-1) is an animal lectin involved in regulation of inflammatory responses, angiogenesis, and tumor progression. Recent studies suggest that high Gal-1 expression in H/RSCs of CHL correlate with poorer event-free survival in young patients with HL.163
Macrophages and Prognosis In classic HL, numerous macrophages and expression of CD68 and CD163 antigens correlates with adverse outcome and presence of EBV in the tumor cell population.164-166
Summary The diagnosis of HL from routine hematoxylin and eosin sections is often readily made. However, increasing recognition of new lymphoma types with overlapping morphologies indicates the value of IHC to avoid incorrect diagnoses. Moreover, because some lineageassociated markers can be aberrantly expressed in other tumors, a panel of IHC stains is often needed for clarification. Among the non-Hodgkin lymphomas that may be confused with HL are T-cell rich B-cell lymphoma, primary mediastinal large B-cell lymphoma,
146
Immunohistology of Hodgkin Lymphoma
CD45–
CD30+ CD15+
CD20–/+, PAX5+, Fascin+
CHL
CD30+ CD15–
CD20+
CD20–, PAX5–, T-antigens+
Atypical T-cell background T-cell clonality
CD30– CD15+/–
CHL (1-2%)
Classic HL
CD20–
PAX5/BSAP–
CD43, T-antigens: all – ALK–
CD43, T-antigens: any+ ALK+(–)
Keratin– S-100–
Keratin+ or S-100+
ALCL
Classic HL
Embryonal CA; pancreatic CA; malignant melanoma
Keratin+
PAX5/BSAP+
Carcinoma
Classic HL
Keratin–
PTCL Sarcoma (including granulocytic sarcoma)
Figure 5-24 Classic Hodgkin lymphoma (HL). The diagnosis rests on a combination of morphologic and immunophenotypic findings. A diagnostic algorithm for immunophenotypic findings helpful in the differential diagnosis of classic Hodgkin lymphoma is presented. ALCL, Anaplastic large cell lymphoma; CA, carcinoma; PTCL, peripheral T-cell lymphoma.
Large cells CD45
CD20
CD30 CD15
CD30 CD15
Background nodular CD21 FDC B-rich EMA CD57 CD4 rosettes NLPHL
Background Background diffuse atypical T T-rich, with TIA-1 , mod/high CD8 MIB TCRBCL
CD20
No T-Ag loss Polyclonal TCR NLPHL, variant
LMP or EBER
Anaplastic morphology
DLBCL, anaplastic
PAX5 T antigens
Mediastinal
Inflammatory cells scant; CD30 weak; CD30 /
CD30 , CD20 with CHL morph
PMBCL
Gray zone
R/O Rituximabtreated NLPHL
CD30 CD15
PAX5 T antigens
ALCL or PTCL
Gray zone
Figure 5-25 Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). The diagnosis requires an assessment of morphologic and immunophenotypic features. A diagnostic algorithm based on immunhistochemical findings in the differential diagnosis of NLPHL is presented. ALCL, Anaplastic large cell lymphoma; CHL, classic Hodgkin lymphoma; EBER, Epstein-Barr virus–encoded small RNAs; EMA, epithelial membrane antigen; DLBCL, diffuse mediastinal B-cell lymphoma; LMP, latent membrane protein; MIB, minimally invasive biopsy; PMBCL, primary mediastinal B-cell lymphoma; PTCL, peripheral T-cell lymphoma; R/O, rule out; TCRBCL, T-cell–rich B-cell lymphoma.
Summary
posttransplant lymphoproliferative disorders, anaplastic large cell lymphoma, peripheral T-cell lymphoma not otherwise specified, and angioimmunoblastic T-cell lymphoma. The clinician must be aware of antigens shared by H/RSCs and non-HL cells. Although silencing of B-cell antigens by ID2 and possibly other mechanisms makes the diagnosis of HL difficult in some cases, at the same time it can be useful in distinguishing H/RSCs from B cells in most non-HLs. The clinician must always be aware of pseudoneoplastic conditions, such as infectious mononucleosis, that mimic HL, wherein IHC is useful to arrive at the correct diagnosis. IHC is also important to distinguish nodular
147
lymphocyte-predominant HL from lymphocyte-rich CHL. Recognition of certain antigens expressed or not expressed by H/RSCs informs us of the biology of these cells. Overall, IHC contributes significantly to our knowledge of HL and to better patient management. One possible immunohistologic approach to resolve differential diagnostic considerations is presented in the diagnostic algorithm shown in Figures 5-24 and 5-25. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References
1. Lukes RJ, Butler JJ: The pathology and nomenclature of Hodgkin’s disease. Cancer Res. 26:1063–1083, 1966. 2. Harris NL, Jaffe ES, Stein H, et al: A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 84:1361–1392, 1994. 3. Mason DY, Banks PM, Chan J, et al: Nodular lymphocyte predominance Hodgkin’s disease. A distinct clinicopathological entity. Am J Surg Pathol. 18:526–530, 1994. 4. Poppema S: The nature of the lymphocytes surrounding ReedSternberg cells in nodular lymphocyte predominance and in other types of Hodgkin’s disease. Am J Pathol. 135:351–357, 1989. 5. Marafioti T, Hummel M, Anagnostopoulos I, et al: Origin of nodular lymphocyte-predominant Hodgkin’s disease from a clonal expansion of highly mutated germinal-center B cells. N Engl J Med. 337:453–458, 1997. 6. Kanzler H, Kuppers R, Hansmann ML, et al: Hodgkin and ReedSternberg cells in Hodgkin’s disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J Exp Med. 184:1495–1505, 1996. 7. von Wasielewski R, Werner M, Fischer R, et al: Lymphocytepredominant Hodgkin’s disease. An immunohistochemical analysis of 208 reviewed Hodgkin’s disease cases from the German Hodgkin Study Group. Am J Pathol. 150:793–803, 1997. 8. Dorfman RF, Gatter KC, Pulford KA, et al: An evaluation of the utility of anti-granulocyte and anti-leukocyte monoclonal antibodies in the diagnosis of Hodgkin’s disease. Am J Pathol. 123:508–519, 1986. 9. Chittal SM, Caveriviere P, Schwarting R, et al: Monoclonal antibodies in the diagnosis of Hodgkin’s disease. The search for a rational panel. Am J Surg Pathol. 12:9–21, 1988. 10. Pinkus GS, Said JW: Hodgkin’s disease, lymphocyte predominance type, nodular–further evidence for a B cell derivation. L & H variants of Reed-Sternberg cells express L26, a pan B cell marker. Am J Pathol. 133:211–217, 1988. 11. Epstein AL, Marder RJ, Winter JN, et al. Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffinembedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors. J Immunol. 133:1028–1036, 1984. 12. Marder RJ, Variakojis D, Silver J, et al: Immunohistochemical analysis of human lymphomas with monoclonal antibodies to B cell and Ia antigens reactive in paraffin sections. Lab Invest. 52:497–504, 1985. 13. Korkolopoulou P, Cordell J, Jones M, et al: The expression of the B-cell marker mb-1 (CD79a) in Hodgkin’s disease. Histopathology. 24:511–515, 1994. 14. von Wasielewski R, Mengel M, Fischer R, et al: Classical Hodgkin’s disease. Clinical impact of the immunophenotype. Am J Pathol. 151:1123–1130, 1997. 15. Zukerberg LR, Collins AB, Ferry JA, et al: Coexpression of CD15 and CD20 by Reed-Sternberg cells in Hodgkin’s disease. Am J Pathol. 139:475–483, 1991. 16. Schmid C, Pan L, Diss T, et al: Expression of B-cell antigens by Hodgkin’s and Reed-Sternberg cells. Am J Pathol. 139:701–707, 1991. 17. Bhargava P, Pantanowitz L, Pinkus GS, et al: Utility of fascin and JunB in distinguishing nodular lymphocyte predominant from classical lymphocyte-rich Hodgkin lymphoma. Appl Immunohistochem Mol Morphol. 18:16–23, 2010. 18. Nam-Cha SH, Montes-Moreno S, Salcedo MT, et al: Lymphocyterich classical Hodgkin’s lymphoma: distinctive tumor and microenvironment markers. Mod Pathol. 22:1006–1015, 2009. 19. Foss HD, Reusch R, Demel G, et al: Frequent expression of the B-cell-specific activator protein in Reed-Sternberg cells of classical Hodgkin’s disease provides further evidence for its B-cell origin. Blood. 94:3108–3113, 1999. 20. Steimle-Grauer SA, Tinguely M, Seada L, et al: Expression patterns of transcription factors in progressively transformed germinal centers and Hodgkin lymphoma. Virchows Arch. 442:284–293, 2003. 21. Stein H, Mason DY, Gerdes J, et al: The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and
147.e1
histiocytic malignancies are derived from activated lymphoid cells. Blood. 66:848–858, 1985. 22. Durkop H, Latza U, Hummel M, et al: Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease. Cell. 68:421– 427, 1992. 23. Smith CA, Gruss HJ, Davis T, et al: CD30 antigen, a marker for Hodgkin’s lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF. Cell. 73:1349–1360, 1993. 24. Bargou RC, Leng C, Krappmann D, et al: High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells. Blood. 87:4340–4347, 1996. 25. Bargou RC, Emmerich F, Krappmann D, et al: Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin’s disease tumor cells. J Clin Invest. 100:2961–2969, 1997. 26. Stein H, Uchanska-Ziegler B, Gerdes J, et al: Hodgkin and Sternberg-Reed cells contain antigens specific to late cells of granulopoiesis. Int J Cancer. 29:283–290, 1982. 27. Hsu SM, Jaffe ES: Leu M1 and peanut agglutinin stain the neoplastic cells of Hodgkin’s disease. Am J Clin Pathol. 82:29– 32, 1984. 28. Pinkus GS, Thomas P, Said JW: Leu-M1–a marker for ReedSternberg cells in Hodgkin’s disease. An immunoperoxidase study of paraffin-embedded tissues. Am J Pathol. 119:244–252, 1985. 29. Gooi HC, Feizi T, Kapadia A, et al: Stage-specific embryonic antigen involves alpha 1 goes to 3 fucosylated type 2 blood group chains. Nature. 292:156–158, 1981. 30. Kishimoto T, Goyert S, Kikutani H, et al: CD antigens 1996. Blood. 89:3502, 1997. 31. Mason DY, Cordell JL, Brown MH, et al: CD79a: a novel marker for B-cell neoplasms in routinely processed tissue samples. Blood. 86:1453–1459, 1995. 32. Kuzu I, Delsol G, Jones M, et al: Expression of the Ig-associated heterodimer (mb-1 and B29) in Hodgkin’s disease. Histopathology. 22:141–144, 1993. 33. Browne P, Petrosyan K, Hernandez A, et al: The B-cell transcription factors BSAP, Oct-2, and BOB.1 and the pan-B-cell markers CD20, CD22, and CD79a are useful in the differential diagnosis of classic Hodgkin lymphoma. Am J Clin Pathol. 120:767–777, 2003. 34. Tzankov A, Zimpfer A, Pehrs AC, et al: Expression of B-cell markers in classical hodgkin lymphoma: a tissue microarray analysis of 330 cases. Mod Pathol. 16:1141–1147, 2003. 35. Schwering I, Brauninger A, Klein U, et al: Loss of the B-lineagespecific gene expression program in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood. 101:1505–1512, 2003. 36. Renne C, Martin-Subero JI, Eickernjager M, et al: Aberrant expression of ID2, a suppressor of B-cell-specific gene expression, in Hodgkin’s lymphoma. Am J Pathol. 169:655–664, 2006. 37. Laumen H, Nielsen PJ, Wirth T: The BOB.1 / OBF.1 co-activator is essential for octamer-dependent transcription in B cells. Eur J Immunol. 30:458–469, 2000. 38. Stein H, Marafioti T, Foss HD, et al: Down-regulation of BOB.1/ OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription. Blood. 97:496–501, 2001. 39. Hertel CB, Zhou XG, Hamilton-Dutoit SJ, et al: Loss of B cell identity correlates with loss of B cell-specific transcription factors in Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma. Oncogene. 21:4908–4920, 2002. 40. Marafioti T, Ascani S, Pulford K, et al: Expression of B-lymphocyteassociated transcription factors in human T-cell neoplasms. Am J Pathol. 162:861–871, 2003. 41. Jundt F, Kley K, Anagnostopoulos I, et al: Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease. Blood. 99:3060–3062, 2002. 42. Torlakovic E, Tierens A, Dang HD, et al: The transcription factor PU.1, necessary for B-cell development is expressed in lymphocyte predominance, but not classical Hodgkin’s disease. Am J Pathol. 159:1807–1814, 2001. 43. Mathas S, Hinz M, Anagnostopoulos I, et al: Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells,
147.e2
Immunohistology of Hodgkin Lymphoma
stimulate proliferation and synergize with NF-kappa B. Embo J. 21:4104–4113, 2002. 44. Watanabe M, Sasaki M, Itoh K, et al: JunB induced by constitutive CD30-extracellular signal-regulated kinase 1/2 mitogenactivated protein kinase signaling activates the CD30 promoter in anaplastic large cell lymphoma and reed-sternberg cells of Hodgkin lymphoma. Cancer Res. 65:7628–7634, 2005. 45. Poppema S: The diversity of the immunohistological staining pattern of Sternberg-Reed cells. J Histochem Cytochem. 28:788– 791, 1980. 46. Stein H, Hansmann ML, Lennert K, et al: Reed-Sternberg and Hodgkin cells in lymphocyte-predominant Hodgkin’s disease of nodular subtype contain J chain. Am J Clin Pathol. 86:292–297, 1986. 47. Prakash S, Fountaine T, Raffeld M, et al: IgD positive L&H cells identify a unique subset of nodular lymphocyte predominant Hodgkin lymphoma. Am J Surg Pathol. 30:585–592, 2006. 48. Payne SV, Wright DH, Jones KJ, et al: Macrophage origin of Reed-Sternberg cells: an immunohistochemical study. J Clin Pathol. 35:159–166, 1982. 49. Mir R, Kahn LB: Immunohistochemistry of Hodgkin’s disease. A study of 20 cases. Cancer. 52:2064–2071, 1983. 50. Carbone A, Gloghini A, Gaidano G, et al: Expression status of BCL-6 and syndecan-1 identifies distinct histogenetic subtypes of Hodgkin’s disease. Blood. 92:2220–2228, 1998. 51. Falini B, Bigerna B, Pasqualucci L, et al: Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin’s disease. Blood. 87:465–471, 1996. 52. Wlodarska I, Nooyen P, Maes B, et al: Frequent occurrence of BCL6 rearrangements in nodular lymphocyte predominance Hodgkin lymphoma but not in classical Hodgkin lymphoma. Blood. 101:706–710, 2003. 53. Jardin F, Buchonnet G, Parmentier F, et al: Follicle center lymphoma is associated with significantly elevated levels of BCL-6 expression among lymphoma subtypes, independent of chromosome 3q27 rearrangements. Leukemia. 16:2318–2325, 2002. 54. Carbone A, Gloghini A, Aldinucci D, et al: Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin’s disease. Br J Haematol. 117:366–372, 2002. 55. Buettner M, Greiner A, Avramidou A, et al: Evidence of abortive plasma cell differentiation in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma. Hematol Oncol. 23:127–132, 2005. 56. Carbone A, Gloghini A, Gattei V, et al: Expression of functional CD40 antigen on Reed-Sternberg cells and Hodgkin’s disease cell lines. Blood. 85:780–789, 1995. 57. Banchereau J, Bazan F, Blanchard D, et al: The CD40 antigen and its ligand. Annu Rev Immunol. 12:881–922, 1994. 58. Kim LH, Eow GI, Peh SC, et al: The role of CD30, CD40 and CD95 in the regulation of proliferation and apoptosis in classical Hodgkin’s lymphoma. Pathology. 35:428–435, 2003. 59. Kadin ME, Muramoto L, Said J: Expression of T-cell antigens on Reed-Sternberg cells in a subset of patients with nodular sclerosing and mixed cellularity Hodgkin’s disease. Am J Pathol. 130:345–353, 1988. 60. Casey TT, Olson SJ, Cousar JB, et al: Immunophenotypes of Reed-Sternberg cells: a study of 19 cases of Hodgkin’s disease in plastic-embedded sections. Blood. 74:2624–2628, 1989. 61. Dallenbach FE, Stein H: Expression of T-cell-receptor beta chain in Reed-Sternberg cells. Lancet. 2:828–830, 1989. 62. Oka K, Mori N, Kojima M: Anti-Leu-3a antibody reactivity with Reed-Sternberg cells of Hodgkin’s disease. Arch Pathol Lab Med. 112:139–142, 1988. 63. Oudejans JJ, Kummer JA, Jiwa M, et al: Granzyme B expression in Reed-Sternberg cells of Hodgkin’s disease. Am J Pathol. 148:233–240, 1996. 64. Krenacs L, Wellmann A, Sorbara L, et al: Cytotoxic cell antigen expression in anaplastic large cell lymphomas of T- and null-cell type and Hodgkin’s disease: evidence for distinct cellular origin. Blood. 89:980–989, 1997. 65. Felgar RE, Macon WR, Kinney MC, et al: TIA-1 expression in lymphoid neoplasms. Identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation. Am J Pathol. 150:1893–1900, 1997.
66. Tzankov A, Bourgau C, Kaiser A, et al: Rare expression of T-cell markers in classical Hodgkin’s lymphoma. Mod Pathol. 18:1542– 1549, 2005. 67. Seitz V, Hummel M, Marafioti T, et al: Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease. Blood. 95:3020–3024, 2000. 68. Muschen M, Rajewsky K, Brauninger A, et al: Rare occurrence of classical Hodgkin’s disease as a T cell lymphoma. J Exp Med. 191:387–394, 2000. 69. Willenbrock K, Ichinohasama R, Kadin ME, et al: T-cell variant of classical Hodgkin’s lymphoma with nodal and cutaneous manifestations demonstrated by single-cell polymerase chain reaction. Lab Invest. 82:1103–1109, 2002. 70. Eberle FC, Song JY, Xi L, et al: Nodal involvement by cutaneous CD30-positive T-cell lymphoma mimicking classical Hodgkin lymphoma. Am J Surg Pathol. 36:716–725, 2012. 71. Weiss LM, Movahed LA, Warnke RA, et al: Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin’s disease. N Engl J Med. 320:502–506, 1989. 72. Wang D, Liebowitz D, Kieff E: An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell. 43:831–840, 1985. 73. Pallesen G, Hamilton-Dutoit SJ, Rowe M, et al: Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin’s disease. Lancet. 337:320–322, 1991. 74. Ambinder RF, Browning PJ, Lorenzana I, et al: Epstein-Barr virus and childhood Hodgkin’s disease in Honduras and the United States. Blood. 81:462–467, 1993. 75. Gulley ML, Eagan PA, Quintanilla-Martinez L, et al: EpsteinBarr virus DNA is abundant and monoclonal in the ReedSternberg cells of Hodgkin’s disease: association with mixed cellularity subtype and Hispanic American ethnicity. Blood. 83:1595–1602, 1994. 76. Herndier BG, Sanchez HC, Chang KL, et al: High prevalence of Epstein-Barr virus in the Reed-Sternberg cells of HIV-associated Hodgkin’s disease. Am J Pathol. 142:1073–1079, 1993. 77. Pinkus GS, Pinkus JL, Langhoff E, et al: Fascin, a sensitive new marker for Reed-Sternberg cells of hodgkin’s disease. Evidence for a dendritic or B cell derivation? Am J Pathol. 150:543–562, 1997. 78. Bakshi NA, Finn WG, Schnitzer B, et al: Fascin expression in diffuse large B-cell lymphoma, anaplastic large cell lymphoma, and classical Hodgkin lymphoma. Arch Pathol Lab Med. 131:742–747, 2007. 79. Fan G, Kotylo P, Neiman RS, et al: Comparison of fascin expression in anaplastic large cell lymphoma and Hodgkin disease. Am J Clin Pathol. 119:199–204, 2003. 80. Sahin U, Neumann F, Tureci O, et al: Hodgkin and ReedSternberg cell-associated autoantigen CLIP-170/restin is a marker for dendritic cells and is involved in the trafficking of macropinosomes to the cytoskeleton, supporting a functionbased concept of Hodgkin and Reed-Sternberg cells. Blood. 100:4139–4145, 2002. 81. Rosenwald IB, Koifman L, Savas L, et al: Expression of the translation initiation factors eIF-4E and eIF-2* is frequently increased in neoplastic cells of Hodgkin lymphoma. Hum Pathol. 39:910–916, 2008. 82. Zheng B, Fiumara P, Li YV, et al: MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival. Blood. 102:1019–1027, 2003. 83. Oelmann E, Herbst H, Zuhlsdorf M, et al: Tissue inhibitor of metalloproteinases 1 is an autocrine and paracrine survival factor, with additional immune-regulatory functions, expressed by Hodgkin/Reed-Sternberg cells. Blood. 99:258–267, 2002. 84. Juszczynski P, Ouyang J, Monti S, et al: The AP1-dependent secretion of galectin-1 by Reed Sternberg cells fosters immune privilege in classical Hodgkin lymphoma. Proc Natl Acad Sci U S A. 104:13134–13139, 2007. 85. Yamamoto R, Nishikori M, Kitawaki T, et al: PD-1-PD-1 ligand interaction contributes to immunosuppressive microenvironment of Hodgkin lymphoma. Blood. 111:3220–3224, 2008. 86. Kadin ME, Leibowitz D: Cytokines and cytokine receptors in Hodgkin’s disease. In: Mauch P, editor: Hodgkin’s disease, Philadelphia, 1999, Lippincott Williams & Wilkins, pp 139–157.
References 87. Gruss HJ, Kadin ME: Pathophysiology of Hodgkin’s disease: functional and molecular aspects. Baillieres Clin Haematol. 9:417–446, 1996. 88. Skinnider BF, Mak TW: The role of cytokines in classical Hodgkin lymphoma. Blood. 99:4283–4297, 2002. 89. Hopken UE, Foss HD, Meyer D, et al: Up-regulation of the chemokine receptor CCR7 in classical but not in lymphocytepredominant Hodgkin disease correlates with distinct dissemination of neoplastic cells in lymphoid organs. Blood. 99:1109–1116, 2002. 90. Hsu SM, Tseng CK, Hsu PL: Expression of p55 (Tac) interleukin-2 receptor (IL-2R), but not p75 IL-2R, in cultured H-RS cells and H-RS cells in tissues. Am J Pathol. 136:735–744, 1990. 91. Levi E, Butmarc J, Kourea HP, et al: Detection of interleukin-2 receptors on tumor cells in formalin-fixed, paraffin-embedded tissues. Appl Immunohistochem. 5:234, 1997. 92. Horie R, Watanabe T, Ito K, et al: Cytoplasmic aggregation of TRAF2 and TRAF5 proteins in the Hodgkin-Reed-Sternberg cells. Am J Pathol. 160:1647–1654, 2002. 93. Kadin ME, Agnarsson BA, Ellingsworth LR, et al: Immunohistochemical evidence of a role for transforming growth factor beta in the pathogenesis of nodular sclerosing Hodgkin’s disease. Am J Pathol. 136:1209–1214, 1990. 94. Ohshima K, Sugihara M, Suzumiya J, et al: Basic fibroblast growth factor and fibrosis in Hodgkin’s disease. Pathol Res Pract. 195:149–155, 1999. 95. Skinnider BF, Elia AJ, Gascoyne RD, et al: Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood. 97:250– 255, 2001. 96. Fichtner-Feigl S, Strober W, Kawakami K, et al: IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med. 12:99–106, 2006. 97. Samoszuk M, Nansen L: Detection of interleukin-5 messenger RNA in Reed-Sternberg cells of Hodgkin’s disease with eosinophilia. Blood. 75:13–16, 1990. 98. Teruya-Feldstein J, Jaffe ES, Burd PR, et al: Differential chemokine expression in tissues involved by Hodgkin’s disease: direct correlation of eotaxin expression and tissue eosinophilia. Blood. 93:2463–2470, 1999. 99. Dukers DF, Jaspars LH, Vos W, et al: Quantitative immunohistochemical analysis of cytokine profiles in Epstein-Barr viruspositive and -negative cases of Hodgkin’s disease. J Pathol. 190:143–149, 2000. 100. Scheeren FA, Diehl SA, Smit LA, et al: IL-21 is expressed in Hodgkin lymphoma and activates STAT5: evidence that activated STAT5 is required for Hodgkin lymphomagenesis. Blood. 111:4706–4715, 2008. 101. Skinnider BF, Elia AJ, Gascoyne RD, et al: Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood. 99:618– 626, 2002. 102. Delsol G, Blancher A, al Saati T, et al: Antibody BNH9 detects red blood cell-related antigens on anaplastic large cell (CD30+) lymphomas. Br J Cancer. 64:321–326, 1991. 103. al Saati T, Tkaczuk J, Krissansen G, et al: A novel antigen detected by the CBF.78 antibody further distinguishes anaplastic large cell lymphoma from Hodgkin’s disease. Blood. 86:2741–2746, 1995. 104. Ree HJ, Teplitz C, Khan A: The Lewis X antigen. A new paraffin section marker for Reed-Sternberg cells. Cancer. 67:1338–1346, 1991. 105. Facchetti F, Alebardi O, Vermi W: Omit iodine and CD30 will shine: a simple technical procedure to demonstrate the CD30 antigen on B5-fixed material. Am J Surg Pathol. 24:320–322, 2000. 106. Feldman AL, Law ME, Inwards DJ, et al: PAX5-positive T-cell anaplastic large cell lymphomas associated with extra copies of the PAX5 gene locus. Mod Pathol. 23:593–602, 2010. 107. Chu WS, Abbondanzo SL, Frizzera G: Inconsistency of the immunophenotype of Reed-Sternberg cells in simultaneous and consecutive specimens from the same patients. A paraffin section evaluation in 56 patients. Am J Pathol. 141:11–17, 1992.
147.e3
108. Kadin ME, Sako D, Berliner N, et al: Childhood Ki-1 lymphoma presenting with skin lesions and peripheral lymphadenopathy. Blood. 68:1042–1049, 1986. 109. Nakamura S, Takagi N, Kojima M, et al: Clinicopathologic study of large cell anaplastic lymphoma (Ki-1-positive large cell lymphoma) among the Japanese. Cancer. 68:118–129, 1991. 110. Gascoyne RD, Aoun P, Wu D, et al: Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma. Blood. 93:3913–3921, 1999. 111. Falini B, Pileri S, Zinzani PL, et al: ALK+ lymphoma: clinicopathological findings and outcome. Blood. 93:2697–2706, 1999. 112. Haybittle JL, Hayhoe FG, Easterling MJ, et al: Review of British National Lymphoma Investigation studies of Hodgkin’s disease and development of prognostic index. Lancet. 1:967–972, 1985. 113. Strickler JG, Michie SA, Warnke RA, et al: The “syncytial variant” of nodular sclerosing Hodgkin’s disease. Am J Surg Pathol. 10:470–477, 1986. 114. Rudiger T, Jaffe ES, Delsol G, et al: Workshop report on Hodgkin’s disease and related diseases (‘grey zone’ lymphoma). Ann Oncol. 9 Suppl 5:S31–S38, 1998. 115. Shiota M, Fujimoto J, Takenaga M, et al: Diagnosis of t(2;5) (p23;q35)-associated Ki-1 lymphoma with immunohistochemistry. Blood. 84:3648–3652, 1994. 116. Pulford K, Lamant L, Morris SW, et al: Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1. Blood. 89:1394–1404, 1997. 117. Suchi T, Lennert K, Tu LY, et al: Histopathology and immunohistochemistry of peripheral T cell lymphomas: a proposal for their classification. J Clin Pathol. 40:995–1015, 1987. 118. Sohani AR, Jaffe ES, Harris NL, et al: Nodular lymphocytepredominant hodgkin lymphoma with atypical T cells: a morphologic variant mimicking peripheral T-cell lymphoma. Am J Surg Pathol. 35:1666–1678, 2011. 119. Quintanilla-Martinez L, Fend F, Moguel LR, et al: Peripheral T-cell lymphoma with Reed-Sternberg-like cells of B-cell phenotype and genotype associated with Epstein-Barr virus infection. Am J Surg Pathol. 23:1233–1240, 1999. 120. Paulli M, Strater J, Gianelli U, et al: Mediastinal B-cell lymphoma: a study of its histomorphologic spectrum based on 109 cases. Hum Pathol. 30:178–187, 1999. 121. Higgins JP, Warnke RA: CD30 expression is common in mediastinal large B-cell lymphoma. Am J Clin Pathol. 112:241–247, 1999. 122. Miles RR, Mankey CC, Seiler CE, 3rd, et al: Expression of Grb2 distinguishes classical Hodgkin lymphomas from primary mediastinal B-cell lymphomas and other diffuse large B-cell lymphomas. Hum Pathol. 40:1731–1737, 2009. 123. Chittal SM, Brousset P, Voigt JJ, et al: Large B-cell lymphoma rich in T-cells and simulating Hodgkin’s disease. Histopathology. 19:211–220, 1991. 124. Rudiger T, Ott G, Ott MM, et al: Differential diagnosis between classic Hodgkin’s lymphoma, T-cell-rich B-cell lymphoma, and paragranuloma by paraffin immunohistochemistry. Am J Surg Pathol. 22:1184–1191, 1998. 125. Chevrel G, Berger F, Miossec P, et al: Hodgkin’s disease and B cell lymphoproliferation in rheumatoid arthritis patients treated with methotrexate: a kinetic study of lymph node changes. Arthritis Rheum. 42:1773–1776, 1999. 126. Kamel OW, Weiss LM, van de Rijn M, et al: Hodgkin’s disease and lymphoproliferations resembling Hodgkin’s disease in patients receiving long-term low-dose methotrexate therapy. Am J Surg Pathol. 20:1279–1287, 1996. 127. Pitman SD, Huang Q, Zuppan CW, et al: Hodgkin lymphomalike posttransplant lymphoproliferative disorder (HL-like PTLD) simulates monomorphic B-cell PTLD both clinically and pathologically. Am J Surg Pathol. 30:470–476, 2006. 128. Mao Z, Quintanilla-Martinez L, Raffeld M, et al: IgVH mutational status and clonality analysis of Richter’s transformation: diffuse large B-cell lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution. Am J Surg Pathol. 31:1605–1614, 2007.
147.e4
Immunohistology of Hodgkin Lymphoma
129. de Leval L, Vivario M, De Prijck B, et al: Distinct clonal origin in two cases of Hodgkin’s lymphoma variant of Richter’s syndrome associated With EBV infection. Am J Surg Pathol. 28:679–686, 2004. 130. Fong D, Kaiser A, Spizzo G, et al: Hodgkin’s disease variant of Richter’s syndrome in chronic lymphocytic leukaemia patients previously treated with fludarabine. Br J Haematol. 129:199– 205, 2005. 131. Ohno T, Smir BN, Weisenburger DD, et al: Origin of the Hodgkin/Reed-Sternberg cells in chronic lymphocytic leukemia with “Hodgkin’s transformation”. Blood. 91:1757–1761, 1998. 132. Schwarting R, Gerdes J, Durkop H, et al: BER-H2: a new antiKi-1 (CD30) monoclonal antibody directed at a formol-resistant epitope. Blood. 74:1678–1689, 1989. 133. Polski JM, Janney CG: Ber-H2 (CD30) immunohistochemical staining in malignant melanoma. Mod Pathol. 12:903–906, 1999. 134. Sheibani K, Battifora H, Burke JS, et al: Leu-M1 antigen in human neoplasms. An immunohistologic study of 400 cases. Am J Surg Pathol. 10:227–236, 1986. 135. Strickler JG, Fedeli F, Horwitz CA, et al: Infectious mononucleosis in lymphoid tissue. Histopathology, in situ hybridization, and differential diagnosis. Arch Pathol Lab Med. 117:269–278, 1993. 136. Reynolds DJ, Banks PM, Gulley ML: New characterization of infectious mononucleosis and a phenotypic comparison with Hodgkin’s disease. Am J Pathol. 146:379–388, 1995. 137. Louissaint A, Jr, Ferry JA, Soupir CP, et al: Infectious mononucleosis mimicking lymphoma: distinguishing morphological and immunophenotypic features. Mod Pathol. 25:1149–1159, 2012. 138. Fellbaum C, Hansmann ML, Lennert K: Lymphadenitis mimicking Hodgkin’s disease. Histopathology. 12:253–262, 1988. 139. Doggett RS, Colby TV, Dorfman RF: Interfollicular Hodgkin’s disease. Am J Surg Pathol. 7:145–149, 1983. 140. Chan JKC, Tsang WYW: Reactive lymphadenopathies. In Weiss LM, editor: Pathology of Lymph Nodes Contemporary Issues in Surgical Pathology, Philadelphia, 1996, Churchill Livingstone. 141. Kadin ME, Donaldson SS, Dorfman RF: Isolated granulomas in Hodgkin’s disease. N Engl J Med. 283:859–861, 1970. 142. Ohshima K, Kikuchi M, Shibata T, et al: Clonal analysis of Hodgkin’s disease shows absence of TCR/Ig gene rearrangement, compared with T-cell-rich B-cell lymphoma and incipient adult T-cell leukemia/lymphoma. Leuk Lymphoma. 15:469–479, 1994. 143. Chute DJ, Cousar JB, Mahadevan MS, et al: Detection of immunoglobulin heavy chain gene rearrangements in classic hodgkin lymphoma using commercially available BIOMED-2 primers. Diagn Mol Pathol. 17:65–72, 2008. 144. Davis TH, Morton CC, Miller-Cassman R, et al: Hodgkin’s disease, lymphomatoid papulosis, and cutaneous T-cell lymphoma derived from a common T-cell clone. N Engl J Med. 326:1115–1122, 1992. 145. Feuerhake F, Kutok JL, Monti S, et al: NFkappaB activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes. Blood. 106:1392–1399, 2005. 146. Savage KJ, Monti S, Kutok JL, et al: The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood. 102:3871–3879, 2003. 147. Eckerle S, Brune V, Doring C, et al: Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma. Leukemia. 23:2129–2138, 2009. 148. Willenbrock K, Kuppers R, Renne C, et al: Common features and differences in the transcriptome of large cell anaplastic
lymphoma and classical Hodgkin’s lymphoma. Haematologica. 91:596–604, 2006. 149. Garcia JF, Camacho FI, Morente M, et al: Hodgkin and ReedSternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays. Blood. 101:681–689, 2003. 150. Schulz H, Rehwald U, Morschhauser F, et al: Rituximab in relapsed lymphocyte-predominant Hodgkin lymphoma: longterm results of a phase 2 trial by the German Hodgkin Lymphoma Study Group (GHSG). Blood. 111:109–111, 2008. 151. Younes A, Romaguera J, Hagemeister F, et al: A pilot study of rituximab in patients with recurrent, classic Hodgkin disease. Cancer. 98:310–314, 2003. 152. Annunziata CM, Safiran YJ, Irving SG, et al: Hodgkin disease: pharmacologic intervention of the CD40-NF kappa B pathway by a protease inhibitor. Blood. 96:2841–2848, 2000. 153. Wahl AF, Klussman K, Thompson JD, et al: The anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin’s disease. Cancer Res. 62:3736–3742, 2002. 154. Francisco JA, Cerveny CG, Meyer DL, et al: cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood. 102:1458–1465, 2003. 155. Falini B, Bolognesi A, Flenghi L, et al: Response of refractory Hodgkin’s disease to monoclonal anti-CD30 immunotoxin. Lancet. 339:1195–1196, 1992. 156. Borchmann P, Schnell R, Fuss I, et al: Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma. Blood. 100:3101–3107, 2002. 157. Younes A, Gopal AK, Smith SE, et al: Results of a pivotal phase II study of brentuximab vedotin for patients with relapsed or refractory Hodgkin’s lymphoma. J Clin Oncol. 30:2183–2189, 2012. 158. Bollard CM, Aguilar L, Straathof KC, et al: Cytotoxic T lymphocyte therapy for Epstein-Barr virus+ Hodgkin’s disease. J Exp Med. 200:1623–1633, 2004. 159. Savoldo B, Rooney CM, Di Stasi A, et al: Epstein Barr virus specific cytotoxic T lymphocytes expressing the anti-CD30zeta artificial chimeric T-cell receptor for immunotherapy of Hodgkin disease. Blood. 110:2620–2630, 2007. 160. Tepler I, Schwartz G, Parker K, et al: Phase I trial of an interleukin-2 fusion toxin (DAB486IL-2) in hematologic malignancies: complete response in a patient with Hodgkin’s disease refractory to chemotherapy. Cancer. 73:1276–1285, 1994. 161. Merz H, Malisius R, Mannweiler S, et al: ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab Invest. 73:149–156, 1995. 162. Ishida T, Ishii T, Inagaki A, et al: The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma. Leukemia. 20:2162–2168, 2006. 163. Kamper P, Ludvigsen M, Bendix K, et al: Proteomic analysis identifies galectin-1 as a predictive biomarker for relapsed/ refractory disease in classical Hodgkin lymphoma. Blood. 117:6638–6649, 2011. 164. Ree HJ, Kadin ME: Macrophage-histiocytes in Hodgkin’s disease. The relation of peanut-agglutinin-binding macrophagehistiocytes to clinicopathologic presentation and course of disease. Cancer. 56:333–338, 1985. 165. Steidl C, Lee T, Shah SP, et al: Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N Engl J Med. 362:875–885, 2010. 166. Kamper P, Bendix K, Hamilton-Dutoit S, et al: Tumor-infiltrating macrophages correlate with adverse prognosis and Epstein-Barr virus status in classical Hodgkin’s lymphoma. Haematologica. 96:269–276, 2011.
C H A P T E R 6
IMMUNOHISTOLOGY LYMPHOMA
OF
NON-HODGKIN
DENNIS P. O’MALLEY, KATE E. GRIMM, PETER M. BANKS
Overview 148 Antigens for Evaluation of Hematologic Disorders 148 Establishing the Diagnosis of Lymphoma: Differential Diagnostic Considerations 154 Immunohistochemical Evaluation of Small B-Cell Lymphomas 157 Large B-Cell Lymphomas and Other Aggressive B-Cell Lymphomas 168 T-Cell Lymphomas 177 Pitfalls in the Diagnosis of Lymphoma: Mimicry 184 Summary 188
Overview Only a little more than 30 years ago, a popular, widely used classification system for non-Hodgkin lymphomas was initiated based solely on hematoxylin and eosin (H&E) stained slide interpretation. The “Working Formulation for Clinical Usage” was used for 12 years, before the mounting evidence for immunophenotyping and genetic characterization of lymphoid neoplasms was recognized as necessary for a more precise and scientific system.1,2 With the benefit of highly specific antibodies against human lymphoid biomarkers, both flow cytometry and immunohistochemistry (IHC) can be used to identify neoplastic lymphoid cells in relation to their normal cellular counterparts in the immune system.3 Some of the most useful antibodies for diagnosis actually represent “trickle down” products, which derive from markers originally identified by genetic methods, such as Bcl-2 and ALK.4,5 Today such methods are requisite for a lymphoma diagnosis and, increasingly, are of predictive value in guiding directed therapies, for example, tumor-cell expression of CD20 for the use of rituximab.6 148
For pathologists too young to remember how revolutionary the advent of IHC was for the study of lymphoid tissues by microscopy, one need only prepare a familiar IHC slide without a nuclear counterstain (Fig. 6-1). Although the pattern of test antigen may allow an educated guess as to the diagnosis, with the addition of nuclear staining, the power of simultaneous histomorphology and antigen detection can be appreciated. To confidently interpret IHC in diagnosing lymphoma, the clinician must first be familiar with the distribution of test antigens in normal and hyperplastic benign lymphoid tissues (Fig. 6-2). Only then can deviations from the norm be gauged in relation to a diagnosis of malignancy. Built-in positive controls are, for this reason, particularly appropriate in lymphoid samples because most markers, but not all, are expressed in reactive elements usually included in tumor biopsies. It is critical to remember that the same good practice procedures required for quality conventional sections are all the more necessary for the production of good IHC.7 Tissue allocation must avoid the introduction of surface drying artifact, and blocks should be adequately thin to allow satisfactory fixation and processing. Selection of the proper fixative, sufficient fixation time, fluid processing, sectioning, and deparaffinization all require constant attention.8 When dealing with small tissue samples, a laboratory protocol calling for a number (6 to 10) of unstained sections mounted on coated slides is an important insurance policy against the misery of inadequate tissue samples available for diagnostically essential IHC.
Antigens for Evaluation of Hematologic Disorders B-Cell–Associated Antigens CD20
CD20 is a nonglycosylated membranous phosphoprotein weighing 35 kD.9-11 CD20 is acquired by late pre-B cells as they mature, and it is typically lost when the B cells become plasma cells.11 Although the exact
Antigens for Evaluation of Hematologic Disorders
149
Figure 6-1 Left, Immunohistochemistry (IHC) for CD45 without nuclear counterstain. Right, IHC for CD45 with nuclear counterstain; the absence selectively among the large tumor cells in a case of classic Hodgkin lymphoma can be appreciated.
function of the CD20 antigen is unknown, it is thought to be involved in B-cell regulation, differentiation, and calcium flux.9 L26 is the most commonly used commercial antibody. CD20 staining is membranous. Currently, CD20 is the first-line B-cell lineage defining antibody.11 Therapeutic strategies that involve the monoclonal antibody directed against CD20 (i.e., rituximab-containing chemotherapeutic regimens) have necessitated the use of alternate B-cell lineage to define
CD20
CD3
markers in patients who have received such therapy and are suspected of relapse.12 Although rarely identified, CD20 expression has been noted on a small subset of nonneoplastic T cells.13 CD20 expression has also been identified in occasional cases of Hodgkin lymphoma, precursor-B acute lymphoblastic leukemia, plasma cell neoplasms, and, rarely, in T-cell lymphomas.6,14 CD19 and CD22 are also available for staining in paraffin sections and are useful adjuncts to determine
bcl-2
Figure 6-2 Comparison of staining distribution in a benign lymph node with immunohistochemistry for B-cell marker CD20, T-cell marker CD3, and Bcl-2 protein. Familiarity with normal cellular compartmental reactivity is essential for application of these markers for lymphoma studies.
150
Immunohistology of Non-Hodgkin Lymphoma
B-cell lineage in patients who have received treatment with rituximab. Pax-5
Pax-5 (B-cell–specific activator protein) is a nuclear protein that belongs to the paired-box containing (PAX) family of transcription factors.15 Pax-5 is thought to commit B-cell progenitors to the B-cell lineage by suppressing non–B-cell–associated genes and activating B-cell–specific genes.16,17 A broader regulatory role has been described that includes regulation of cell adhesion and migration, inducing V-DJ immunoglobulin (Ig) heavy-chain recombination and facilitation of early B-cell receptor signaling to promote development to the mature B-cell stage.17 The staining pattern in B cells is strong and nuclear; Reed-Sternberg cells show a characteristic weak, variable pattern of nuclear expression. Pax-5 is expressed by early B-cell precursors and by mature B cells, and it is lost as B cells mature to plasma cells.18 It is expressed on normal B cells and on their malignant counterparts, and it has become a valuable adjunct marker of B-cell lineage. Rare reports of Pax-5–expressing non–B-cell–lineage neoplasms have been described in anaplastic large cell lymphoma (ALCL) and lymphomatoid papulosis.18 Expression occurs in nonhematolymphoid malignancies; Pax-5 expression has been identified in atypical carcinoids, small cell lung carcinomas, and large cell neuroendocrine carcinomas.19 CD79a
CD79a is associated with the immunoglobulin receptor complex in the B-cell membrane.20 It is the B-cell marker with the broadest sensitivity, because it is expressed in early B-cell precursors, even before immunoglobulin heavy-chain gene rearrangement, and throughout B-cell maturity and is eventually lost only in the late plasma cell stage.21 The staining pattern is cytoplasmic. B-cell–lineage antigen CD79a expression has also been reported in cases of T-cell lymphoblastic lymphoma and acute myeloid leukemia, often in acute promyelocytic leukemia.22,23 Bcl-6
The BCL6 gene encodes a 79-kD zinc finger-binding protein thought to play a role in B-cell differentiation within the germinal center.24 This Bcl-6 protein is expressed on B cells of germinal center origin with a nuclear staining pattern. Expression of Bcl-6 protein, demonstrated through IHC, does not necessarily correlate with the presence of a BCL6 gene rearrangement. BCL6 rearrangements are one of the more common genetic abnormalities and are found in diverse lymphomas not restricted to those that arise from a B-cell of germinal center origin. In contrast, Bcl-6 protein is normally expressed in nonneoplastic germinal center B cells. Lymphomas of germinal center origin, such as follicular lymphoma or Burkitt lymphoma, express Bcl-6 protein demonstrated by IHC,
however, this does not imply the presence of a BCL6 translocation. Although more often seen in cells of the B-cell lineage, Bcl-6 expression is reported in occasional ALCLs and in peripheral T-cell lymphomas.25,26 MUM1/IRF4
The melanoma-associated antigen (mutated) 1 (MUM1) gene belongs to a larger group, termed the interferon regulatory family (IRF4), and it encodes a transcription factor responsible for development in B, T, plasma, dendritic, and myeloid cells.19 Two antibodies against human MUM1/IRF4 protein, Mum-1p (monoclonal) and ICSTAT (polyclonal), are commercially available.27 Staining is both nuclear and cytoplasmic.28 MUM1 was originally recognized by its upregulation in multiple myeloma with a translocation (t) of (6;14) (p25;q32); however, it was soon found not to be specific for plasmacytic differentiation.29 MUM1 protein expression has been described in numerous neoplasms that include lymphoplasmacytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, Hodgkin lymphoma, ALCL, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), adult T-cell lymphoma/ leukemia, and melanoma.27,29 MUM1 expression is also seen in nonneoplastic “activated” T cells, a subset of germinal center B cells, and normal melanocytes.27-29 In general, MUM1 immunostaining is thought to parallel CD30 staining.27,28 OCT-2 AND BOB.1
OCT-2 and its coactivator BOB.1 are transcription factors of the POU homeo-domain family that bind to the conserved octamer sites in the promoters of the Ig genes involved in B-cell differentiation and regulation.30,31 Staining is nuclear. BOB.1 is a novel 256–amino acid proline-rich protein seen predominantly in the B-cell lineage expressed in precursor and mature B cells.32 BOB.1 interacts with either OCT-1 or OCT-2 to coactivate gene transcription by binding to a number of octamer sites throughout the genome. BOB.1 is also known as OBF1, OCT Binding Factor 146, OCA-B47, and Bob-148.32 Given the postulated mechanisms of action and origins of classic Hodgkin lymphoma (CHL), OCT-2 and BOB.1 expression were thought to be absent in Reed-Sternberg cells, a fact that was largely used to differentiate the lymphocyte predominant cells (“popcorn cells”) of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) from Reed-Sternberg cells of CHL. However, weak expression of OCT-2 and BOB.1 in the Reed-Sternberg cells of CHL has been reported in a subset of cases.33,34 Strong consistent expression of OCT-2 and BOB.1 is reported in NLPHL and in non-Hodgkin lymphomas.34 OCT-2 and BOB.1 are also expressed in a subset of acute myeloid leukemias, in which their expression may have a prognostic relevance.35
Antigens for Evaluation of Hematologic Disorders
Other Important Markers CD138
CD138 (syndecan-1) is a 200-kD member of the transmembrane family of heparin sulfate proteoglycan proteins.36 Located on chromosome 2p23-24, CD138 is thought to be a receptor for matrix proteins and a cofactor for growth factors.36,37 The anti-syndecan 1 monoclonal antibody Mi15 is frequently used.36 The staining pattern is membranous.36 CD138 is most commonly associated with plasmacytic differentiation and is seen on both benign plasma cells and their malignant counterparts (plasma cell myeloma). Among hematolymphoid neoplasms, expression of CD138 is interpreted as evidence of plasmacytic differentiation; however, caution should be used because non-Hodgkin lymphomas, as well as many nonhematolymphoid neoplasms, may express this marker. CD138 expression is seen on nonneoplastic epithelial surfaces and correspondingly has been reported among various types of carcinomas.38 Nonneoplastic early precursor B cells and posttransplant lymphoproliferative disorders express CD138. Plasmablastic lymphoma, lymphoplasmacytic lymphoma, and a small subset of cases of chronic lymphocytic leukemia express CD138.38 Expression of CD138 on mesenchymal neoplasms and tumors of melanocytic origin has also been reported.38 CD30
CD30 is a membrane-bound phosphorylated glycoprotein weighing 120 kD. It is a member of the tumor necrosis factor receptor superfamily 8 (TNFRSF8).39,40 Monoclonal antibodies used in paraffin include Ber-H2; HeFi; HRS-1, -2, and-3; M44 and M67; and C10.40 CD30 is expressed on normal activated T and B cells and on virally transformed B and T cells (Epstein-Barr virus [EBV], human T-cell lymphotropic virus 1 and 2, and human immunodeficiency virus [HIV]). Monocytes/ macrophages and granulocytes also constitutively express CD30.40 In lymph node and tonsil sections, a small subset of lymphocytes in the parafollicular areas express CD30,40 and CD30 is thought to transduce a cell survival signal and to be involved in the T-cell– dependent portion of the immune response.39,40 CD30 staining may be membranous, or it may be concentrated in the Golgi zone outside the nucleus (paranuclear). CD30 is consistently overexpressed on Hodgkin/ Reed-Sternberg cells. However, CD30 expression is seen in a number of settings that include lymphomatoid papulosis, ALCL, adult T-cell lymphomas, some cutaneous T-cell lymphomas, natural killer neoplasms, a subset of B-cell lymphomas (DLBCL, Burkitt lymphoma), and embryonal carcinoma.39-41 CD30 expression in neoplasms has become increasingly important with the development and successful treatment with anti-CD30 monoclonal antibodies. Brentuximab vedotin is an antiCD30 chimeric IgG1 monoclonal antibody used to target CD30 neoplasms, particularly in the setting of a relapse after first-line therapy has failed.39
151
ANAPLASTIC LYMPHOMA KINASE
Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that belongs to the insulin receptor superfamily, and it weighs 220 kD.42 Initially described in ALCL, t(2;5)(p23;q35) creates a fusion gene from the nucleophosmin gene (NPM1) and the tyrosine kinase receptor gene (ALK); the resulting chimeric gene encodes a constitutively activated tyrosine kinase that is a potent oncogene.43 Additional translocations were subsequently discovered; at least 15 different ALK fusion proteins have been described.44 Although initially described in ALCL, numerous other malignancies express ALK, either as activated fusion proteins derived from chromosomal rearrangements or as mutationally activated ALK proteins, such as the activating mutations described in neuroblastoma.44 Staining may be cytoplasmic, nuclear, or membranous, and different staining patterns correlate with specific translocations. Normal ALK expression is seen immunohistochemically in rare scattered neural cells, endothelial cells, and pericytes in the brains of adults.44 ALK expression has been reported in cases of B-cell lymphomas, non–small cell lung cancers (NSCLCs), rhabdomyosarcomas, glioblastomas, melanomas, inflammatory myofibro blastic tumors (IMTs), esophageal squamous cell carcinomas, and systemic histiocytosis.44 Crizotinib (PF02341066), a receptor kinase inhibitor, has been used to treat ALCL and NSCLC cell lines that harbor ALK translocations.45 TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that catalyzes the addition of deoxynucleotides to the 3′-hydroxyl terminus.46,47 TdT is expressed in precursor B and precursor T lymphocytes during early differentiation; it generates antigen receptor diversity in both cell lines by synthesizing nongermline elements at the ends of rearranged Ig heavy-chain and T-cell receptor gene segments, respectively.46 Staining may be nuclear, as well as membranous, or it may occur with paranuclear dotlike positivity.47 Nonmalignant TdT expression is seen in sections of the thymus that contain precursor T cells and in bone marrow sections that contain early B-cell precursors, so-called hematogones. The malignant blasts of acute T- or B-lymphoblastic leukemia/lymphoma express TdT, as do a subset of acute myeloid leukemias and hematodermic CD56-positive/CD4-positive neoplasms.47 Nonhematopoietic malignancies that express TdT include some pediatric small round blue cell tumors, Merkel cell carcinoma, and small cell lung carcinoma.47 CD34
CD34 is a 115-kD transmembrane sialomucin encoded on chromosome 1q32.1.48-50 Proposed functions include inhibition of differentiation, proliferation, and adhesion.48 Most of the studies to evaluate CD34 expression have used the My10 or the QBEND/10 monoclonal
152
Immunohistology of Non-Hodgkin Lymphoma
antibodies. Staining in blasts is described as membranous with some cytoplasmic staining. Numerous benign and neoplastic proliferations express CD34, which is not lineage specific and has different meanings in the various contexts. CD34 expression is found on both lymphoid and myeloid blasts, and in the context of hematopoietic elements, it implies immaturity. CD34 expression in nonneoplastic cells may be seen in vascular and rare stromal elements. Neoplastic proliferations with CD34 expression include most vascular tumors, some spindle cell tumors, and diverse other hematopoietic-derived tumors.49-51 CD43
CD43 is a sialomucin expressed on hematopoietic precursors, and it is thought to play a role in regulation of hematopoiesis.52 In adults, CD43 occurs both on bone marrow hematopoietic stem cells and on mature white blood cells in the periphery, with the exception of resting B lymphocytes. CD43 is also found on tissue macrophages, dendritic cells, smooth muscle cells, epithelium, and endothelium. CD43 expression has been reported on myeloblasts, lymphoma cells, and metastases of solid neoplasms.53 Staining is cytoplasmic. CYCLIN D1
Cyclin D1 (BCL1/PRAD1) is a transcriptional regulator protein that complexes with the cyclin-dependent kinases that maintain regulation of G1 to the S phase of the cell cycle.54,55 The CCND1 gene is found at chromosome 11q13. Most notably, mantle cell lymphoma (MCL) is associated with the translocation involving this gene and the Ig heavy-chain locus t(11 : 14)(q13;q32).56 In addition to MCL, occasional cases of plasma cell myeloma and hairy cell leukemia have been found to express cyclin D1, and focal areas of weak cyclin D1 expression have been described in the proliferation centers of chronic lymphocytic leukemia.57 Nonneoplastic lymphoid cells do not express cyclin D1.54 Overexpression of cyclin D1 is not restricted to hematopoietic malignancies and has been associated with breast carcinoma and NSCLC.55 Scattered nonneoplastic endothelial cells will often express cyclin D1 and can be used as an internal positive control. Staining is nuclear. BCL2
IHC antibodies detect an increase in the antiapoptotic Bcl-2 protein, usually resulting from the translocation of the BCL2 gene to a position behind the enhancer elements of the Ig heavy-chain gene t(14;18)(q32;q21).58 In addition to inhibiting apoptosis, the resulting BCL2 oncogene may also block chemotherapy-induced cell death.59 Staining is membranous. Bcl-2 protein expression may be used as one means of differentiating benign follicular hyperplasia from its neoplastic counterpart, follicular lymphoma; however, Bcl-2 expression should not be interpreted as evidence of malignancy.60 Intrafollicular T cells, T and B cells in the interfollicular areas, primary follicles, and mantle zone B cells normally express Bcl-2. Expression of Bcl-2
is not limited to lymphomas and is commonly encountered in nonhematopoietic malignancies as well. CD117
CD117 (c-kit) is a 145-kD transmembrane tyrosine kinase receptor that is the product of the kit gene located on chromosome 4 (4q11-q12).61,62 CD117 is not lineage specific and is seen on numerous tissues throughout the body, including hematopoietic precursors, mast cells, and melanocytes. Staining on myeloid and erythroid precursors and the neoplastic cells of plasma cell myeloma is weak and cytoplasmic. Staining in mast cells is strong and cytoplasmic. CD117 immunoreactivity has become increasingly important, because it is the basis for treatment of gastrointestinal stromal tumors (GISTs) with the tyrosine kinase inhibitor imatinib mesylate.61,63
T-Cell–Associated Antigens CD2
CD2 is a 50- to 55-kD transmembrane glycoprotein found on both T cells and natural killer (NK) cells.64,65 The CD2 genes are found on chromosome 1 and are thought to play a role in antigen-independent adhesion and in T-cell activation.64,66,67 In T-cell development, CD2 is thought to appear after CD7. The staining is cytoplasmic. CD2 expression is seen on both immature (precursor) T cells as well as on mature (peripheral) T cells. Aberrant loss of CD2 expression is seen in T-cell lymphomas. CD2 expression is usually seen in T-acute lymphoblastic leukemia and, more rarely, can be seen on the myeloblasts of acute myeloid leukemia.68 CD2 expression in mast cells is considered aberrant and supports a diagnosis of systemic mastocytosis.69 CD3
CD3 is a T-cell antigen composed of four distinct subunits (ε, γ, δ, and ζ) that span the membrane and are associated with the T-cell receptor (TCR).70,71 CD3 staining is membranous and cytoplasmic. CD3 is first found in the cytoplasm of developing T cells as cytoplasmic CD3 (cCD3). As T cells mature, the CD3 antigen moves to the surface. Similarly, the neoplastic counterparts show a similar distinction with cCD3 on precursor T-cell neoplasms and with surface CD3 seen on peripheral, “mature” T-cell neoplasms. Although most widely used as a T-cell lineage–specific antigen, CD3 expression (cytoplasmic and membranous) has been reported on B-cell lymphomas, particularly those with expression of EBV.72 CD4/CD8
CD4 and CD8 T-cell surface molecules play a role in T-cell recognition and activation by binding to their respective class II and class I major histocompatibility complex (MHC) ligands on an antigen-presenting cell (APC).73 CD4 has the additional role of stabilization of
Antigens for Evaluation of Hematologic Disorders
the TCR complex; it is also a major target of HIV.74 Staining for both CD4 and CD8 is both cytoplasmic and membranous. Early T-cell precursors express both CD4 and CD8 simultaneously, but with maturation, they lose one of these markers. CD4 staining is seen in the T-helper class of T cells, the predominant population in the T-cell compartment; CD8 staining is seen in the cytotoxic T-cell population and in nonneoplastic sinusoids of the spleen. The majority of T-cell lymphomas express CD4, not CD8, and are thought to be derived from the T-helper lineage. Cytotoxic T-cell lymphomas that express CD8, not CD4, are a proportionally smaller group of lymphomas. Rarely, lymphomas may have aberrant loss of both of these antigens; however, a small subset of nonneoplastic T cells will be “double negative” for these two markers, as well as for the γ/δ T cells, and this finding should not be misinterpreted as aberrant antigen loss. CD5
CD5 is a 67-kD type I glycoprotein that is thought to attenuate signals that arise from the cross-linking of the T-cell receptor and its antigen on the MHC of antigenpresenting cells.75,76 Staining is membranous. CD5 is expressed by the majority of peripheral (mature) T cells; the loss of this marker may be seen as one of the first findings in a developing T-cell lymphoma.76,77 However, this finding has been reported in reactive populations of T cells as well. Expression of CD5 has also been used to distinguish the neoplastic thymocytes of thymic carcinoma from a benign thymoma.78 Although a T-cell lineage antigen, CD5, is famously coexpressed aberrantly on certain B-cell lymphomas that include chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/SLL) and MCL. More rarely, cases of DLCBL and cases of follicular lymphoma may express this T-cell antigen as well. In addition, a small nonneoplastic subset of B cells, the B1a cells, normally expresses this T-cell marker. CD7
CD7 is a 40-kD glycoprotein member of the Ig gene family. CD7 antigen is thought to be involved in signal transduction, proliferation, and adhesion. The CD7 monoclonal antibody CBC.37 shows expression on the majority of peripheral T cells (i.e., mature T cells), NK cells, and precursor T cells (immature T cells). Staining is membranous. CD7 is one of the earliest markers in T-cell development.79 Although a T-cell lineage antigen, CD7 expression may be seen in small populations of fetal bone marrow B cells and myeloid precursor cells, this is subsequently lost early with differentiation.79 As with CD5, loss of CD7 antigen may be seen in the setting of a T-cell lymphoproliferative process as well as in a benign reactive setting. Aberrant CD7 expression has been described in the myeloblasts of acute myeloid leukemia, where it has been associated with Fms-like tyrosine kinase-3 internal tandem duplication (FLT3/ITD) mutation and significantly shorter disease-free postremission survival.80,81
153
TIA1, GRANZYME B, AND PERFORIN
The T-cell compartment is composed of CD4-expressing T-helper cells and CD8-expressing T-cytotoxic cells. T-cell intracytoplasmic antigen 1 (TIA1), granzyme B, and perforin are markers used to identify the cytotoxic T cells, which induce lysis of their targets by using these granule-associated cytotoxic proteins. Expression of TIA1 can be detected in all cytotoxic cells, whereas granzyme B and perforin expression can be detected in high levels only in activated cytotoxic cells.82 Expression is cytoplasmic. CD56/CD57
CD56 and CD57 are frequently used to identify the NK cell lineage; however, they are frequently found expressed in unrelated tissues. CD56, or neuronal cell adhesion molecule (N-CAM), is an NK cell marker that is also expressed in the central and peripheral nervous systems.83 Some cases of plasma cell myeloma also express this marker.84 Similarly, CD57 is a marker expressed on NK cells and other T cells; it is often used in the diagnosis of nodular lymphocyte– predominant Hodgkin lymphoma (NLPHL) to visualize the small T cells that ring the LP cells. The staining of CD56 (monoclonal antibody 123C3) and CD57 are membranous.85 NK cells express CD2, CD7, CD8, CD56, and CD57. They are positive for cytoplasmic CD3, but not surface CD3, and do not typically express CD5. The neoplastic counterpart, extranodal NK/T-cell lymphomas, express CD2, cytoplasmic CD3, CD56, and, in most cases, EBV.83,86 Additional markers for NK cells include killer inhibitory receptors (KIRs). KIR expression is identified by using monoclonal antibodies specific for CD94, CD158a, and CD158b.87 β-F1
β-F1 staining identifies a portion of the β-subunit of the T cells that carry the α/β T-cell receptor.88 T cells with the α/β T-cell receptor normally represent the majority of T cells (95%). Similarly, the majority of T-cell lymphomas will express the α/β T-cell receptor. Staining is membranous. Negativity for β-F1 stain implies that the T cells carry the alternative T-cell receptor subunits gamma and delta (γ/δ). Small subsets of normal γ/δ T cells are found in the skin, splenic red pulp, mucosal-associated lymphoid tissue (MALT), and in the medulla of the thymus. These cells have a distinct immunophenotypic profile, such that CD2 and CD3 are negative, as are CD4 and CD8 (double-negative T cells), and CD5 is likewise usually negative. The neoplastic counterparts, hepatosplenic T-cell lymphoma and subcutaneous panniculitic T-cell lymphoma, also express the γ/δ T-cell receptor. However, caution should be used in interpreting this negative staining as evidence of a γ/δ T-cell derivation, because NK cells will also be negative for β-F1; staining with CD56 in this instance will help to identify these cells.
154
Immunohistology of Non-Hodgkin Lymphoma
Establishing the Diagnosis of Lymphoma: Differential Diagnostic Considerations Conventional microscopic findings, coupled with the clinical history, is still the bulwark for a lymphoma diagnosis. Although modern lymphoma classification emphasizes relatedness of the neoplastic process to normal cell counterparts of the immune system (i.e., B cell vs. T cell vs. NK cell), microscopic cellular features and patterns of proliferation frame the differential diagnosis. This starting point for pathologists is all important, because the cost-effective selection of definitive special study methods, in particular IHC, depends on their acumen. It is usually more critical for the patient that the diagnosis of lymphoma per se be correct than that its classification be precise. There are many processes, both benign and malignant, that can microscopically, and sometimes even clinically, simulate lymphoma.89 IHC can be a very powerful tool in resolving differential diagnostic puzzles; however, the correct markers must be brought to bear.
Low-Grade Lymphoma Versus Chronic Lymphoid Hyperplasia The most common differential diagnostic quandary consists of the often difficult distinction between low-grade (B-cell) malignant lymphoma and chronic immune hyperplasia, usually in an older patient. The overriding principle in this setting is not to make the diagnosis of lymphoma unless absolutely certain, because in most cases, early detection is not effective in eradicating such lymphomas, and an erroneous diagnosis of malignancy may take a long time, if ever, to be suspected clinically. As mentioned in the overview, the pathologist’s strength is in recognizing the microscopic and IHC hallmarks of a benign immune reaction. Functional immune compartments are distinct and recognizable and include follicle centers, mantle zones, paracortex, sinuses, and medullary cords. Special cellular compartments may be recruited and expanded as well, such as with plasmacytoid monocytes, granulomas, abscesses, and so on. Appropriate IHC markers to label these compartments may be of use in confirming the diagnosis of benign hyperplasia; for example, B-cell markers CD20 and CD10, or Bcl-6 for follicle centers, and CD3 for the T cells of the paracortex. A diffuse, uniform cellular composition is the hallmark of a neoplastic process. Various combinations of IHC markers can be used to confirm the diagnosis, for example, expression of one or more B-cell markers with only a small minority of interspersed T cells. Abnormal coexpression of certain markers, such as CD43 and/or CD5, or demonstration of immunoglobulin light-chain restriction can add certainty to the diagnosis and assist further in proper classification of the lymphoma (Fig. 6-3). Even with such evidence, caution must be exercised, because rare benign conditions have been identified with light-chain restriction and CD43 coexpression.90
When select compartments are disproportionately expanded, it raises the question of the early appearance of a particular form of low-grade lymphoma. Such cases can be extremely difficult to diagnose. For example, follicular lymphoma versus follicular hyperplasia is perhaps the commonest and most recognized conundrum within this grouping.91 When even meticulous evaluation of conventionally stained sections fails to resolve this issue, IHC for Bcl-2 protein and for Ki-67 is almost always conclusive (Fig. 6-4). As is always the case, the interpretation of such IHC studies requires experience and skill. It is not a matter of just positive or negative staining; rather it presents a complex pattern-recognition challenge (Fig. 6-5). In rare cases, even further study is warranted, such as polymerase chain reaction (PCR) for clonality testing. A related differential diagnosis addresses the sometimes subtle distinction between follicular hyperplasia with the variant features of progressively transformed germinal centers and the early appearance of NLPHL. Here, IHC is the definitive ancillary method. CD20 and Bcl-6 highlight scattered individual tumor cells against a background of small mantle-zone B-lymphocytes, and collarettes of small T cells surround the tumor cells (see Fig. 6-5).92 The IHC studies indicated depend on the differential diagnosis (Table 6-1). For example, when medullary cord regions of a lymph node are selectively expanded by a plasmacytoid cellular proliferation, staining for κ and λ light chains is likely to be informative. In situ hybridization (ISH) for light-chain mRNA has become a widely popular substitute for IHC, because this method eliminates the problem of background plasma immunoglobulin as a visually interfering phenomenon (Fig. 6-6). The recognition of the earliest involvement of sampled lymphoid tissues by marginal zone lymphoma (MZL) or chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) can be almost impossible without powerful ancillary studies. Sometimes the effort is triggered by detection of a small population of light-chain restricted cells in flow cytometry or by suspicious clinical findings. Indeed, the distinction of these two processes can itself be difficult.93 Coexpression of CD5 and CD23 weighs strongly in favor of CLL/SLL (see Table 6-1). Because MZL has essentially no disease-specific markers, in comparison with other types of low-grade B-cell lymphoma, its diagnosis is often based on exclusion of alternative types. In some cases of very subtle, early nodal involvement by MZL, only molecular probe determinations can detect the process.94
High-Grade Malignant Lymphoma Versus Acute Immune Hyperplasia Fortunately, only rarely do benign immune reactions simulate high-grade lymphoma, because when they do, it represents an emergency for clinical management. The histologic hallmark of aggressive lymphomas is the predominance of large, sometimes bizarre-appearing cells with high proliferation. In the setting of normal host immunity, only a few processes can instigate benign
+
+
+
+
+
+
+
+
+
+
−
−
−
−
−
+/−
−/+
−
CLL/SLL
MCL
FL
HCL
LPL
EMZL
B-PLL
SMZL
NMZL
−
−
−
−
−
−/+
−
+
−
Cyclin D1
+
+
+
+
+
−
+/−
+
+
Bcl-2
−
−
−
−
−
−
+
−
−
Bcl-6
−
del 7q
−
Various
t(11;14)
BRAF
t(14;18)
t(11;14)
Various
Genetics
−
−
−
−
−
+
−
−
−
DBA.44
−
+/−
−/+
−
−
−
+/−
−/+
+
CD23
−
+
+
+/−
+ −
+
−
+
+
+
−
FMC7
−
−
+
−
−
−
CD25
−
+/−
−/+
−
−
+
−
−
−/+
CD11c
Additional Evaluation
−
−
−
−
−
−/+
+
−
−
CD10
−
−/+
−
−
−
+
−
−/+
−
TRAP
+
−/+
+/−
−/+
−/+
+
−
+
+
CD43
B-PLL, B-cell prolymphocytic leukemia; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; EMZL, extranodal marginal zone lymphoma; FL, follicular lymphoma; HCL, hairy cell leukemia; LPL, lymphoplasmacytic lymphoma; MCL, mantle cell lymphoma; NMZL, nodal marginal zone lymphoma; SMZL, splenic marginal zone lymphoma.
CD20
CD5
Diagnosis
Primary Evaluation
TABLE 6-1 Comparison of Immunohistochemical Features of Common B-Cell Lymphomas and Other Lymphoma Types
Establishing the Diagnosis of Lymphoma: Differential Diagnostic Considerations
155
156
Immunohistology of Non-Hodgkin Lymphoma
H&E
CD20
CD3
CD43
Figure 6-3 Atypical small lymphocytic infiltrate in gastric biopsy is shown to be strongly positive for B-cell marker CD20. Only scattered T cells stain for CD3, whereas strong coexpression of CD43 is apparent. In this setting, this finding is considered strong evidence of an aberrant immunophenotype, in this case most consistent with extranodal marginal zone (mucosal-associated lymphoid tissue [MALT]) lymphoma. H&E, Hematoxylin and eosin.
mimics of this type. Viral infections, in particular those of the Herpesviridae family, can induce an intense immune proliferation that features expanses of large immunoblastic cells. Primary EBV infection (infectious mononucleosis) is the classic example, most often encountered in tonsillectomy specimens. Detection of this virus with IHC for EBV latent membrane protein (LMP) or with ISH for Epstein-Barr encoded RNA (EBER) is effectively conclusive (Fig. 6-7).95 Herpes simplex, human herpesvirus-6 (HHV-6), and varicella viruses can also less commonly be the culprit for mimicry of high-grade lymphoma.96 In the setting of abnormal host immunity, a number of processes, some yet only poorly understood, can microscopically mimic high-grade lymphoma. EBV-driven B-cell proliferations in the setting of immunosuppression can mimic either high-grade B-cell non-Hodgkin lymphoma or CHL.97,98 Zones of necrosis and plasma-cellular maturation are features that may tip off the wary pathologist as to the
benign nature of such cases; however, clinical history is the most essential means to avoid an erroneous diagnosis of lymphoma. Highly atypical expanses of T-cell proliferation are seen in Kikuchi-Fujimoto histiocytic necrotizing lymphadenitis. The demonstration of a CD8-positive population of large T-cell immunoblasts in combination with histiocytes in the absence of neutrophils is the key to recognizing this mysterious entity, which may be a relatively mild, self-limited autoimmune disorder.99 The autoimmune lymphoproliferative disorder is a congenital disorder related to the absence of the cellular Fas receptor, which permits T cells to escape normal apoptotic destruction. The resulting accumulation of these T cells in both peripheral blood and lymphoid tissues produces a microscopic appearance that resembles a peripheral T-cell lymphoma; however, these cells are negative for both CD4 and CD8, but they do stain for pan–T-cell markers, such as CD3 and CD2.100
Immunohistochemical Evaluation of Small B-Cell Lymphomas
BCL-2
BCL-2
Ki-67
Ki-67
157
Figure 6-4 A comparison of patterns of immunohistochemistry (IHC) staining in benign follicular hyperplasia (left) and follicular lymphoma (right). The neoplastic follicles are positive for Bcl-2 protein and show a low labeling for Ki-67 with poor delineation of the follicles in this stain. Pattern recognition is critical for the interpretation of IHC in lymphoid proliferations.
Cases that leave no doubt as to their neoplastic nature may yet pose a challenge to the pathologist with a different form of mimicry—that of other forms of malignancy that can be mistaken for lymphoma. This differential diagnostic grouping is the subject of the final section in this chapter.
Immunohistochemical Evaluation of Small B-Cell Lymphomas In most circumstances, initial evaluation of lymph nodes can determine whether the histologic and architectural pattern represents general categories of lymphoid processes. In general, these can be divided into groups that
include reactive processes, small B-cell lymphomas, large-cell lymphoma, Hodgkin lymphoma, or other considerations. This categorization is simplified, because there may be circumstances in which an overlap between these groups must be considered. In this section, an overview of small B-cell lymphomas, predominantly with indolent clinical behavior, is presented; these account for approximately 40% of all B-cell lymphoma.101 The most common lymphomas considered in this evaluation include follicular lymphoma (FL), grades 1 and 2; CLL/SLL; MZL; and MCL (see Table 6-1). Other, more rare, subtypes of small B-cell lymphoma include hairy cell leukemia, lymphoplasmacytic lymphoma (LPL), and plasma cell disorders; these will be covered in the latter portion of this section.
158
Immunohistology of Non-Hodgkin Lymphoma
CD20 Benign PTGC
CD20 Early NLPHL
Figure 6-5 A comparison of staining for CD20 in benign follicular hyperplasia (left) compared with the early appearance of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL, right). The separation of the large positive tumor cells from the surrounding small B cells allows the diagnosis. Pattern recognition is critical for this interpretation. PTGC, progressively transformed germinal center.
The diagnosis of lymphoma should be arrived at by a combination of findings that include clinical, cytologic, histologic, genetic, and other laboratory results. In many cases, the combination of clinical and histologic features are distinctive enough to make a diagnosis; however, immunophenotype may occasionally reveal unusual antigen expression, uncover important clinical or clinicopathologic features, or add clarity to a difficult diagnostic problem.
kappa
General Immunohistochemical Approach to Diagnosis of Small B-Cell Lymphoid Lesions Histologic findings are the cornerstone of diagnosis of small B-cell lymphoid proliferations. However, because of considerable overlap between entities and small samples with limited architectural features, a panel of
lambda
Figure 6-6 A dense plasmacytic infiltrate of the skin shows selective cytoplasmic staining for λ light chain with focal Golgi concentration. Because pericellular staining and staining for κ light chain indicates plasma immunoglobulin, this is a difficult interpretation that requires the skill of a pathologist with extensive experience in the application of this method.
Immunohistochemical Evaluation of Small B-Cell Lymphomas
159
EBV LMP
A
B
C
Figure 6-7 Acute primary Epstein-Barr virus (EBV) infection of the tonsil microscopically mimics an aggressive lymphoma. A, Low magnification shows an expansive, diffuse cellular proliferation. B, High magnification shows large, bizarre immunoblastic cells. C, Immunohistochemistry for EBV-latent membrane protein (LMP) demonstrates strong staining in scattered large, atypical cells.
IHC stains is of considerable benefit in the evaluation of lymphoid lesions. Foremost is evaluation of CD20 and CD3. These stains allow identification of amount and distribution of B and T cells in almost all cases. In cases of small B-cell lymphomas, CD20 will be markedly increased and will likely label much of the tissue present. In partial involvement or unusual cases, CD3-positive T cells may be more numerous. Staining for CD5 allows identification of abnormal CD5-coexpressing B cells and also acts as a secondary pan–T-cell antigen, in case of a possible T-cell neoplasm. The combination of Bcl-2 and Bcl-6 staining adds several benefits. Most classically, distinction of reactive from neoplastic follicles can be determined, with normal follicles positive for Bcl-6 and negative for Bcl-2, whereas the majority of abnormal follicles of low-grade FL are positive for Bcl-6 and Bcl-2. Cyclin D1 is another critical stain and is primarily used to identify or exclude MCL. CD43 can be of benefit in evaluation of small B-cell lymphomas. Normal B cells do not express CD43. However, many small B-cell lymphomas are positive for CD43, including most cases of CLL/SLL and MCL. Approximately 20% to 30% of MZLs are positive for CD43, and approximately 50% of LPLs are positive. CD43 can also be useful in the differential diagnosis of lymphoma types: first, FLs are almost never positive; and second, if the differential diagnosis includes CLL/ SLL or MCL, a negative result would favor other diagnoses, such as MZL or FL. In the authors’ experience, CD10 is only of limited benefit and is better replaced by Bcl-6 in most cases. In addition, CD23 expression is also considered of limited benefit. Although its primary use is to distinguish CLL/ SLL from MCL, there are subsets of cases of each type
that show aberrant expression patterns, which reduces the utility of this stain. The expression or lack of cyclin D1 would trump the results of CD23 expression in any case. In addition, variable expression of CD23 is seen in other small B-cell lymphomas (FL, MZL), which does not add diagnostic clarity but rather adds another potentially confusing result. The use of light-chain staining for κ and λ in small B-cell lymphomas is controversial. In most cases, if evidence of lymphoma is clear, demonstration of light-chain restriction is unnecessary. If no plasma cell differentiation is evident, light-chain expression and interpretation is positive only in a limited number of cases by using the standard IHC staining in most laboratories. It can be useful in cases with plasma cell differentiation because it can demonstrate the presence or absence of light-chain restriction in such a population. Finally, Ki-67 can be quite useful in the evaluation of lymphoid lesions, including small B-cell lymphomas.91 The overall pattern of staining can be instructive and quite distinctive for a specific diagnosis. The low proliferation seen in follicles is characteristic of lowgrade FL; it can even be used when abnormal Bcl-2 expression is lacking. Furthermore, it can be used to highlight the follicular colonization in MZL and the proliferation centers of CLL/SLL.
General Prognostic and Therapeutic Issues Prognosis and therapy in small B-cell lymphomas is typically well defined. In most cases, the prognosis is relatively good, with indolent disease and a gradual increased risk over time of transformation to a more aggressive disease. Histologic evidence of transformation to a large
160
Immunohistology of Non-Hodgkin Lymphoma
B-cell lymphoma, a blastic B-cell malignancy, or other histologic transformations is associated with more aggressive disease and a poor outcome. In general, genetic abnormalities, except as defining the disorder, are not specifically associated with outcome. However, certain general findings do apply. Abnor malities associated with p53 tumor suppressor gene (deletion at 17p13) have been associated with a poor outcome. Likewise, a complex karyotype beyond the typical genetic abnormalities implies genetic instability in a lymphoma and can portend a stormy clinical course. For almost all B-cell lymphoma patients, rituximab, an anti-CD20 humanized monoclonal antibody, is presently included as part of their therapy. This therapeutic option is based on the almost universal expression of CD20 by mature B-cell malignancies. As a secondary agent, or in rare cases as a primary agent, anti-CD22 therapies such as epratuzumab and inotuzumab ozogamicin have also been used. Although not in general use at present, anti–Bcl-2 therapies—such as oblimersen, an antisense Bcl-2 oligodeoxynucleotide—have been used in some trials and may find use as primary or salvage therapies for CLL/SLL and other lymphoma types.
Follicular Lymphoma Follicular lymphoma (FL) is a distinctive B-cell lymphoma of abnormal cells of follicular origin. In cases of low-grade FL (grades 1 and 2), an architectural arrangement of abnormal follicular structures is typically present. However, in small samples, the characteristic architecture may not be appreciated. In exceptional cases, a partly or predominantly diffuse pattern may be apparent in otherwise unremarkable FL. The immunophenotype is distinctive and helpful in the diagnosis. The lymphoma cells express pan–B-cell antigens (CD19, CD20, CD22, Pax-5) and germinal center–associated antigens such as Bcl-6. It should be noted that CD10 can also be seen in benign and malignant germinal centers, although the loss of CD10 expression in the neoplastic cells of FL is not uncommon. Other stains associated with the germinal center, with high degrees of sensitivity and specificity, include human germinal center–associated lymphoma (HGAL) and germinal center B-cell–expressed transcript 1 (GCET-1). HGAL may be of benefit in identifying FL in bone marrow samples, because it retains more robust staining compared with Bcl-6 in decalcified marrow samples. The vast majority of low-grade FL is positive for Bcl-2, and this expression is most often due to the presence of the t(14;18) (IGH/BCL2) translocation with overexpression of Bcl-2. Proliferation rate in low-grade FL will be less than 5% to 25% within follicles. Similar to normal follicles, follicular dendritic cells (FDCs) will be highlighted by CD21 staining. FDC cells will also be variably positive for CD23, and the FL cells may also express CD23. FL lacks expression of cyclin D1, CD5, and CD43, and it only rarely expresses cytoplasmic light chains by IHC or in situ staining with clear evidence of monoclonality. Atypical immunophenotypic findings in low-grade FL include expression of CD5 or CD43 (rare cases; less
than 1% to 2%) or an increased proliferation rate with an apparent low-grade histology. These latter cases may have a more aggressive clinical course than usual lowgrade FL, and a diagnosis of FL grade 1-2/3 with increased proliferation rate should be noted.102 PROGNOSTIC AND THERAPEUTIC STUDIES
Follicular lymphoma international prognostic index (FLIPI) has been shown to strongly correlate with prognosis in FL. Histologic progression in FL can occur in 5% to 60% of cases and is associated with a poor prognosis.101 These include genetic instability with secondary genetic and molecular events, associated with a poor prognosis, including chromosomal gains of 7, 12q13-14, and 18q and chromosomal losses del6q, 9p21, and 17p13 by array comparative genomic hybridization (aCGH).101 Poor prognosis is associated with gains of 7, 12q13-14, and 18q (oncogenes or dosage effects) as well, and transformation of FL to large cell lymphoma is of critical clinical significance in overall prognosis.101 PITFALLS
Potential pitfalls in FL arise from some inconsistent IHC findings as well as variations with different grades of FL. It is important to be advised that not all FL will be Bcl-2 protein–expression positive. These will typically have other standard features of FL, including an abnormal low-proliferation rate by Ki-67. IgH PCR can be negative in a significant number of cases of FL. In particular, FL grade 3 can be challenging, because it is more commonly Bcl-2 negative and will have a high proliferation rate, comparable to hyperplastic processes.
Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a relatively indolent B-cell lymphoma that occurs with frequent bone marrow and peripheral blood involvement. The appearance of CLL/SLL cells is that of small round lymphocytes with dense mature chromatin and scant cytoplasm. Almost always, a subset of cells is seen with large round nuclei, prominent nucleoli, and increased amounts of cytoplasm (prolymphocytes or paraimmunoblasts). Mitotic figures are very rare. Larger cells will sometimes cluster and form vague nodules known as proliferation centers or pseudofollicles. The typical immunophenotype for CLL/SLL includes expression of CD5, CD23, CD19, CD43, and Bcl-2. Most cases express CD20, although weakly, and focal expression may be seen in a subset of cases. CLL/SLL most typically has a proliferation rate of less than 10%, although increased proliferation may be seen in pseudofollicles/proliferation centers by Ki-67 staining. CLL/SLL lacks expression of CD10, cyclin D1, and only rarely express cytoplasmic light chains by IHC or in situ staining with clear evidence of monoclonality. Typically, no appreciable FDC networks are apparent, unless preserved normal follicles are present.
Immunohistochemical Evaluation of Small B-Cell Lymphomas
Atypical immunophenotypic findings in CLL/SLL include lack of staining for CD5 or CD23, focal expression of cyclin D1, and, rarely, evidence of plasma cell differentiation. CLL/SLL without CD5 expression is estimated to be present in approximately 2% of cases.103 It should be noted that other CD5-negative lymphomas should be ruled out carefully before diagnosing CD5negative CLL/SLL. Lack of CD23 expression is rare. In this circumstance, because of the close overlap with the immunophenotype of MCL, it is appropriate to exclude MCL carefully, by either cyclin D1 staining or genetic studies for t(11;14) of MCL. Likewise, CD5-positive MZL may not express CD23 and should be considered in the differential diagnosis. Focal expression of cyclin D1 has been noted in CLL/SLL. Typically seen in proliferation centers, it is a rare occurrence that has no specific impact on diagnosis. Likewise, these cases will not have evidence of t(11;14) (IGH/CCND1) of MCL. PROGNOSTIC AND THERAPEUTIC STUDIES
Immunoglobulin heavy-chain variable region (IGHV) mutation status has been shown to be associated with prognosis in CLL. Cases with unmutated heavy-chain variable gene regions are associated with a poor prognosis compared with those with mutated IGHV genes. CD38 expression correlates partly with IGVH status and prognosis but may be independent of IGHV in some cases. IHC or flow cytometric staining for ζchain–associated protein kinase 70 (ZAP-70), a tyrosine kinase that plays a role in signal transduction, has been shown to correlate with prognosis in CLL/SLL.103 ZAP-70 expression by both flow cytometry and IHC has been shown to correlate with IGHV mutation status and with prognosis. Overexpression of p53 by ICH suggests an aggressive clinical course. Poor-risk cytogenetics include 17p deletions (5% to 8%), 11q deletions (20%), trisomy 12 (15%), complex karyotype, mutated IGHV genes, and expression of CD38.101,104,105 NOTCH1 mutations (10%) are associated with a poor prognosis, and deletion 17p is associated with rapid disease progression, drug resistance, and overall short survival.105 One rare finding is t(14;19),
A
161
which is associated with a relatively aggressive clinical course. Deletion 13q14, no expression of ZAP-70, CD38 negativity, and mutated IGHV are genetic and laboratory findings associated with a good prognosis.105 Splicing factor 3b subunit 1 (SF3B1) is associated with fludaribine-refractory disease in some cases.105 PITFALLS
Pitfalls in the diagnosis of CLL/SLL are mostly due to variations in immunophenotype, as mentioned above. It should be recognized that variation in CD5, CD23, CD20, and Bcl-2 may be seen in individual cases. Likewise, focal staining for cyclin D1 and some cases with high proliferation by Ki-67 can be seen (Fig. 6-8). In all cases, exclusion of other lymphoma types such as MCL, CD5-positive MZL, CD5-positive LPL, and other rare lymphoma types is prudent.
Mantle Cell Lymphoma Of the predominantly small B-cell lymphomas, MCL is distinctive. Although exceptions do exist, in most cases MCL has a decidedly poor prognosis despite more aggressive therapeutic approaches. Histologic patterns of diffuse, nodular, and mantle zone types are described. In conventional cases, the cytologic appearance is that of small lymphocytes with slightly irregular nuclei; dense, mature chromatin; and scant cytoplasm. Notably, the cells typically lack nucleoli, and in most cases, large lymphocytes are rare. In the background, bland histiocytes with round or ovoid nuclei and ample pale or pink cytoplasm are seen. Occasional mitotic figures may be seen. MCL expresses pan–B-cell antigens (CD19, CD20, and CD22), CD5, CD43, Bcl-2, and cyclin D1 (Fig. 6-9). The cornerstone of diagnosis of MCL, cyclin D1 staining, is seen in almost all abnormal cells; variation in intensity of this nuclear stain is typical in conventional MCL, with more uniform strong staining seen in the blastoid variant (see below). MCL lacks staining for CD10, Bcl-6, and CD23 and only rarely expresses clear evidence of monoclonality by using cytoplasmic light chains by IHC or in situ staining. No FDC networks are seen with CD21 staining in diffuse cases, although the
B
Figure 6-8 A, Hematoxylin and eosin stain of proliferation center in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). B, Cyclin D1 staining in proliferation center of CLL/SLL is a rare finding, but the staining is not typical for mantle cell lymphoma (MCL), and these cases do not have evidence of the (11;14) translocation characteristic of MCL.
162
Immunohistology of Non-Hodgkin Lymphoma
A
B
Figure 6-9 A, Typical histology of mantle cell lymphoma (MCL). B, Cyclin-D1 staining is shown. Note that although positive in the majority of cells, staining is of varying intensity. This is a usual finding in typical cases of MCL. Compare with the focal staining seen in rare cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (see Fig. 6-8).
nodular or mantle zone patterns may have residual FDC networks in follicles. Atypical IHC findings include coexpression of CD10 (very rare), and CD23 can be seen in as many as 25% of cases, with mostly focal and/or weak staining.106 Lack of CD5 expression may be seen in very rare cases. Literature reports of “cyclin D1–negative MCL”107,108 are thankfully rare (<1%); these will often coexpress cyclins D2, D3, or E, and they have a histologic appearance and clinical course comparable to that of typical MCL. Most of these cases are SOX11 positive, which can be helpful in their diagnosis. Bcl-6 expression can be seen in approximately 12% of cases of MCL, and MUM1 expression is seen in 35%.106 PROGNOSTIC AND THERAPEUTIC STUDIES
The proliferation rate in MCL by Ki-67 is often less than 30%, and cases with more than 30% are associated with a worse prognosis; in addition, p53 overexpression, as a surrogate for loss of p53 tumor suppressor activity, is associated with poor prognosis in MCL. SOX11 staining in MCL lymphoma has been suggested as a prognostic marker, but this is controversial. Although most indolent MCL cases are SOX11 positive, this marker does not adequately distinguish between indolent and aggressive cases.109 PITFALLS
Not all cyclin D1–positive neoplasms are MCL. A subset of plasma cell neoplasms is cyclin D1 positive, and some are positive for t(11;14). Hairy cell leukemia lacks t(11;14) but has weak cyclin D1 expression in most cases. A small subset of diffuse large B-cell lymphoma (DLBCL) expresses cyclin D1, but in the absence of t(11;14). Although most cases of MCL are associated with aggressive clinical behavior, a small subset has indolent behavior. The basis for recognition of these cases has not been established. Cases of in situ MCL have also been reported, but these have only limited lymph node involvement and are confined to mantle
zones, which are not always associated with progressive disease.110
Lymphoplasmacytic Lymphoma Lymphoplasmacytic lymphoma (LPL) is a rare and indolent B-cell lymphoma with proliferations of small lymphocytes, lymphoplasmacytic cells, and plasma cells in varying proportion. In almost all cases, bone marrow and peripheral blood involvement is apparent with varying degrees of involvement of spleen and lymph nodes.111 The morphologic and immunophenotypic distinction of LPL from marginal zone lymphoma (both nodal and extranodal) can be difficult. When associated with a monoclonal protein of the IgM type, the preferred diagnosis is Waldenström macroglobulinemia (WM)/LPL. The immunophenotype of LPL is not distinctive; it will express pan–B-cell antigens CD19, CD20, CD79a, and Pax-5, and Bcl-2 is positive in almost all cases. Expression of IgM by neoplastic lymphoid and plasma cells is often seen. Both plasma cells and lymphoplasmacytic cells will express plasma-cell–associated antigens CD38, VS38c, and CD138 with monoclonal expression of light chains. Rare cases are positive for CD5 (17% to 43%), some are positive for CD10 (16%), and many are positive for CD23 (58%).112 LPL does not express cyclin D1 or Bcl-6, but most cases of LPL are positive for CD25. Although not entirely specific, an increase in mast cells has been noted in WM/LPL. Methods to highlight increases in mast cells, including CD117 and/or tryptase staining, could provide diagnostic support in difficult cases. PROGNOSTIC AND THERAPEUTIC STUDIES
No specific IHC findings are associated with prognosis in LPL, although a variety of genetic findings can be seen. Cases with l6q deletions have been associated with a worse prognosis.112-114 Gains in 4q and 8q occur in 12% and 10% of WM/LPL and are not commonly reported in MZL.
Immunohistochemical Evaluation of Small B-Cell Lymphomas
PITFALLS
The primary pitfall in management of LPL and WM/ LPL is the clear diagnosis of these entities, which can be difficult; correlation of clinical, histologic, immunophenotypic, and genetic information is critical.
Nodal Marginal Zone Lymphoma Nodal marginal zone lymphoma (NMZL) is a low-grade B-cell lymphoma that is predominantly composed of small, mature-appearing B cells.113,115 It is expected to have predominantly lymph node involvement, to distinguish it from extranodal marginal zone lymphoma (covered below); it does not have prominent splenic involvement, because this would suggest a diagnosis of splenic marginal zone lymphoma (see below). NMZL may have evidence of follicular colonization, a marginal zone pattern, monocytoid B-cell differentiation, and plasma cell differentiation, although these features are variable in individual cases. The immunophenotypic findings in NMZL are not entirely specific and are mostly derived from a lack of staining seen in other specific types. Distinction from lymphoplasmacytic lymphoma is difficult.115,116 NMZL will typically express pan–B-cell antigens that include CD19, CD20, Pax-5, and CD79a; expression of Bcl-2 is seen in almost all cases, and CD43 coexpression by B cells is seen in 50% of cases.117 Proliferation assessment by Ki-67 is generally low (<10%); if it is greater than 30% to 40%, this would raise the possibility of a variant of DLBCL. NMZL lacks expression of CD5, CD10, Bcl-6, and cyclin D1 in contrast to cases of MCL, CLL/SLL, and follicular lymphoma. Rare cases may express CD5, but without a careful correlation of clinical, histologic, and genetic features, distinction from CLL/SLL can be difficult.115 Pediatric NMZL is a distinctive variant associated with young boys (2 to 18 years).118 These cases are almost always stage I at presentation, are seen in the head and neck, and have an excellent prognosis, even with conservative management. Although trisomies of some chromosomes can be identified, none of the translocations seen in extranodal marginal zone lymphoma (EMZL) are seen in NMZL. Genetic findings include trisomies 3, 7, 12, and 18; structural rearrangement of chromosome 1; and gains in several regions of chromosome 3.117 The presence of translocations specific for other lymphoma types would preclude a diagnosis of NMZL. PROGNOSTIC AND THERAPEUTIC STUDIES
No specific pathologic testing to predict prognosis is known for NMZL. Features that are prognostic include age and stage of disease. The FLIPI has been shown to strongly correlate with prognosis in NMZL.117
Extranodal Marginal Zone Lymphoma Extranodal marginal zone lymphoma (EMZL) is an indolent lymphoma with variable morphology found in diverse sites. These arise at a number of extranodal sites
163
that include stomach, lung, small intestine, salivary gland, thyroid, skin, and other sites. EMZL recapitulates some features of mucosal-associated lymphoid tissue (MALT) and are often referred to as MALT lymphomas. EMZL often arises in a background of chronic antigenic stimulation.119 As such, the presence of reactive germinal centers and polyclonal plasmacytosis may be seen in association with EMZL. A distinctive feature of MZL is follicular colonization characterized by invasion of reactive germinal centers by neoplastic lymphoid cells. The cellular composition of EMZL is predominantly small lymphocytes that will often have increased amounts of pale cytoplasm (e.g., monocytoid B-cell appearance). Larger, admixed, transformed lymphocytes typically number less than 30% of the overall cellularity. In addition, varying degrees of plasma cell differentiation may be present; Dutcher bodies and Russell bodies may be seen in these plasma cells.115 When extensive, the degree of plasma cell differentiation may raise the possibility of plasmacytoma. It should be noted that some authors believe that many extramedullary plasmacytomas are simply localized manifestations of EMZL with extensive plasma cell differentiation. The immunophenotype of EMZL is often based on exclusion of other lymphoma types. The neoplastic lymphocytes express pan–B-cell antigens (CD20, CD79a, and Pax-5), and are positive for Bcl-2 in the vast majority of cases. They have a low proliferation rate (<10% in most cases) by Ki-67 and lack expression of CD10, cyclin D1, and Bcl-6. Rare cases (<5%) will express CD5 and tend to be in nongastric sites.120 Follicular colonization can be highlighted by use of BCL-6 staining or Ki-67, highlighting follicles (Bcl-6 positive, Ki-67 high) disrupted by lymphoma cells (Bcl-6 negative, Ki-67 low; Fig. 6-10). EMZL can be difficult to distinguish from LPL, in which increased mast cells, marrow and peripheral blood involvement, and large amounts of serum IgM are more common and may be helpful in distinguishing the two. In addition, clear evidence of CD20- and Pax-5– positive plasma cells or lymphoplasmacytic cells would favor a diagnosis of LPL. A broad range of genetic abnormalities, including trisomies, and a variety of translocations are seen in MZLs (Table 6-2). These have different frequencies depending on the sites.121,122 The presence of t(11;18) (API2/MALT1) or t(1;14)(BCL10/IGH) are seen in gastric EMZL, and these cases tend not to respond to Helicobacter pylori eradication therapy.123 Trisomies of chromosome 3 and 18 are also seen frequently in EMZL, but these are not entirely specific to EMZL. Lymphoepithelial lesions are prominent in many EMZLs because of homing present in the neoplastic cells to epithelial structures. The use of pankeratin stains, such as AE1/AE3, may be useful in highlighting the lymphoepithelial lesions of EMZL (Fig. 6-11). H. pylori has been linked to the development of EMZL in the stomach. It serves as the initial and sustaining antigenic event in both the reactive lymphoid proliferation and subsequent proliferation of lymphoma cells. This critical pathophysiologic mechanism in a number of the cases is also critical, because a large
164
Immunohistology of Non-Hodgkin Lymphoma
A
B
C
D
E
F
G
H
Figure 6-10 Follicular colonization in marginal zone lymphoma (MZL). With histologic image (A), Bcl-2 staining (B), Bcl-6 staining (C), and Ki-67 (D). Contrast this to a normal follicle from the same case: histology (E), Bcl-2 (F), Bcl-6 (G), and Ki-67 (H). In the MZL, disruption is evident in the follicular structure by abnormal lymphocytes, which are positive for Bcl-2, negative for Bcl-6, and have a low proliferation rate by Ki-67. Compared with the normal follicle, these cells stand out, and the immunohistochemical staining highlights their presence among the residual reactive follicle-center cells.
Immunohistochemical Evaluation of Small B-Cell Lymphomas
165
TABLE 6-2 Cytogenetic Alterations, Frequencies, and Etiologic Associations in Extranodal Marginal Zone Lymphoma Anatomic Site
Infectious Agent
Translocation
Gene
Frequency
Stomach
Helicobacter pylori
t(11;18)(q21;q21) t(1;14)(p22;q32)
API2-MALT1 BCL10
22% 3%
Lung
−
t(11;18)(q21;q21) t(1;14)(p22;q32)
API2-MALT1 BCL10
42% 7%
Intestine
Campylobacter jejuni*
t(11;18)(q21;q21) t(1;14)(p22;q32)
API2-MALT1 BCL10
15% 10%
Ocular adnexa
Chlamydia psittaci*
t(3;14) (p14.1;q32) t(14;18)(q32;q21)
FOXP1 MALT1
20% 13%
Skin
Borrelia burgdorferi*
t(14;18)(q32;q21) t(3;14) (p14.1;q32)
MALT1 FOXP1
14% 10%
Salivary gland
Autoimmune?
t(14;18)(q32;q21)
MALT1
5%
Thyroid
Autoimmune?
t(3;14) (p14.1;q32)
FOXP1
50%
From Gascoyne RD: Hematopathology approaches to diagnosis and prognosis of indolent B-cell lymphomas. Hematology Am Soc Hematol Educ Program. 2005:299–306. *Evidence supportive of a definitive role for these organisms is lacking.
portion of these lymphomas will respond favorably to H. pylori eradication therapy. The literature has stated that IHC staining for H. pylori is not necessary in routine cases (Fig. 6-12).124 However, given the prevalence and relative impact on diagnostic and prognostic decisions, the use of the IHC stains for H. pylori in cases with a suspicion of lymphoma is warranted.
Hairy Cell Leukemia Hairy cell leukemia (HCL) is a rare, low-grade B-cell lymphoma with prominent involvement of spleen, bone marrow, and peripheral blood. It only rarely involves lymph nodes, most commonly in the splenic hilum or intraabdominal region. The morphology is of small to intermediate-sized lymphocytes with round nuclei and moderate to large amounts of pale cytoplasm. In peripheral blood, the abnormal lymphocytes will have delicate, hairlike projections that give the disorder its name. In
A
histologic preparations, HCL will often have a round, centrally placed nucleus with a rim of pale or clear cytoplasm; this has been referred to as a “fried egg” appearance traditionally. In most circumstances, HCL is identified by flow cytometry of either blood or bone marrow samples. The flow cytometric immunophenotype is distinctive and shows expression of pan–B-cell antigens, which includes CD20 (bright) and CD19, with restricted light-chain expression. HCL will also have a characteristic pattern of expression of CD103, CD11c, CD22, and CD25. FMC7 is positive, as is CD123. Most cases lack CD5 and CD10 expression, although rare cases have been reported that show the presence of either or both. Primary diagnostic IHC studies of HCL in tissue include expression of tartrate-resistant acid phosphatase (TRAP) and DBA.44 (Fig. 6-13). Annexin-A1 is positive in almost all cases but can be difficult to interpret as a result of extensive normal staining of
B
Figure 6-11 Infiltration of gastric submucosa by extranodal marginal zone lymphoma (A). The gastric glands have lymphoepithelial lesions, which are highlighted by negative-staining lymphocytes within glands stained by pankeratin (AE1/AE3; B).
166
Immunohistology of Non-Hodgkin Lymphoma
A
B
Figure 6-12 Gastric mucosa with mild acute gastritis (A) and immunohistochemical (IHC) staining for Helicobacter pylori (B). Although H. pylori can be visualized by hematoxylin and eosin or special stains (Giemsa, etc.), IHC staining is accurate, fast, and highly reproducible.
background elements. Cyclin D1 is positive in a large percentage of cases of HCL and can be useful in discriminating between HCL and other lymphoma types; it stains less intensely and less uniformly than in cases of MCL.125 Expression of the protein encoded by T-cell– specific T-box transcription factor (T-bet) lacks sensitivity and specificity, but if positive, this could support a diagnosis of HCL. No specific cytogenetic abnormalities have been associated with HCL. Recently, it has been noted that most cases of HCL will harbor a BRAF V600E mutation.126 Although not necessary for primary diagnosis of HCL, it may be of benefit in cases with a differential diagnosis of similar or related entities, such as splenic marginal zone lymphoma (SMZL) and HCL variant or splenic, diffuse red-pulp small B-cell lymphoma.
A
C
Splenic Marginal Zone Lymphoma Splenic marginal zone lymphoma (SMZL) is uncommon and accounts for less than 1% of all non-Hodgkin lymphoma (NHL). It is an indolent lymphoma of small B cells that prominently involves splenic white pulp, bone marrow, and peripheral blood. It is purported to arise from cells of the splenic marginal zone. The morphology has features of MZL in other sites that include monocytoid differentiation, follicular colonization, plasma cell differentiation, and admixed large cells. Immunophenotype in SMZL includes expression of pan–B-cell antigens that include CD20, Pax-5, and CD19. Generally, SMZL lacks expression of CD5, CD10, CD23, cyclin D1, CD43, and CD123. Some cases show TRAP expression but will lack expression of
B
Figure 6-13 Extensive bone marrow infiltration by hairy cell leukemia (A) with granular cytoplasmic staining for tartrateresistant acid phosphatase (B) and granular cytoplasmic staining for DBA.44 (C).
Immunohistochemical Evaluation of Small B-Cell Lymphomas
DBA.44, in contrast to HCL. CD103 is positive by flow cytometry but is not presently available as for paraffin IHC. Most cases coexpress IgM and IgD, and Annexin-A1 is uniformly negative. CD5 expression can be seen in approximately 5% of cases, and Ki-67 staining will reveal a low proliferation rate; however, follicular colonization is highlighted by Bcl-6 or Ki-67 staining. Normal residual follicular structures will show staining for both Bcl-6 and Ki-67, with disruption of normal follicular architecture by SMZL cells that are negative for both markers. Cases with overexpression of p53 may be associated with an aggressive clinical course. Deletion 7q is a relatively common genetic abnormality in SMZL, but its frequency varies with the method of detection.122,127 The presence of 7q deletion makes diagnoses of CLL/SLL, MCL, HCL, and FL unlikely. However, other less well-defined splenic B-cell lymphomas (SDRPL, hairy cell leukemia variant; below) have 7q deletion rates comparable to SMZL, making distinction by that criterion alone impossible.127
Hairy Cell Leukemia Variant Hairy cell leukemia variant (HCL-V) is an exceedingly rare lymphoma with prominent splenic, bone marrow, and peripheral blood involvement.128 Although relatively indolent in clinical course, it is more aggressive than conventional HCL, and some cases may be aggressive and refractory to typical treatment. In peripheral blood, HCL-V cells will be intermediate to large in size with prominent nucleoli, and moderate to large amounts of pale cytoplasm are seen. Cytoplasmic projections may be apparent in peripheral blood smears. In tissue, the cells will be intermediate to large in size, although the nucleoli may be less apparent than in peripheral blood. As with conventional HCL, they will have a diffuse red-pulp pattern of involvement in the spleen. Most cases of HCL-V are identified by flow cytometry of peripheral blood or bone marrow. HCL-V will be positive for B-cell markers such as CD20, CD19, and Pax-5. They also express CD11c, CD22, CD103, DBA.44, and FMC7 with bright expression of immunoglobulin light chains and CD20.129,130 In contrast to most cases of conventional HCL, HCL-V lacks expression of cyclin D1, CD123, CD25, TRAP, and Annexin-A1.131 No specific genetic abnormalities are identified in HCL-V. At present, in contrast to conventional HCL, HCL-V does not show evidence of BRAF mutation.
Splenic Diffuse Red-Pulp Small B-Cell Lymphoma Splenic diffuse red-pulp small B-cell lymphoma (SDRPL) has only recently been identified as a distinctive entity.128 Traditionally, because of the overlapping features with SMZL, it has been considered a “red-pulp variant” of SMZL. Histologic and clinical findings are similar to those of SMZL; however, because of comparable peripheral blood and bone marrow findings, SDRPL can only be diagnosed by splenectomy, because
167
the diagnosis is based on the architectural pattern in spleen. IHC staining highlights SDRPL as a B-cell lymphoma (CD20 positive), lacking specific markers of other lymphoma types. Bcl-6, TRAP, CD10, CD23, Annexin-A1, and cyclin D1 are negative. DBA.44 is positive in most cases (88%), suggesting that this marker is not useful in distinguishing small B-cell lymphomas of the spleen.130 CD123 is reported as being negative in SDRPL.113 Most cases are positive for IgG (67%), and a minority are positive for IgD (27%). CD5 has been noted in rare cases; proliferation rate, as measured by Ki-67, is less than 10%; and CD43 is reported as negative.132 In addition, p53 is seen in a subset of cases (29%), but its overall impact on prognosis is not clear. SDRPL has similar frequency of 7q deletion to SMZL.
Plasma Cell Neoplasms Plasma cell disorders encompass a broad range of neoplasms of differing clinical aggressiveness.133 A number of types of lymphomas may have plasmacytic or plasmablastic differentiation and are covered in other sections. This section will cover manifestations of plasma cell myeloma (PCM) and extraosseous plasmacytomas. PCM is an aggressive disorder of post germinal center B-cell derivation with end differentiation into plasma cells. It is notable for patchy systemic distribution with prominent involvement in bone and bone marrow. The term plasmacytoma can be used in two ways: first, for a localized extraosseous tissue manifestation of PCM; second, for a single isolated mass without associated systemic disease. These two can be difficult to distinguish with only pathology findings. In this context, the term isolated plasmacytoma will be used to refer to the non–PCM-related type. Plasma cell differentiation by IHC can be evaluated by the presence of expression of CD79a, CD138, CD38, VS38c, and MUM1. Another hallmark is the strong expression of restricted cytoplasmic light chains (κ or λ) in almost all cases. Evaluation of heavy chains (IgA, IgG, IgM, IgD) may be of benefit in some cases but is not highly specific in a diagnostic or therapeutic context. In most cases, mature neoplastic plasma cells will not express pan–B-cell antigens CD20, Pax-5, or CD19, nor will they express CD45. Most cases of PCM (~70%) express CD56. Isolated plasmacytoma will typically not express CD56, p53, or cyclin D1. If any of these markers were present, it would be more suggestive of tissue involvement by PCM. A subset of PCM will express CD20. In some of these cases, there is concurrent presence of t(11;14) identical to that seen in MCL. PCM with t(11;14) will express cyclin D1, CD19, CD20, and Pax-5, along with expression of plasma cell markers CD138 and MUM1. Differentiation from MCL can be difficult, and in many cases, the presence of clinical features of myeloma may be necessary to make a conclusive diagnosis. Occasional cases of myeloma without t(11;14) will express cyclin D1; in a case with clear plasma cell differentiation, this marker can exclude other alternative diagnoses, such as that of LPL.
168
Immunohistology of Non-Hodgkin Lymphoma
KEY DIAGNOSTIC POINTS Small Cell Lymphoid Neoplasms • The lymphoma cells express pan–B-cell antigens (CD19, CD20, CD22, PAX-5). • The vast majority of low-grade follicular lymphoma (FL) is positive for Bcl-2, which is negative in reactive follicles. • Grade 3 FL is more commonly Bcl-2 negative and will have a high proliferation rate. • Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) includes expression of CD5, CD23, CD19, CD43, and Bcl-2 and has a proliferation rate of less than 10%. • CLL/SLL ZAP-70 expression by both flow cytometry and IHC has been shown to correlate with immunoglobulin heavy-chain variable region (IGHV) mutation status and with prognosis. • Mantle cell lymphoma (MCL) expresses pan–B-cell antigens (CD19, CD20, CD22), CD5, CD43, Bcl-2, and cyclin D1. • MCL lacks staining for CD10, Bcl-6, and CD23. • Proliferation rate in MCL by Ki-67 is often less than 30%; cases with more than 30% are associated with a worse prognosis. • Lymphoplasmacytic lymphoma will express pan–B-cell antigens CD19, CD20, CD79a, and PAX5. Bcl-2 is positive in almost all cases, and there is often expression of immunoglobulin M, CD38, VS38c, and CD138, with monoclonal expression of light chains. • Nodal marginal zone lymphoma (NMZL) will typically express pan–B-cell antigens that include CD19, CD20, PAX5, and CD79a; coexpression with Bcl-2 and CD43 is common and occurs in 50%. • NMZL will lack expression of CD5, CD10, Bcl-6, and cyclin D1. • Extranodal MZL diagnosis is often based on exclusion of other lymphoma types. The neoplastic lymphocytes express pan–B-cell antigens (CD20, CD79a, PAX5) and are positive for Bcl-2 in the vast majority of cases. • Hairy cell leukemia (HCL) is frequently identified by flow cytometry of either blood or bone marrow samples. • Splenic marginal zone lymphoma (MZL) has features of MZLs in other sites and lacks expression of CD5, CD10, CD23, cyclin D1, CD43, and CD123. • Plasma cell differentiation can be evaluated by the presence of expression of CD79a, CD138, CD38, VS38c, and MUM1.
Large B-Cell Lymphomas and Other Aggressive B-Cell Lymphomas The 2008 World Health Organization (WHO) classification includes at least 26 types of large B-cell lymphomas, which includes classification of clinicopathologic entities based on morphology, histologic features, IHC features, genetics, and combinations of the above.113 Sorting of this large category often requires careful correlation of a variety of clinical and pathologic findings. In some cases, therapeutic differences are not specific, but a number of subcategories are associated with differences in prognosis. IHC is a keystone in identifying many of the different types of large B-cell lymphomas. In almost all cases, primary identification is made by a histologic pattern along with the presence of pan–Bcell antigens, most often CD20. In the appropriate context, the combination of large cells in a diffuse pattern with clear expression of CD20 may be adequate for identification of diffuse large B-cell lymphoma (DLBCL). Subclassification of specific types is dependent on a variety of additional antibody combinations, which allow for specific subtyping. Modern lymphoma classification relies on the presence of subsets within the general category of DLBCL. In older systems, morphologic findings such as immunoblastic, centroblastic, or anaplastic features were noted. However, groupings based on morphologic findings alone lacked sufficient reproducibility to prognosticate in this large group of disorders. More recent classifications have taken gene expression array findings into consideration to group DLBCL into categories of germinal center–derived (GC), activated–B-cell type
(ABC), and a third group that encompasses primary mediastinal B-cell lymphoma.134,135 GC-type lymphomas are associated with a better prognosis by using current therapeutic regimens such as R-CHOP (cyclophosphamide, hydroxydaunorubicin, oncovorin, and prednisone). ABC types of lymphomas have a more aggressive clinical course and appear to be dependent on dysregulation of nuclear factor κ-B (NF-κB) pathways.135 Gene-expression arrays are not currently applied in routine diagnosis, and a number of IHC systems have been proposed to act as surrogates for gene-expression arrays. Although the Hans classifier was considered to be the benchmark for some time,136 subsequent studies have shown that it is lacking in the necessary sensitivity and specificity to accurately predict gene-expression array patterns.137 Other systems (tally classifier, Choi classifier) have been proposed as being more accurate (Fig. 6-14).138,139 In most cases, the goal is to evaluate the expression of a group of germinal center–associated markers in the lymphoma (CD10, Bcl-6, GCET1) and to compare those with the expression of non–germinal center markers (MUM1, FOXP1). Natkunam and colleagues proposed HGAL/GCET2 expression as another robust marker of GC derivation.140,141 In combination, DLBCL that are of germinal center origin have been shown to have an overall better prognosis in both the pre-rituximab and post-rituximab eras. Those DLBCL that are not germinal center derived have been shown to have an overall worse prognosis. Other markers of benefit in evaluation of DLBCL are Bcl-2, Ki-67, CD5, CD30, cyclin D1, and in situ staining for EBV (EBER). These add diagnostic, and in some
Large B-Cell Lymphomas and Other Aggressive B-Cell Lymphomas
169
HANS CLASSIFIER Germinal center type
Non-GC type
CD10
MUM1
Germinal center type
BCL-6
Non-GC type
A
TALLY CLASSIFIER GC
NGC
CD10 (+ or –) GCET1 (+ or –) Score (0, 1, 2)
MUM1 (+ or –) FOXP1 (+ or –) Score (0, 1, 2)
GC > NGC = GC NGC > GC = NGC
GC = NCG, then LMO2 > 30% = GC LMO2 < 30% = NGC
B Figure 6-14 Classification of diffuse large B-cell lymphoma. GC, germinal center; MUM, melanoma-associated antigen; NGC, non–germinal center.
cases prognostic, information to subclassify lymphomas in this group. For example, Bcl-2 expression is associated with a relatively poor prognosis in DLBCL. Although the tendency is to suggest that a high proliferation rate by Ki-67 is associated with a poor prognosis, this is controversial; some literature reports support a prognostic role, but others suggest no specific prognostic implications.
MYC in Aggressive B-Cell Lymphomas Because of its association with high proliferation, MYC has been of considerable interest in DLBCL. In most cases, the presence of an MYC translocation— identified by classic cytogenetics, fluorescence in situ hybridization (FISH), or other molecular studies—has been of benefit in evaluating lymphomas.142 The presence of MYC translocations is most often associated with a diagnosis of Burkitt lymphoma (BL). However, DLBCL may have MYC translocations as well (8% to 16%), as does a subset of DLBCL, which has features
intermediate between BL and DLBCL, so-called doublehit lymphomas (lymphomas with the combination of IGH/MYC translocations and either IGH/BCL2 or BCL6 translocation), and rare B-lymphoblastic lymphomas.143 It is generally accepted that whereas BL and lymphoblastic lymphomas have distinctive prognostic and therapeutic characteristics, DLBCL with MYC translocations also have a poor prognosis.143 IHC expression of MYC does not indicate the presence of an MYC translocation. However, recent studies have suggested that lymphomas with high levels of MYC expression (70% of cells or more) by IHC are more likely to have these translocations (Fig. 6-15).144 As such, IHC could be used as a screening test for FISH or other testing for MYC translocations.
CD5-Positive Diffuse Large B-Cell Lymphoma CD5-positive DLBCL was described in the 2008 WHO classification (Fig. 6-16).113 CD5-positive DLBCLs have
170
Immunohistology of Non-Hodgkin Lymphoma
dependent on an antecedent history of the low-grade disorder with subsequent transformation.
Diffuse Large B-Cell Lymphoma: Cyclin D1 Cyclin D1 expression can be seen in some DLBCL (Fig. 6-17).146 However, in contrast to that seen in MCL, it is only weakly positive and is seen in a minority of lymphoma cells. It is not known to be specifically associated with differences in prognosis but is more commonly seen in lymphomas of GC derivation. Figure 6-15 Immunohistochemical staining for MYC in a Burkitt lymphoma. MYC staining can be seen in a large number of B-cell lymphomas and does not necessarily indicate the presence of an MYC translocation. However, current literature suggests that expression of MYC protein in 70% or more of lymphoma cells is associated with the presence of MYC translocations.
some noted differences from other typical DLBCLs. In general, they are more often associated with a more aggressive clinical course and poor outcome and are seen at a higher frequency in patients with HIV/AIDS. They express Bcl-2; the majority express MUM1, and half express Bcl-6.145 The expression of CD5 raises the differential diagnosis of MCL and CLL/SLL, although MCL can be distinguished by morphology in most cases of conventional MCL; in cases of blastoid MCL, the uniform expression of cyclin D1 would distinguish this from DLBCL. The possibility of CD5 expression in a large B cell, or Richter transformation of CLL/SLL, is
Diffuse Large B-Cell Lymphoma: CD30 CD30 expression by itself has no specific impact on the diagnosis of DLBCL. It is more often seen in specific lymphoma types such as lymphomatoid granulomatosis and large cell lymphomas with overlapping features of CHL. At present, its evaluation may be most relevant because of brentuximab, an anti-CD30 monoclonal antibody used in the treatment of some lymphomas.
Diffuse Large B-Cell Lymphoma: Epstein-Barr Virus Expression of EBV in lymphomas has been of interest for some time. However, only in the 2008 WHO classification was the specific entity EBV-associated DLBCL of the elderly codified. These lymphomas are always associated with EBV infection and, by definition, are seen in patients 50 years or older.147,148 In addition, they are more common in Asian patients (8% to 10%) and
A
B
C
Figure 6-16 High-magnification image of an aggressive B-cell lymphoma (A). CD5-positive diffuse large B-cell lymphoma. B, CD5. C, CD20.
Large B-Cell Lymphomas and Other Aggressive B-Cell Lymphomas
A
171
B
Figure 6-17 A, Diffuse large B-cell lymphoma composed of a population of large, abnormal lymphocytes. B, In this case, cyclin D1 is expressed in a subset of cells. Note that only a small number of the cells are positive and weakly express cyclin D1. Cyclin D1–positive DLBCL does not show evidence of the typical (11;14) translocation seen in mantle cell lymphoma.
are less common in Western populations (5% or less). The morphologic findings include monomorphous large transformed B cells. Most of the lymphomas are associated with areas of geographic necrosis, although this is not a universal feature. EBV-associated DLBCL of the elderly is associated with a poor prognosis compared with typical DLBCL and is identified most reliably by in situ staining for EBV, such as EBER (Fig. 6-18).149 In the diagnosis of EBV-positive DLBCL, exclusion of impairment of the immune system secondary to immunosuppression, transplantation, autoimmune disease, medications, or previous lymphoma is necessary. The immunophenotype of EBV-associated DLBCL of the elderly is positive for pan–B-cell antigens CD79a,
A
C
CD20, CD19, and Pax-5. They are often positive for CD30 and MUM1 and are negative for CD15 and CD10. Although no specific cutoff has been identified, most studies use at least 50% of cells positive for EBV.
Large B-Cell Lymphoma Subtypes Some specific subtypes of large B-cell lymphomas are worthy of note, with particular expression patterns that are distinctive from other types. PLASMABLASTIC LYMPHOMA
Plasmablastic lymphoma is an aggressive B-cell neoplasm that has immunophenotypic features of plasma
B
Figure 6-18 A, Diffuse large B-cell lymphoma (DLBCL) in lymph node shows diffuse infiltration of large abnormal lymphocytes adjacent to an area of coagulative necrosis. B, The abnormal lymphocytes are positive for CD20. C, In situ staining is extensive for Epstein-Barr virus (EBV) by using EBV encoded RNA. In an older patient, this finding would be compatible with EBVassociated DLBCL of the elderly.
172
Immunohistology of Non-Hodgkin Lymphoma
A
B
C
Figure 6-19 Plasmablastic lymphoma is composed of large atypical lymphoid cells (A), which lack staining for CD20 (not shown) and CD138 (B) and have a high proliferation rate by Ki-67 (C).
cells with large cell morphology and varying degrees of associated, more typical mature plasma cells.113,150 Most cases are associated with immunodeficiency, particularly HIV/AIDS, although a subset of cases is seen in immunocompetent patients. Plasmablasts are large and round with round nuclei, prominent nucleoli, and small or minimal amounts of cytoplasm. The immunophenotypic features of plasmablastic lymphoma include expression of CD138, CD38, and CD79a (50% to 85%), but they usually lack expression of CD45, CD20, and Pax-5 (Fig. 6-19). They will also show monoclonal cytoplasmic light-chain expression in approximately 50% to 70% of cases, and the proliferation rate by Ki-67 is greater than 90%. They lack expression of CD56, which is seen more frequently in high-grade transformation of plasma cell myeloma. EMA and CD30 are often positive. Importantly, the majority of cases are positive for EBV by in situ staining (EBER; 70% to 85%). Human herpesvirus 8 (HHV-8) is not seen, in contrast to some plasma cell neoplasms associated with HIV/AIDS.150 Expression of CD56 or cyclin D1 or bone marrow involvement would suggest plasmablastic plasma cell myeloma rather than plasmablastic lymphoma.151 T-CELL/HISTIOCYTE–RICH LARGE B-CELL LYMPHOMA
T-cell/histiocyte–rich large B-cell lymphoma (TCHRLBCL) has distinctive morphologic and clinicopathologic findings.152 It is a rare, large, B-cell lymphoma most
commonly seen in middle-aged men, and it is associated with an aggressive clinical course. By morphology, rare, large, abnormal B cells (10% or less of the overall cellularity) are seen in a background of varying numbers of small lymphocytes (T cells) or histiocytes. Because of this distinctive appearance, the differential diagnosis of CHL is frequently a consideration. The large B cells are positive for pan–B-cell antigens that include CD19, CD20, CD79a, and Pax-5 with expression of CD45 (Fig. 6-20). CD30 expression is seen in many cases, but the large cells do not express CD15. They are positive for OCT-2 and BOB.1 but do not express CD5, CD43, or CD138. They are usually positive for Bcl-6 and Bcl-2, without expression of CD10. EMA may be variably positive in some cases. In contrast to many cases of CHL, staining for EBV is exceedingly rare in TCHRLBCL. If EBV is present, other diagnoses should be considered, including CHL or EBV-positive DLBCL of the elderly. The background small lymphocytes are usually T cells that are positive for pan–T-cell antigens, with more CD8-positive T cells compared with CD4. Histiocytes are positive for CD68 and CD163. Staining with follicular dendritic cell (FDC) markers (CD21, CD23, etc.) may be of benefit; FDC networks are absent in TCHRLBCL but would be present in cases of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL). IgD is expressed in many of the small lymphocytes associated with NLPHL, and these cells are typically lacking in the background of TCHRLBCL.
Large B-Cell Lymphomas and Other Aggressive B-Cell Lymphomas
A
173
B
Figure 6-20 The abnormal large lymphocytes of T-cell/histiocyte–rich large B-cell lymphoma share features of Hodgkin cells (A), but in contrast to Hodgkin cells show uniform strong staining for CD20 (B).
ALK-POSITIVE LARGE B-CELL LYMPHOMA
ALK-positive large B-cell lymphoma (ALK-LBCL) is exceedingly rare, more common in men (M : F 5 : 1), and is seen in a wide age range (14 to 85 years).153 It is composed of large cells with large round nuclei with prominent nucleoli and often with large amounts of pale-staining cytoplasm. These cells show expression of the ALK protein but lack the T-cell antigen expression of T-cell receptor gene rearrangements seen in anaplastic large (T) cell lymphoma.154 ALK staining is typically granular and cytoplasmic, although rare cases with cytoplasmic and nuclear staining can be seen. These lymphomas will often express plasma cell–associated markers that include CD138, CD38, and IgA (88%) and cytoplasmic restricted light chains. They often lack expression of pan–B-cell antigens (CD19, CD20 [9%], and Pax-5).155 CD45 is seen in most cases (88%), and epithelial membrane antigen (EMA) is seen in almost all cases. CD30 is rarely expressed (6%), and no association has been found with HHV-8 or EBV (EBER) as is seen in plasmablastic lymphoma and other HIV/AIDSrelated lymphomas. ALK-LBCL is likely susceptible to the relatively recently developed anti-ALK therapy, crizotinib. PRIMARY EFFUSION LYMPHOMA
Primary effusion lymphoma (PEL) is another exceedingly rare B-lineage lymphoma, most often seen in HIV/ AIDS patients. As its name implies, most cases are seen in fluid effusions of the pleural or peritoneal cavities,113,150 although some rare cases of solid tissue–based PEL have been reported. All cases have evidence of HHV-8 infection, and cases not associated with HIV/ AIDS are typically seen in areas with endemic HHV-8 infection. Coinfection with EBV (as identified by EBER staining) is seen in a number of cases. The immunophenotype includes expression of CD45 but lack of expression of most pan–B-cell markers (CD20, CD19, CD79a). Plasma cell–associated markers that include CD138, CD38, VS38c, MUM1, and EMA are positive. CD30 is often positive. PEL cells lack T-cell–associated antigen expression, although
exceedingly rare cases of PEL of T-cell lineage have been reported. Most cases show evidence of clonal B-cell gene rearrangements by PCR. PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMA
Primary mediastinal large B-cell lymphoma (PMLBCL) is associated with a fairly unique clinicopathologic presentation. It is seen most frequently in middle-aged women, and as the name implies, it is seen in the mediastinal location. The lymphoma cells are large in size, with round to slightly irregular nuclei and often have moderate amounts of pale cytoplasm, imparting a clearcell appearance. This is not seen in all cases and is only focal in some cases. In most cases, the differential diagnosis is between PMLBCL, CHL, and other types of DLBCL. Because of its relatively distinct prognosis and a proposed origin from thymic B cells, it often has a distinctive, although not pathognomonic, immunophenotype. However, it should be noted that gene-expression arrays in PMLBCL show considerable overlap with CHL, and overlapping diagnostic features can be seen (see later in this section). In rare cases, collision tumors of PMLBCL and CHL, and synchronous and metachronous presentations of both, have been noted. The immunophenotype shares features with other typical, large B-cell lymphomas. The lymphoma cells are positive for pan–B-cell antigens that include CD20, CD19, CD22, and Pax-5. Distinctive features include expression of CD23 and MUM1 in most cases, and most cases are also positive for p63 (Fig. 6-21).156 CD30 expression is present in approximately 80% but is usually weak and heterogeneous—and this is important, considering the differential diagnosis of CHL in this site. Expressions of Bcl-2 and Bcl-6 are variable, and CD10 is typically negative. In comparing CHL and PMLBCL, Hoeller and colleagues157 suggest that expression of BOB.1 favors PMLBCL, and expression of cyclin E favors CHL. Although these IHC stains are not routinely available, most cases of PMLBCL express myelin and lymphocyte protein (MAL), CD54, CD95, and TNF receptor-associated factor 1 (TRAF1).
174
Immunohistology of Non-Hodgkin Lymphoma
A
B Figure 6-21 Abnormal lymphocytes of primary mediastinal large B-cell lymphoma (A), which are often positive for p63 (B).
Intravascular large B-cell lymphoma (IVLBCL) is an extremely rare lymphoma. Because of its unusual clinical presentation, the diagnosis is often unexpected. It can be seen in a variety of tissue types that include skin, lung, brain, prostate, and many others. As implied in the name, clusters and aggregates of large, atypical lymphoid cells are seen in an intravascular distribution. IVLBCL expresses pan–B-cell antigens that include CD19, CD20, CD79a, and Pax-5. They express CD5 in 38% and CD10 in 13%. Most cases are positive for MUM1, except those that are CD10 positive. IVLBCL likely has unique markers that account for its intravascular distribution, but the markers known to be abnormal (CD29, CD54) are not routinely evaluated for lymphoma diagnosis.
accurately diagnose BL in approximately 94% of cases (CD20 and CD10 positive, Ki-67 >95%, with CD38 expression and negative Bcl-2).158 In situ staining for EBV (EBER) will be positive in rare cases, more often in those associated with immunosuppression. In current classification, the presence of MYC– associated t(8;14)(MYC/IGH) or variant is necessary to confirm all but the most classic cases. In some cases, Bcl-2 expression may be seen, and when present, other diagnoses that include MYC translocations in DLBCL, “B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL,” and double-hit lymphomas should be excluded (discussed below). In MYC translocation–positive lymphomas, a simple karyotype (IGH/MYC, with one or no other genetic abnormality) is supportive of a diagnosis of BL, whereas higher numbers of genetic abnormalities support a diagnosis of either double-hit lymphoma or DLBCL.159
BURKITT LYMPHOMA
B-LYMPHOBLASTIC LYMPHOMA
Burkitt lymphoma (BL) is a distinctive but relatively rare aggressive B-cell lymphoma. It can be found in both extranodal and nodal sites. Patients are generally younger than 30 years, but BL may be seen in older patients. It has a distinctive morphology of intermediate-sized lymphocytes with round nuclei, mature chromatin, and small to moderate amounts of deeply staining cytoplasm. Mitotic figures and apoptotic bodies are frequently seen. Tingible body macrophages will often impart the classic “starry sky” pattern at low magnification. The characteristic immunophenotype of BL shows pan–B-cell antigen expression (CD20, Pax-5, CD19, CD22, CD79a) with strong expression of CD10 and a very high proliferation rate by Ki-67 (~100%; Fig. 6-22). BL lacks expression of both TdT (to exclude B-lymphoblastic lymphoma) and Bcl-2. BL expresses germinal center–associated markers that include Bcl-6, GCET1, and HGAL. Some cases will express MUM1. BL lacks expression of cyclin D1, CD5, CD23, and CD43, whereas flow cytometry will often show strong expression of CD38, a reflection of the high proliferative cell fraction in BL. Classic immunophenotype can
B-lymphoblastic lymphoma is in most cases analogous to a tissue manifestation of B-cell lymphoblastic leukemia. It is a neoplastic proliferation of precursor B cells (lymphoblasts). The prognosis is highly dependent on genetic findings and age, and adults tend to have a poor prognosis. Although many cases respond well to therapy, those with poor prognostic features can represent an extremely aggressive disease. The lymphoma is composed of intermediate-sized lymphocytes with delicate, open blastic chromatin and scant cytoplasm. Frequent mitotic figures and apoptotic bodies are seen. The immunophenotype of B-cell acute lymphoblastic lymphoma is typically distinctive enough to distinguish it from other lymphomas of mature B-cell origin. Although some pan–B-cell antigens such as CD19, Pax-5, and CD79a are present, CD20 is often absent or is only partially or weakly expressed. Most cases will have expression of markers associated with germinal center origin, including CD10 and Bcl-6. The most distinctive markers expressed are TdT in almost all cases and CD34 in most cases (Fig. 6-23); Bcl-2 is often positive, as is CD43, and the proliferation rate by Ki-67 is often 90% or greater.
Other Aggressive B-Cell Lymphomas INTRAVASCULAR LARGE B-CELL LYMPHOMA
Large B-Cell Lymphomas and Other Aggressive B-Cell Lymphomas
A
175
B
C Figure 6-22 Pancreas with infiltration by Burkitt lymphoma (hematoxylin and eosin, A), with staining for CD10 (B) and a proliferation rate near 100% by Ki-67 (C). Markers for B-cell lineage (Pax-5, CD20) and germinal-center derivation (Bcl-6) would also be positive (not shown). No expression of terminal deoxynucleotidyl transferase would be seen, and Bcl-2 expression is seen only rarely.
A
B
C Figure 6-23 Extensive infiltration of testicular parenchyma by B-cell acute lymphoblastic leukemia (hematoxylin and eosin, A) with strong staining for CD79a (B), confirming B-cell lineage, and nuclear staining for terminal deoxynucleotidyl transferase (C), confirming the precursor nature of the cells.
176
Immunohistology of Non-Hodgkin Lymphoma
BLASTOID MANTLE CELL LYMPHOMA
A subtype of MCL, the blastoid type, warrants discussion among large cell and aggressive lymphomas. The blastoid type of MCL is typically divided into two morphologic variants, pleomorphic and blastic. By morphology, the pleomorphic type has significant numbers of large cells, compared with the small cells of conventional MCL. They may be multinucleated or anaplastic in appearance and may be uniform or admixed with small abnormal lymphocytes. The blastic variant of blastoid MCL is composed of intermediate-sized lymphoid cells with open chromatin, comparable to lymphoblasts. Blastoid MCL is associated with a more aggressive clinical course than conventional MCL. Blastoid MCL has an immunophenotype comparable to conventional MCL, including coexpression of pan–Bcell antigens (CD20, Pax-5, CD79a) and coexpression of CD5 in the majority of cases. In contrast to conventional MCL, stronger and more uniform expression of cyclin D1 is often seen. In most cases, the proliferation rate by Ki-67 exceeds 30% and may approach 100% in some cases. A subset of these cases may express p53, a marker associated with poor prognosis in MCL. Expression of other markers is typical of MCL, including expression of CD43 and Bcl-2 and lack of CD23, CD10, and Bcl-6 in most cases.
Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma As mentioned previously, some cases of DLBCL have features that overlap with those of BL. Features of overlap can be considered to represent the following diagnostic problems: 1) morphologic features that overlap, including intermediate-sized cells with relatively uniform cytologic appearance; 2) immunophenotypic overlaps, such as proliferation rate near 100% in DLBCL or Bcl-2 expression as an atypical feature in an otherwise typical case of BL; or 3) genetic findings that support BL in a case with other features of DLBCL. The resolution of these problems can be quite difficult, and it is important to note that without careful correlation of morphologic, immunophenotypic, and genetic findings, the designation of B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL may be appropriate. However, because of the often different therapeutic approaches used for BL and DLBCL, considerable effort must be made to distinguish these two entities. When possible, diagnoses should be made of either DLBCL, BL, or double-hit lymphoma. If this distinction is not possible, the diagnosis of B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL should be applied. The term double-hit lymphoma refers to a group of aggressive B-cell lymphomas that share features of DLBCL and BL. They have a distinctly poor prognosis in spite of aggressive therapy.143 By definition, these lymphomas have the presence of a Burkitt-associated
translocation (t[8;14] or variants) as well as either an IGH/BCL6 or BCL6 translocation. Rare cases have also been identified with all of these translocations, so-called triple-hit lymphomas. A more rare variant is one with an MYC translocation and the addition of t(11;14) (CCND1/IGH); these likely represent an aggressive transformation of MCL. It has been proposed that some double-hit lymphomas may be an aggressive transformation of previous follicular lymphomas, with t(14;18) occurring first, followed by t(8;14). In any case, the combination of these lymphoma-associated translocations makes for an especially potent tumor cell with high proliferation and resistance to apoptosis. These lymphomas will often have immunophenotypic features of BL, including CD10 (88%) and Bcl-6 (75%) expression with a high proliferation rate by Ki-67 (median 90%, range of 50% to 100%).143 Features less typical of BL and more typical of DLBCL would include a more pleomorphic, largecell morphology and expression of Bcl-2 (95%). MUM1 is seen in some cases (17%).
Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classic Hodgkin Lymphoma A subset of large cell lymphomas, mostly in the mediastinum, has a combination of features intermediate between DLBCL and CHL. These cases tend to have certain general features that include 1) strong expression of B-cell markers (CD20, Pax-5, CD79a, OCT1, BOB2) typically not seen in CHL, 2) lack of CD15 expression, 3) expression of CD45 in otherwise typical CHL, and 4) other combinations of findings. In general, a hierarchy of findings will strengthen or weaken a diagnosis of CHL versus DLBCL (Table 6-3). Some of these features are more strongly supportive of one diagnosis versus the other. In cases in which there is clear evidence of “borderline features,” a diagnosis of B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and CHL should be rendered; these have also been referred to as gray-zone lymphomas in previous literature. Because important therapeutic differences exist between DLBCL and CHL, a careful attempt should be made to make the distinction between the two diagnoses. Features that make a diagnosis of CHL less likely are expression of B-cell–associated markers that include CD79a, BOB.1, and OCT-2.156 CD45 expression makes a diagnosis of CHL unlikely. In addition, the strong and uniform expression of either CD20 and/or Pax-5 is not typical for CHL. However, weak staining for Pax-5 is typical, and weak and variable staining for CD20 is common in cases of CHL. The lack of CD15 staining in CHL is not uncommon; it can be seen in 20% to 40% of cases. Conversely, the expression of CD15 in DLBCL is highly unlikely. Also, p63 and cyclin E do not help to reliably distinguish PMBCL from so-called gray-zone lymphoma or CHL.160 CD23 is seen in mediastinal DLBCL in 80% of cases in one series.161
T-Cell Lymphomas
177
TABLE 6-3 Distinction between Diffuse Large B-Cell Lymphoma (DLCBL) and Classic Hodgkin Lymphoma (CHL) Feature
Favors DLBCL
Favors CHL +
CD15 expression
Notes Up to 30% of CHL may be negative for CD15 expression.
CD30 expression weak/rare
+
CD30 staining is expected to be strong in CHL.
CD45 expression
+
CD45 expression is rare in CHL (≪5%). Loss of CD45 expression may be seen in some DLBCL.
PAX5 strong, uniform
+
PAX5 expression is usually weak in CHL.
CD20 strong, uniform
+
Uniform strong expression of CD20 is seen only rarely in CHL. However, up to 30% to 40% of CHL will have weak and variable expression of CD20.
+
CD20 weak, variable OCT.2/BOB.1 both strong
+
CD79a expression
+
Epstein-Barr virus (EBV)/ EBER expression MUM1
–/+
B-cell clone by IGH polymerase chain reaction (PCR)
+
p63
+
Most cases of CHL lack both OCT.2 and BOB.1 expression, but some cases (10% to 15%) may express one or the other; rare cases (<5%) express of both. CD79a expression is quite rare in CHL. +
Overall, approximately 40% of all CHL cases express EBV. It is a rare finding in DLBCL, especially in those that are morphologic mimics of CHL.
+
MUM1 is positive in virtually all cases of CHL but is variable in DLBCL. Clonal IGH peaks are seen in almost all cases of DLBCL. However, only a minority of CHL cases will have clonal B-cell PCR.
–
More likely positive in mediastinal lymphomas, including gray-zone lymphomas
EBER, Epstein-Barr encoded RNA; MUM1, melanoma-associated antigen 1.
KEY DIAGNOSTIC POINTS Large B-Cell Lymphoid Neoplasms • Gene-expression arrays classify diffuse large B-cell lymphoma (DLBCL) into germinal center–derived (GC), activated–B-cell type (ABC), and primary mediastinal B-cell lymphoma. • DLBCLs of germinal center origin have been shown to have an overall better prognosis than those of non–germinal center origin. • Plasmablastic lymphoma is an aggressive B-cell neoplasm associated with immunodeficiency; it includes expression of CD138, CD38, and CD79a (50% to 85%) but usually lacks expression of CD45, CD20, and PAX5. • Double-hit lymphoma refers to a group of aggressive B-cell lymphomas that share features of DLBCL and Burkitt lymphoma. • A subset of large-cell lymphomas, mostly in the mediastinum, has a combination of features intermediate between DLBCL and classic Hodgkin lymphoma.
T-Cell Lymphomas T-cell lymphomas are frequently more difficult to diagnose compared with B-cell lymphomas. This difficulty is in part due to their rarity; they account for approximately 10% of all lymphomas. In addition, the broad
spectrum of histologic and immunophenotypic findings and the overlap of some subtypes with other lymphomas and reactive conditions further confounds diagnosis. A subset of T-cell lymphomas has distinctive clinical and pathologic features that render them slightly easier to characterize (Fig. 6-24). However, it should be noted that in almost all circumstances, when specific immunophenotypic features are mentioned in T-cell lymphoma types, almost always exceptional cases exist that lack these features or have other, different features.162 Some general hints to the diagnosis of T-cell lymphomas can be applied. In general, T cells are present in a mixture of CD4- and CD8-positive cells. Although the ratio of these cells is 2-4 : 1 in peripheral blood, this ratio can vary widely in tissue types and in different immune responses. If there were a massive preponderance of either CD4-positive or CD8-positive T cells, this would suggest a diagnosis of T-cell lymphoma. Likewise, loss of pan–T-cell antigen expression is atypical and may support a diagnosis of T-cell lymphoma. Loss of CD3, CD2, CD5, CD43, or CD45 may be seen in some T-cell lymphomas and would be a supportive finding. CD7 is somewhat more problematic. Although it is present in the majority of T cells and is lost in many T-cell lymphomas (such as mycosis fungoides), it may also be downregulated or lost completely in some immune responses, and so its lack provides less robust support for a T-cell lymphoma. EBV positivity, as assessed by in situ stains for EBER, is seen in approximately 30% of
178
Immunohistology of Non-Hodgkin Lymphoma
2.5% 0.9% 1.4% 1.7%
12.2% 25.9% 4.7% 5.5% 6.6%
18.5% 9.6% 10.4%
Peripheral T-cell lymphoma Angioimmunoblastic lymphoma Natural killer/T-cell lymphoma Adult T-cell leukemia/lymphoma Anaplastic large cell lymphoma, ALK Anaplastic large cell lymphoma, ALK Enteropathy-type T-cell lymphoma Primary cutaneous ALCL Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like lymphoma Unclassifiable PTCL Other disorders
Figure 6-24 Frequency of T-cell lymphomas. ALCL, anaplastic large cell lymphoma; PTCL, peripheral T-cell lymphoma. From International PTCL and Natural Killer/T cell lymphoma study: pathology findings and outcomes. J Clin Oncol 2008;26[25]4124-4130.
T-cell lymphomas.163 Although not a distinguishing feature from B-cell lymphomas or reactive conditions, the presence of EBV positivity in the appropriate context can support a diagnosis of T-cell lymphoma. Loss of Bcl-2 expression is another example of a finding supportive of T-cell lymphoma.164,165 The large majority of normal T cells are positive for α/β T-cell receptors (TCRs). If staining for TCR β-F1 is negative, it suggests the T cells are not of the α/β type and are γ/δ positive; this finding would be suggestive of a diagnosis of a T-cell lymphoma of a γ/δ type. Aberrant expression of some antigens can also suggest or support a diagnosis of T-cell lymphoma. The expression of CD56 in more than rare T cells would be suggestive of a T cell, NK/T cell, or NK cell neoplasm. Several monoclonal antibody–based therapies have been applied in T-cell lymphomas. These include alemtuzumab, an anti-CD52 molecule that has been shown to have efficacy in some T- and B-cell lymphomas.166 Denileukin diftitox (Ontak) is a fusion protein that attaches diphtheria toxin to IL-25 (CD25 receptor); it has been used as a therapy for selected T-cell lymphomas. Brentuximab is a monoclonal antibody that targets CD30. It has shown effectiveness in ALCL. Crizotinib is an anti-ALK monoclonal antibody that has been shown to have considerable effectiveness in ALKpositive ALCL. The immunophenotype of normal and abnormal NK cells is distinct from that of T cells. NK cells do not express surface CD3, but they do express cytoplasmic CD3. As such, NK cells will be positive for CD3 staining by IHC. NK cells do not express CD4 or CD8. Likewise, they will not express TCR α/β or γ/δ. Normal NK cells will be positive for CD16, CD56, and CD57 with variation of these markers, depending on the exact NK subset and degree of maturation. CD16 staining is not available in paraffin, but CD56 and CD57 are available. In evaluating CD56 and CD57, it should be noted that only small numbers of NK cells are usually present in normal tissues, including lymph nodes. Significant increased staining for these markers in lymphocytes
should raise the possibility of an NK- or T-cell proliferation. NK cells, like cytotoxic T cells, are positive for markers of cytotoxic granules that include TIA1, perforin, and granzyme B.
Peripheral T-Cell Lymphoma Peripheral T-cell lymphoma (PTCL) represents the largest category of T-cell lymphomas. It is heterogeneous, and doubtless several specific diagnostic entities exist within the group. However, at present, the group remains a catch-all for those T-cell lymphomas without welldefined, specific clinical or pathologic characteristics. General IHC features of PTCL are that the majority are CD4 positive compared with CD8. Almost all cases express pan–T-cell antigens CD3, CD2, and CD43 (Fig. 6-25). Occasional cases will show loss of CD5 and/or CD7. An α/β derivation (TCR β−F1 positive) is considerably more common than those of γ/δ derivation. PTCL typically lacks expression of CD10, Bcl-6, CXCL13, or PD1, all of which are typically seen in AITL.164 CD30 may be positive in a subset of cells;163 however, if more than 75% are positive, with strong and uniform expression, a diagnosis of ALCL (see below) is more likely. Rarely, coexpression of CD20 and/or CD79a may be seen. However, even in these rare cases, other pan–B-cell antigens are lacking. In general, about 30% of cases of PTCL are positive for EBV.163 Adverse IHC findings include Ki-67 greater than 25%, significant numbers of EBV-positive cells, CD56 expression, and CD30 expression by more than 20% of cells.167 Numerous genetic abnormalities have been reported, but none are entirely specific for a diagnosis of PTCL. PCR studies for T-cell clonality are positive in the large majority of cases.
Anaplastic Large Cell Lymphoma (ALK+, ALK−) Anaplastic large cell lymphoma (ALCL) is a category of T-cell lymphoma defined by its immunophenotype.
T-Cell Lymphomas
A
C
Although it has been historically identified by its striking morphology, its definitive description is based on its expression of CD30 and other various T-cell antigens. ALCL is further subdivided by the presence of a group of cytogenetic abnormalities that involve the ALK gene, which is commonly identified by the overexpression of the ALK protein in the lymphoma. Significant prognostic differences have been noted in ALK-positive versus ALK-negative cases of systemic ALCL. Other differences based on systemic versus cutaneous ALCL will be discussed below. By definition, ALCL is positive for CD30, and the expression should be strong and in at least 75% of the abnormal lymphocytes present (Fig. 6-26). Many T-cell lymphomas and some B-cell lymphomas express CD30, but ALCL must have strong expression in the majority
A
179
B
Figure 6-25 A, Histology of peripheral T-cell lymphoma (PTCL). Immunohistochemical staining for CD3 (B) and CD8 (C) are shown. The majority of PTCL is CD4 positive, but the group has heterogeneous immunophenotypic features.
of cells. In both ALK-positive and ALK-negative types, the cell derivation and immunophenotype of ALCL is of T-cell lineage. However, in most cases the expression of T-cell markers is incomplete, and in some cases, termed a null cell type, no identifiable T-lineage– associated markers are evident. CD3 is negative in as many as 75% to 80% of cases of ALCL, with a lack of CD45 (leukocyte common antigen [LCA]) and CD45RO in many cases. CD43 is positive in the majority of cases, and many cases will express CD2, CD5, and CD4 (~70% each). In addition, cytotoxic granules, TIA1, perforin, and granzyme B are positive in most cases. Only a small subset of cases has been described with CD8 expression, and CD7 is only rarely positive. ALK staining is a defining characteristic of the subtype of systemic ALCL with a better prognosis
B
Figure 6-26 A, Hematoxylin and eosin staining in the large, pleomorphic lymphoid cells of anaplastic large cell lymphoma. B, CD30 staining is positive on the membrane and cytoplasm with occasional bright, nodular staining in the Golgi apparatus.
180
Immunohistology of Non-Hodgkin Lymphoma
A
B
Figure 6-27 Hemtoxylin and eosin staining in anaplastic large cell lymphoma (A) with strong nuclear and cytoplasmic staining for ALK (B). This pattern of ALK staining is seen with a (2;5)(NPM-ALK) translocation.
(ALK-positive ALCL). This IHC staining is associated with translocations of the ALK gene with various partners. Interestingly, the pattern of expression of ALK by IHC shows some correlation with the translocation partner. The most common translocation by far has a pattern of nuclear and cytoplasmic ALK staining, seen in t(2;5)(p23;q35) (Fig. 6-27). This translocation involves the NPM gene, nucleophosmin, which is a nuclear transporter protein that leads to nuclear expression of the ALK protein. Other translocations are rare, and some different staining patterns have been observed. Cytoplasmic staining is seen with t(1;2)(TPM3/ALK), t(2;3)(TGF-ALK), and inv2(ATIC/ALK, EML4 or RANBP2/ALK). Cytoplasmic/granular staining is seen with a translocation of ALK and the clathrin gene [t(2;17)(CLTC/ALK)]. Membranous staining is seen with t(2;X)(MSN/ALK), and other rare translocations include t(2;19)(TPM4/ALK), t(2;22)(MYH9/ALK), and t(2;11;2)(CARS/ALK).168 The presence of the ALK translocation can also be evaluated by FISH studies in addition to IHC. Unusual IHC staining patterns can be seen in ALCL. Markers not typically associated with T cells can be seen in a small subset of cases that include Bcl-6 and Pax-5 reactivity. EMA staining can be seen in up to 80% of cases of ALK-positive ALCL, and it was historically used as a surrogate for ALK staining. Clusterin and fascin have been reported as positive in ALCL but are not sufficiently specific to support the diagnosis when considering a typical differential diagnosis. EBV is not typically associated with ALCL. CD15 expression in a CD30-positive lymphoma is most often associated with CHL (see Chapter 5). However, a small subset of CD15-positive T-cell lymphomas, including ALCL, has been well described in the literature.169 The pattern of CD15 staining these rare cases has been described as punctate and cytoplasmic, in contrast to Hodgkin cases.165 Although not specific to the diagnosis, most cases of ALCL express CD25. Ontak, a targeted therapy for CD25, has been used effectively in ALCL therapy.
Angioimmunoblastic T-Cell Lymphoma Angioimmunoblastic T-cell lymphoma (AITL) is distinctive based on its combination of clinical, histologic,
and immunophenotypic features.170 AITL is often associated with immune deficiencies, hypergammaglobulinemia, rashes, infections, and systemic symptoms.168 Because of its relatively distinctive immunophenotype, AITL is thought to derive from follicular T-helper cells. Histologic findings in lymph nodes include proliferation of a polymorphous infiltrate of lymphocytes. The abnormal cells present are frequently intermediate in size with irregular nuclei and moderate amounts of pale cytoplasm, admixed smaller lymphocytes, and some larger transformed cells. The neoplastic cells of AITL are positive for pan–Tcell antigens CD3, CD2, and CD5 and are CD4 positive. However, in all cases, numerous reactive CD8 cells are present. Focal expression of CD10, CXCL13, Bcl-6, and PD1 (60% to 100%) is seen in the abnormal T cells of AITL. The presence of this staining is variable, but it can be prominent in abnormal lymphocytes adjacent to vascular structures. The T cells present are α/β positive, and CD56 is typically not expressed. Focal staining for EBV by in situ methods is seen in the majority of cases (>70%).168,170 No significant immunophenotypic differences have been found between nasal and extranasal types of AITL.171 A characteristic feature of AITL is the proliferation and expansion of FDC meshworks (Fig. 6-28). These may be associated with germinal centers or may arise in other nodal areas, and they do not maintain the typical nodular shape of follicles. Staining for FDC markers such as CD21, CD23, CD35, CNA42, fascin, and D2-40 can identify them. Residual follicles are often seen in AITL, although by H&E morphology, they may be inapparent. Staining with B-cell markers will reveal islands and clusters of B cells. The normal follicles will often be fragmented or disrupted by T cells, imparting unusual shapes to the usually round nodules of B cells. These follicles will often be positive for Bcl-6 and CD10, increasing the challenge of interpreting expression of these stains in T cells. A subset of AITL (~20%) will be associated with development of concurrent large B-cell lymphomas. These are often positive for EBV and have a high proliferation rate. The presence of these large B-cell lymphomas portends a dismal prognosis.
T-Cell Lymphomas
A
181
B
Figure 6-28 A, An intermediate-magnification image of angioimmunoblastic T-cell lymphoma, with a polymorphous cellularity composed of lymphocytes, histiocytes, plasma cells, and eosinophils. B, CD21 staining shows expanded abnormal proliferations of follicular dendritic cells not associated with germinal centers.
Extranodal Natural Killer/T-cell Lymphoma, Nasal Type Another distinct clinicopathologic entity is extranodal nasal type NK/T-cell lymphoma (ENKTL).171 Its usual presentation is in the nose or sinuses, although other sites are involved, albeit rarely. A polymorphous infiltrate is usually present with numerous plasma cells and eosinophils, and extensive necrosis may be associated with secondary infection (fungal, bacterial), which may complicate the diagnosis. It is important to note that
this disease is often unrecognized in the first biopsy sample, and repeat biopsies may be necessary to yield a diagnosis. There is a predilection for perivascular spaces for the neoplastic cells, with associated necrosis. This accounts for one of the previous names, the so-called angiocentric T-cell lymphoma. The neoplastic cells of ENKTL are intermediate to large, often with irregular nuclear contours. Their immunophenotype is variable, they are positive for cytoplasmic CD3, and they also express CD56, a marker most often associated with NK differentiation (Fig. 6-29). They typically lack surface CD3 expression but express
A
B
C
D
Figure 6-29 A, High-magnification image of extranodal natural killer/T-cell lymphoma, nasal type (ENKTL), with large lymphoid cells and many fragments of necrotic cells. B, CD3 staining is positive in the cytoplasm of ENKTL, but rarely is surface CD3 positive (assessed by flow cytometry). CD56 is positive in the lymphoma cells (C), as is in situ staining for Epstein-Barr virus (Epstein-Barr encoded RNA; D).
182
Immunohistology of Non-Hodgkin Lymphoma
other pan-T antigens (CD2, CD43) and commonly express cytotoxic molecules such as perforin, TIA1, and granzyme B. They are most often negative for CD4, CD5, CD8, CD16, CD57, TCR α/β (TCR β-F1), and TCR γ/δ. They are variably positive for CD7 and CD30. EBV, when assessed by in situ stains (EBER), is positive in essentially all cases, and cases without EBV expression should be evaluated very carefully before rendering a diagnosis of ENKTL. No specific genetic findings are characteristic of ENKTL. High proliferation rate by Ki-67, as well as expression of cyclin D1 and Bcl-2, are associated with a poor prognosis.172,173
Enteropathy-type T-Cell Lymphoma Enteropathy-type T-cell lymphoma (ETCL) is a rare disorder most often characterized by involvement of the small intestine, with aggressive clinical behavior.174,175 A subset of cases is associated with preexisting celiac disease (e.g., gluten enteropathy arising from an abnormal susceptibility to gluten proteins).176 Celiac disease– related ETCL develops when there is an autonomous proliferation of T cells that arise from an antigen-driven process. ETCL can be associated with intestinal obstruction or perforation. Two types of ETCL have been identified and can be differentiated based both on clinical and immunophenotypic differences. ETCL type I (ETCL-I), represents the majority of cases (80% to 90%); these are associated with a specific genetic abnormality, the
expression of human leukocyte antigen (HLA) DQ2/8 in virtually all cases. Both type I and type II ETCL are characterized by expression of pan–T-cell antigens, including CD3 and CD7 (Fig. 6-30). CD2 is not expressed in approximately half of the cases,175 and ETCL of both types is negative for CD4 and CD5. Variable degrees of CD30 expression is seen, and it is more frequently present in type I. ETCL type I lacks expression of CD8 and CD56, in contrast to ETCL type II (ETCL-II). EBV expression is seen only rarely in cases of ETCL. In areas adjacent to ETCL, findings of refractory celiac disease may be seen; the intraepithelial lymphocytes present are T cells that are positive for CD4 and negative for both CD4 and CD8.175 Most cases express TCR α/β. In virtually all cases, PCR studies show clonal T-cell gene rearrangements. No specific genetic findings are characteristic of ETCL, but gains of 1q, 5q, 8q24 (MYC), and 9q or loss of 16q are seen in both types. NOTCH1, located at 9q34, may play a role in ETCL pathogenesis.174
T-Cell Prolymphocytic Leukemia/Lymphoma T-cell prolymphocytic leukemia/lymphoma (T-PLL) is a rare and aggressive T-cell neoplasm.168 It most often presents as disease of the spleen, peripheral blood, and bone marrow. The cells are intermediate to large in size with irregular nuclei, prominent nucleoli, and small to moderate amounts of cytoplasm. Lymph node
A
B
C
D
Figure 6-30 A, Diffuse infiltration of small intestine by enteropathy-type T-cell lymphoma (ETCL) by small to intermediate irregular lymphocytes with small to moderate amounts of clear cytoplasm. Type II ETCL is positive for CD8 (B), CD56, (C) and TIA1 (D), as in this case.
T-Cell Lymphomas
involvement is occasionally seen and typically diffusely effaces lymph nodes. T-PLL is positive for pan–T-cell antigens CD3, CD2, and CD7. It is often negative for CD5. The pattern of CD4/CD8 expression in T-PLL is 60% CD4 positive/ CD8 negative, 25% CD4 positive/CD8 positive, and 15% CD4 negative/CD8 positive. T-PLL is positive for CD52, and alemtuzumab has been used in therapy. T-PLL is almost always positive for TCL1, a protooncogene that acts as an AKT activator. T-PLL is negative for CD1a and TdT, which distinguishes it from T-cell lymphoblastic leukemia/lymphoma, a frequent differential diagnostic consideration, based on morphology.
Hepatosplenic T-Cell Lymphoma A distinctive T-cell lymphoma, hepatosplenic T-cell lymphoma has a characteristic clinicopathologic presentation. Patients often have splenic and hepatic involvement, and bone marrow is typically involved, but neoplastic cells are only rarely identified circulating in peripheral blood. This lymphoma is associated with a poor prognosis. It is seen mostly in younger, predominantly male patients, and an increased incidence is seen in patients after organ transplantation and in those with immunosuppression. The lymphoma cells are of T-cell immunophenotype and in most cases have γ/δ T-cell receptors, which are usually seen in only 5% of circulating T cells. In typical cases, TCR-δ can be identified, with no staining for TCR-β (see below). The lymphoma cells of hepatosplenic T-cell lymphomas are also negative for CD5 and CD4 and are variably positive for CD8 and CD56, with expression of the cytotoxic T-cell marker TIA1. Interestingly, they are negative for perforin and granzyme B. Hepatosplenic T-cell lymphoma is associated with clonal T-cell receptors in most cases. In addition, a characteristic genetic abnormality, isochromosome 7q, is seen in almost all cases. This abnormality can be identified by appropriate FISH studies. Rare cases will have an α/β immunophenotype; these cases would be positive for TCR β-F1 staining by IHC. This immunophenotype does not appear to have a specific effect on prognosis or therapeutic options.
Mycosis Fungoides Mycosis fungoides (MF) is the most common T-cell lymphoma of the skin177 and typically has an indolent clinical behavior. It is composed of small, intermediate, and large atypical T lymphocytes, which most commonly involve the superficial dermis with some involvement of the epidermis (epitheliotropism). These are T cells that express a near complete T-helper cell immunophenotype that includes expression of CD3, CD2, CD4, and CD5. Loss of CD7 is identified in most cases of MF. Although exceedingly rare, a variant of MF that is CD8 positive has been identified, and other rare cutaneous types of T-cell lymphomas with CD8 expression have also been described.178 Most cases are positive for cutaneous lymphocyte antigen (CLA), a skin-selective homing receptor for skin-associated memory T cells,
183
and for β-F1 TCR. They are notably negative for CD56 expression and δ-TCR. Some expression of CD30 may be seen, but diffuse and strong expression would raise the possibility of ALCL (see previous section). Rare variants of MF are CD8 positive; these cases are more often hypopigmented and are mostly seen in pediatric patients.179,180
CD30-Positive Cutaneous Lymphoproliferative Disorders CD30-positive cutaneous lymphoproliferative disorders represent a spectrum of pathologic processes in the skin that range from clinically benign but recurrent disorders (lymphomatoid papulosis [LyP]) to frankly malignant T-cell lymphomas (cutaneous ALCL [C-ALCL]). Histologic variation is considerable, although most cases will have at least some very large abnormal lymphoid cells. Large multinucleated cells with wreathlike nuclei are often seen, also referred to as hallmark cells. These abnormal lymphocytes are T cells that uniformly express CD30. The abnormal T cells express CD4 with variable loss of pan–T-cell antigens CD2, CD3, and CD5. CD45 is variable, but CD43 is expressed in most cases. Cytotoxic markers that include TIA1, perforin, and granzyme B are frequently expressed. ALK expression is not usually seen on C-ALCL, although rare cases have been reported. Clonal T-cell gene rearrangements are seen in most cases, and this is not sufficient to distinguish C-ALCL from LyP. Likewise, pan–T-cell antigenic loss does not reliably distinguish these disorders. In almost all cases, large tumor size is more compatible with C-ALCL, but in each case, careful clinicopathologic correlation is necessary. It should be noted that a single patient may have both C-ALCL and LyP concurrently or dyssynchronously.
Subcutaneous Panniculitic Type T-Cell Lymphoma A rare T-cell lymphoma most often seen involving subcutaneous soft tissue, including adipose tissue, subcutaneous panniculitic type T-cell lymphoma (SPTCL) affects a broad age range and shows a slight female predominance. SPTCL only rarely involves lymph nodes, and the clinical behavior is relatively indolent, especially compared with other T-cell lymphomas. The immunophenotype is of cytotoxic T cells with expression of CD3, CD8, TIA1, perforin, granzyme B, and TCR α/β (e.g., β-F1). By definition, no expression of CD56 or γ/δ is found; presence of these markers would indicate the exceedingly rare cutaneous γ/δ-positive T-cell lymphoma. Loss of other pan–T-cell antigens, CD5 and CD7, is variable.
T-Cell Large Granular Lymphocytic Leukemia/Lymphoma T-cell large granular lymphocytic leukemia/lymphoma (T-LGLL) is a rare disorder that typically involves bone marrow, peripheral blood, and spleen. In most cases it
184
Immunohistology of Non-Hodgkin Lymphoma
A
B
Figure 6-31 Hematoxylin and eosin staining in a thymoma (A), with numerous, admixed, immature T cells that are positive for terminal deoxynucleotidyl transferase (B). In these cases, careful evaluation is required to exclude T-cell acute lymphoblastic leukemia/lymphoma.
is an indolent disorder associated with patients with autoimmune disorders, especially those with rheumatoid arthritis; patients will often have peripheral blood cytopenias. The proliferation of cells seen are most often intermediate-sized lymphocytes with slightly irregular nuclei and moderate to large amounts of pale cytoplasm. Occasional cytoplasmic granules may be seen. These neoplastic cells are comparable in appearance to cytotoxic T cells or NK cells seen in normal peripheral blood. In most cases, evaluation is by flow cytometry of a “wet” specimen, such as bone marrow or peripheral blood. By immunophenotypic analysis, the cells of T-LGLL are CD3 positive with expression of CD2, CD8, CD57 (>80%), CD16 (>80%), and TCR-α/β. Expression of CD5 and/or CD7 is frequently abnormal, dim, or absent. Flow-cytometric evaluation of KIRs shows restricted expression, supporting the neoplastic nature of these lymphocytes.181,182 By IHC evaluation, these abnormal lymphocytes express cytotoxic markers TIA1, granzyme B, and perforin. However, these can be especially difficult to interpret in bone marrow, because normal granulocytes will often express these markers. Immunophenotypic variations of T-LGLL include CD4 expression (very rare), CD4/CD8 negative (very rare), or γ/δ expression.
Adult T-Cell Leukemia/Lymphoma Yet another rare T-cell lymphoma, adult T-cell leukemia/ lymphoma (ATL), is caused by the retrovirus human T-cell lymphotrophic virus type 1 (HLTV-1).183 ATL has a variety of clinical presentations, with different clinical courses; these include smoldering, chronic, acute, and lymphoma. In advanced or clinically aggressive disease, ATL is associated with a poor prognosis, and these have a T-cell immunophenotype that expresses pan–T-cell antigens CD2 and CD5 but lacks CD7. Surface CD3 expression may be dim or negative (by flow cytometry), but IHC expression (e.g., cytoplasm) will be positive. The majority of cases are CD4 positive, with rare CD8positive cases and exceedingly rare double-positive cases (CD4 and CD8 positive); CD25 is strongly expressed in almost all cases. ATL is derived from regulatory T cells that express FoxP3.184 CD30 is expressed
in large cells of ATL, and some cases may be considered for anti-CD30 therapies. HTLV-1 is positive, and testing is usually by serology or PCR-based studies.
Pitfalls in Diagnosis of T-Cell Lymphomas Immature T cells may be double negative (CD4−/ CD8−) or double positive (CD4+/CD8+), or they may express CD4 or CD8 (Fig. 6-31). In addition, they express TdT and have a high proliferation rate by Ki-67. These immature T cells can be seen in normal thymus, ectopic thymus, and thymomas, and their immunophenotype can closely mimic a T-lymphoblastic lymphoma. Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder associated with immunodeficiency and lymphadenopathy. Patients may have proliferations of T cells with aberrant immunophenotypes, most notably being double negative for CD4 and CD8.185 KEY DIAGNOSTIC POINTS T-Cell Lymphoid Neoplasms • Almost all peripheral T-cell lymphomas express pan–T-cell antigens CD3, CD2, and CD43. • Anaplastic large-cell lymphoma (ALCL) is positive for CD30, and the expression should be strong and in at least 75% of the cells. • In both ALK-positive and ALK-negative ALCL, the cell derivation and immunophenotype of ALCL is of the T-cell type. • The neoplastic cells of angioimmunoblastic T-cell lymphoma (AITL) are positive for pan–T-cell antigens CD3, CD2, CD5, and CD4.
Pitfalls in the Diagnosis of Lymphoma: Mimicry Earlier in this chapter, difficulties distinguishing benign from malignant lymphoid proliferations as they relate to the establishment of a diagnosis of lymphoma were discussed. Even when the process is manifestly malignant—that is, with diffuse effacing growth pattern
Pitfalls in the Diagnosis of Lymphoma: Mimicry
185
TABLE 6-4 Malignant Mimics of Lymphoma Type of Neoplasm
Useful Positive Markers
Markers Shared by Lymphomas
Carcinoma (e.g., lymphoepitheliomas)
Cytokeratins
p63, EMA (Muc-1)
Germ cell neoplasm
PLAP, CD117, cytokeratins
CD30
Melanoma
S-100 protein, Melan-A, HMB-45
Vimentin
Myeloid sarcoma
Myeloperoxidase, lysozyme, CD68
CD45
Small-cell blastic tumors
Synaptophysin, chromogranin, cytokeratins, myogenin, actin, PAX2
CD99 and CD56
Thymoma (can mimic either low- or high-grade lymphomas)
Cytokeratins
CD20, CD5, and terminal deoxynucleotidyl transferase
EMA, Epithelial membrane antigen; PLAP, placental alkaline phosphatase.
and aggressive cellular composition—there are pitfalls for which the pathologist must be wary. High-grade malignancies tend to merge in appearance with loss of functional differentiation, therefore it is not surprising that other neoplasms can masquerade as lymphoma and vice versa. The tools available for IHC application can be critical in avoiding misdiagnosis, however, once out of the realm of the immune system, many of the markers used for lymphoma classification are found to be expressed as well by elements of diverse organ systems.89
Other Neoplasms that Express Markers Associated with Lymphoma Table 6-4 lists tumors that most commonly mimic lymphoma microscopically, together with the markers that effectively identify them as nonlymphoid. Also listed are markers that, although commonly associated with lymphomas, are in fact not specific and can be expressed in nonlymphoid neoplasms. A good example
Cohesive
of lymphoma mimicry can be found in the undifferentiated carcinoma, so-called lymphoepithelioma, of nasopharyngeal origin. In Figure 6-32 both cohesive and dispersed large, delicate tumor cells can be appreciated in a background of benign immune cellularity that features small lymphocytes, eosinophils, plasma cells, and some histiocytes. These findings are consistent with either the so-called syncytial variant of nodular sclerosing CHL or metastatic lymphoepithelioma. In Figure 6-33, definite tumor cell staining for CD30 is evident, a marker most would consider specific for lymphoma, most commonly CHL or the anaplastic large cell type. However, IHC for the cytokeratin cocktail AE1/AE3 shows tumor cells to be strongly positive, effectively excluding lymphoma. Until recently it was thought that CD30 was specific for activated lymphoid cells, however, a series of nasopharyngeal carcinoma cases found that about 10% were positive for this marker.186 Other markers have also been found to be expressed by carcinomas, including Bcl-2, Bcl-6, CD138, and clusterin.187-190
Dispersed
Figure 6-32 Left, A case of metastatic nasopharyngeal carcinoma with delicate, large tumor cells showing cohesive growth pattern. Right, The same tumor cells are finely dispersed, suggesting lymphoma rather than carcinoma.
186
Immunohistology of Non-Hodgkin Lymphoma
CD30
Cytokeratin
Figure 6-33 Top, The same case as in Figure 6-32, with tumor cellularity that displays staining for CD30, suggesting the possibility of Hodgkin lymphoma. Bottom, Tumor cells, both cohesive and dispersed, show strong expression of cytokeratin AE1/AE3.
The marker CD15, commonly used within the panel for identification of CHL, is also used in the recognition of adenocarcinoma as distinct from mesothelioma.191 One notable example of lymphoma mimicry is thymoma, unique in its ability to be mistaken for either low-grade or high-grade lymphoma. Although the neoplastic epithelial cells are relatively small and delicate, the associated lymphoid cells vary in their composition from case to case. In thymomas that are lymphocyte-rich and of medullary differentiation, the
small lymphoid elements resemble a low-grade lymphoma (Fig. 6-34). The most effective way to recognize the process as thymoma is with IHC for cytokeratin AE1/AE3. A diffuse, delicate network of positive dendritic cells reveals the underlying epithelial neoplasm. In addition, the neoplastic thymic epithelium can express CD20, a marker usually associated with B-cell lymphomas (Fig. 6-35).192 When lymphocyte-rich thymomas are of the cortical type, their lymphoid elements correspond to precursor T-cells, thus resembling lymphoblastic lymphoma. Again, the demonstration of the delicate network of neoplastic thymic epithelial cells with IHC for cytokeratin is the means to avoid misdiagnosis. Other neoplasms that can be indistinguishable microscopically from lymphoma include malignant melanoma, germ-cell tumors, myeloid sarcomas, and blastic small-cell tumors such as Ewing/PNET, rhabdomyosarcoma, and neuroblastoma.89
Lymphomas That Express Markers Associated with Other Neoplasms Certain types of lymphoma can resemble carcinomas remarkably (Table 6-5). The classic example is ALCL, which often displays a cohesive and sinusoidal growth pattern within lymph nodes that mimics metastatic malignancy. This lymphoma frequently expresses EMA (or MUC-1), giving it the potential for misinterpretation as carcinoma.193 Hodgkin lymphoma, particularly the nodular sclerosing type, can also grow in cohesive clusters, the so-called syncytial variant, which bears a striking resemblance to carcinoma.194 Anaplastic plasmacytomas, in particular those that arise in mucosal sites, may appear very epithelial; lacking expression of
Cytokeratin
Figure 6-34 Thymoma featuring sharply defined, fibrous septa at low magnification; at higher magnification, predominance of small lymphoid cells resembles low-grade lymphoma. A stain for cytokeratin AE1/AE3 reveals the widespread, delicate network of neoplastic epithelial cells diagnostic for thymoma.
Pitfalls in the Diagnosis of Lymphoma: Mimicry
Cytokeratin
CD20
187
CD3
Figure 6-35 The same case as in Figure 6-34, showing staining of neoplastic epithelium for CD20 and cytokeratin. The lymphoid cellularity is almost purely T cell, staining for CD3.
common lymphoid markers, such as CD45 and CD20, and staining for EMA and CD138, these can easily be misdiagnosed as carcinoma. Even conventional large B-cell lymphoma can simulate carcinoma by growing in cohesive sheets, sometimes within sinuses (Fig. 6-36).195 Rare examples of a rosetting growth pattern or “anemone cell” lymphoma have been reported, in which glandlike tumor cell arrays resemble adenocarcinoma.196 CD56 or
the neural cellular adhesion molecule NCAM-1 is expressed in a distinct minority of cases of large B-cell lymphoma, which can create confusion in relation to neuroendocrine type carcinoma.197 Pax-8, a marker used for identification of renal cell carcinoma and a number of other epithelial neoplasms, has been shown to crossreact with Pax-5, leading to Pax-8 expression in lymphomas.198
CD20
Figure 6-36 A lymph node shows cohesive expanses of pink tumor cellularity with anaplastic nuclear features, suggesting metastatic carcinoma. The cohesive tumor cellularity displays strong uniform expression of CD20, revealing that this is a diffuse large B-cell lymphoma mimicking metastatic carcinoma.
188
Immunohistology of Non-Hodgkin Lymphoma
TABLE 6-5 Lymphomas that Can Mimic Other Neoplasms Type
Useful Positive Markers
Markers Not Entirely Specific
Anaplastic large cell lymphoma
ALK, CD43, CD30*
EMA (Muc-1), CD30*, p63
Syncytial Hodgkin lymphoma
Vimentin, PAX5, MUM1, CD30*
CD15, CD30*, Epstein-Barr virus (EBV)
Large B-cell lymphoma
CD20, PAX5, Bcl-6
Bcl-6
Anaplastic plasmacytoma
Immunoglobin components
CD138, EMA (Muc-1)
*CD30 can be positive in as many as 10% of nasopharyngeal lymphoepithelioma-type carcinomas.
Summary The morphologic panorama of lymphoid malignancies is heterogeneous, and immunophenotypic heterogeneity is even more extreme. Proper tissue examination, in concert with imaging and clinical findings, is of paramount importance in each case. Lest we forget, highgrade lymphomas deserve the same diagnostic workup as tumors of unknown origin to exclude the mimics of lymphoid tumors in nodal and extranodal sites. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. The non-Hodgkin’s lymphoma pathologic classification project. National Cancer Institute sponsored study of classifications of non-Hodgkin’s lymphomas. Cancer. 49:2112–2135, 1982. 2. Harris NL, Jaffe ES, Stein H, et al: A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 84:1361–1392, 1994. 3. Foon KA, Gale RP: Immunologic classification of lymphoma and lymphoid leukemia. Blood Rev. 1:77–88, 1987. 4. Ngan BY, Chen-Levy Z, Weiss LM, et al: Expression in nonHodgkin’s lymphoma of the bcl-2 protein associated with the t(14;18) chromosomal translocation. N Engl J Med. 318:1638– 1644, 1988. 5. Benharroch D, Meguerian-Bedoyan Z, Lamant L, et al: ALKpositive lymphoma: a single disease with a broad spectrum of morphology. Blood. 91:2076–2084, 1998. 6. Foran JM, Cunningham D, Coiffier B, et al: Treatment of mantlecell lymphoma with Rituximab (chimeric monoclonal antiCD20 antibody): analysis of factors associated with response. Ann Oncol. 11(Suppl 1):117–121, 2000. 7. Middleton LP, Price KM, Puig P, et al: Implementation of American Society of Clinical Oncology/College of American Pathologists HER2 Guideline Recommendations in a tertiary care facility increases HER2 immunohistochemistry and fluorescence in situ hybridization concordance and decreases the number of inconclusive cases. Arch Pathol Lab Med. 133:775–780, 2009. 8. Banks PM: Technical Factors in the preparation and evaluation of lymph node biopsies. In Knowles D, editor: Neoplastic Hematopathology, ed 2, 2001, Lippincott Williams & Wilkins, pp 467–482. 9. Riley JK, Sliwkowski MX: CD20: a gene in search of a function. Semin Oncol. 27(6 Suppl 12):17–24, 2000. 10. Shan D, Ledbetter JA, Press OW: Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood. 91(5):1644–1652, 1998. 11. Chu PG, Loera S, Huang Q, et al: Lineage determination of CD20- B-Cell neoplasms: an immunohistochemical study. Am J Clin Pathol. 126(4):534–544, 2006. 12. Plosker GL, Figgitt DP: Rituximab: a review of its use in nonHodgkin’s lymphoma and chronic lymphocytic leukaemia. Drugs. 63(8):803–843, 2003. 13. Hultin LE, Hausner MA, Hultin PM, et al: CD20 (pan-B cell) antigen is expressed at a low level on a subpopulation of human T lymphocytes. Cytometry. 14(2):196–204, 1993. 14. Robillard N, Avet-Loiseau H, Garand R, et al: CD20 is associated with a small mature plasma cell morphology and t(11;14) in multiple myeloma. Blood. 102(3):1070–1071, 2003. 15. Feldman AL, Dogan A: Diagnostic uses of Pax5 immunohistochemistry. Adv Anat Pathol. 14(5):323–334, 2007. 16. Medvedovic J, Ebert A, Tagoh H, et al: Pax5: a master regulator of B cell development and leukemogenesis. Adv Immunol. 111:179–206, 2011. 17. Cobaleda C, Schebesta A, Delogu A, et al: Pax5: the guardian of B cell identity and function. Nat Immunol. 8(5):463–470, 2007. 18. Hagiwara M, Tomita A, Takata K, et al: Primary cutaneous CD30 positive T-cell lymphoproliferative disorders with aberrant expression of PAX5: report of three cases. Pathol Int. 62(4):264– 270, 2012. 19. Song J, Li M, Tretiakova M, et al: Expression patterns of PAX5, c-Met, and paxillin in neuroendocrine tumors of the lung. Arch Pathol Lab Med. 134(11):1702–1705, 2010. 20. Mason DY, Cordell JL, Brown MH, et al: CD79a: a novel marker for B cell neoplasms in routinely processed tissue samples. Blood. 86:1453, 1995. 21. Chu PG, Arber DA: CD79: a review. Appl Immunohistochem Mol Morphol. 9(2):97–106, 2001. 22. Li S, Borowitz MJ: CD79a(+) T-cell lymphoblastic lymphoma with coexisting Langerhans cell histiocytosis. Arch Pathol Lab Med. 125(7):958–960, 2001. 23. Arber DA, Jenkins KA, Slovak ML: CD79 alpha expression in acute myeloid leukemia. High frequency of expression in acute promyelocytic leukemia. Am J Pathol. 149(4):1105–1110, 1996. 24. Ohno H, Fukuhara S: Significance of rearrangement of the BCL6 gene in B-cell lymphoid neoplasms. Leuk Lymphoma. 27(1– 2):53–63, 1997.
188.e1
25. Saglam A, Uner AH: Immunohistochemical expression of Mum-1, Oct-2 and Bcl-6 in systemic anaplastic large cell lymphomas. Tumori. 97(5):634–638, 2011. 26. Rodríguez-Pinilla SM, Atienza L, Murillo C, et al: Peripheral T-cell lymphoma with follicular T-cell markers. Am J Surg Pathol. 32(12):1787–1799, 2008. 27. Gualco G, Weiss LM, Bacchi CE: MUM1/IRF4: A Review. Appl Immunohistochem Mol Morphol. 18(4):301–310, 2010. 28. Falini B, Fizzotti M, Pucciarini A, et al: A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood. 95(6):2084–2092, 2000. 29. Natkunam Y, Warnke RA, Montgomery K, et al: Analysis of MUM1/IRF4 protein expression using tissue microarrays and immunohistochemistry. Mod Pathol. 14(7):686–694, 2001. 30. Sáez AI, Artiga MJ, Sánchez-Beato M, et al: Analysis of octamerbinding transcription factors Oct2 and Oct1 and their coactivator BOB.1/OBF.1 in lymphomas. Mod Pathol. 15(3):211–220, 2002. 31. Salas M, Eckhardt LA: Critical role for the Oct-2/OCA-B partnership in Ig-secreting cells. J Immunol. 171(12):6589–6598, 2003. 32. Matthias P: Lymphoid-specific transcription mediated by the conserved octamer site: who is doing what? Semin Immunol. 10(2):155–163, 1998. 33. García-Cosío M, Santón A, Martín P, et al: Analysis of transcription factor OCT.1, OCT.2 and BOB.1 expression using tissue arrays in classical Hodgkin’s lymphoma. Mod Pathol. 17(12):1531–1538, 2004. 34. McCune RC, Syrbu SI, Vasef MA: Expression profiling of transcription factors Pax-5, Oct-1, Oct-2, BOB.1, and PU.1 in Hodgkin’s and non-Hodgkin’s lymphomas: a comparative study using high throughput tissue microarrays. Mod Pathol. 19(7):1010– 1018, 2006. 35. Advani AS, Lim K, Gibson S, et al: OCT-2 expression and OCT-2/BOB.1 co-expression predict prognosis in patients with newly diagnosed acute myeloid leukemia. Leuk Lymphoma. 51(4):606–612, 2010. 36. Costes V, Magen V, Legouffe E, et al: The Mi15 monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsy specimens. Hum Pathol. 30(12):1405–1411, 1999. 37. Sebestyén A, Berczi L, Mihalik R, et al: Syndecan-1 (CD138) expression in human non-Hodgkin lymphomas. Br J Haematol. 104(2):412–419, 1999. 38. O’Connell FP, Pinkus JL, Pinkus GS: CD138 (syndecan-1), a plasma cell marker immunohistochemical profile in hematopoietic and nonhematopoietic neoplasms. Am J Clin Pathol. 121(2):254–263, 2004. 39. Gualberto A: Brentuximab Vedotin (SGN-35), an antibodydrug conjugate for the treatment of CD30-positive malignancies. Expert Opin Investig Drugs. 21(2):205–216, 2012. 40. Gruss H-J, Herrmann F: CD30 ligand, a member of the TNF ligand superfamily, with growth and activation control CD30+ lymphoid and lymphoma cells. Leuk Lymphoma. 20(5–6):397– 409, 1996. 41. Wezler X, Hust M, Helmsing S, et al: Human antibodies targeting CD30 lymphomas. Hum Antibodies. 21(1):13–28, 2012. 42. Gadgeel SM, Bepler G: Crizotinib: an anaplastic lymphoma kinase inhibitor. Future Oncol. 7(8):947–953, 2011. 43. Drexler HG, Gignac SM, von Wasielewski R, et al: Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas. Leukemia. 14(9):1533–1559, 2000. 44. Ardini E, Magnaghi P, Orsini P, et al: Anaplastic Lymphoma Kinase: role in specific tumours, and development of small molecule inhibitors for cancer therapy. Cancer Lett. 299(2):81–94, 2010. 45. Rodig SJ, Shapiro GI: Crizotinib, a small-molecule dual inhibitor of the c-Met and ALK receptor tyrosine kinases. Curr Opin Investig Drugs. 11(12):1477–1490, 2010. 46. Chilosi M, Pizzolo G: Review of terminal deoxynucleotidyl transferase. Biologic aspects, methods of detection, and selected diagnostic applications. Appl Immunohistochem. 3:209–221, 1995.
188.e2
Immunohistology of Non-Hodgkin Lymphoma
47. Sur M, AlArdati H, Ross C, et al: TdT expression in Merkel cell carcinoma: potential diagnostic pitfall with blastic hematological malignancies and expanded immunohistochemical analysis. Mod Pathol. 20(11):1113–1120, 2007. 48. Tardío JC: CD34-reactive tumors of the skin. An updated review of an ever-growing list of lesions. J Cutan Pathol. 36(1):89–102, 2009. 49. Drew E, Merzaban JS, Seo W, et al: CD34 and CD43 inhibit mast cell adhesion and are required for optimal mast cell reconstitution. Immunity. 22(1):43–57, 2005. 50. Nielsen JS, McNagny KM: CD34 is a key regulator of hematopoietic stem cell trafficking to bone marrow and mast cell progenitor trafficking in the periphery. Microcirculation. 16(6):487–496, 2009. 51. Lanza F, Healy L, Sutherland DR: Structural and functional features of the CD34 antigen: an update. J Biol Regul Homeost Agents. 15(1):1–13, 2001. 52. Moore T, Huang S, Terstappen LW, et al: Expression of CD43 on murine and human pluripotent hematopoietic stem cells. J Immunol. 153(11):4978–4987, 1994. 53. Rupniewska ZM, Roliński J, Bojarska-Junak A: Universal CD43 molecule. Postepy Hig Med Dosw. 54(5):619–638, 2000. 54. Hasanali Z, Sharma K, Epner E: Flipping the cyclin D1 switch in mantle cell lymphoma. Best Pract Res Clin Haematol. 25(2):143–152, 2012. 55. Gautschi O, Ratschiller D, Gugger M, et al: Cyclin D1 in nonsmall cell lung cancer: a key driver of malignant transformation. Lung Cancer. 55(1):1–14, 2007. 56. Sander B: Mantle cell lymphoma: recent insights into pathogenesis, clinical variability, and new diagnostic markers. Semin Diagn Pathol. 28(3):245–255, 2011. 57. Gradowski JF, Sargent RL, Craig FE, et al: Chronic lymphocytic leukemia/small lymphocytic lymphoma with cyclin D1 positive proliferation centers do not have CCND1 translocations or gains and lack SOX11 expression. Am J Clin Pathol. 138(1):132–139, 2012. 58. Snuderl M, Kolman OK, Chen YB, et al: B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma and diffuse large B-cell lymphoma. Am J Surg Pathol. 34(3):327–340, 2010. 59. Kramer MH, Hermans J, Wijburg E, et al: Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood. 92(9):3152–3162, 1998. 60. Masir N, Campbell LJ, Goff LK, et al: BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations. Br J Haematol. 144(5):716–725, 2009. 61. Dorfman DM, Bui MM, Tubbs RR, et al: The CD117 immunohistochemistry tissue microarray survey for quality assurance and interlaboratory comparison: a College of American Pathologists Cell Markers Committee Study. Arch Pathol Lab Med. 130(6):779–782, 2006. 62. Dirnhofer S, Zimpfer A, Went P: The diagnostic and predictive role of kit (CD117). Ther Umsch. 63(4):273–278, 2006. 63. de Silva CM, Reid R: Gastrointestinal stromal tumors (GIST): C-kit mutations, CD117 expression, differential diagnosis and targeted cancer therapy with Imatinib. Pathol Oncol Res. 9(1):13–19, 2003. 64. Moingeon P, Chang HC, Sayre PH, et al: The structural biology of CD2. Immunol Rev. 111:111–144, 1989. 65. Choudhuri K, van der Merwe PA: Molecular mechanisms involved in T cell receptor triggering. Semin Immunol. 19(4):255– 261, 2007. 66. de Vries JE, Yssel H, Spits H: Interplay between the TCR/CD3 complex and CD4 or CD8 in the activation of cytotoxic T lymphocytes. Immunol Rev. 109:119–141, 1989. 67. Bierer BE, Peterson A, Gorga JC, et al: Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor. J Exp Med. 168(3):1145–1156, 1988. 68. Khalidi HS, Medeiros LJ, Chang KL, et al: The immunophenotype of adult acute myeloid leukemia: high frequency of lymphoid antigen expression and comparison of immunophenotpye, French-American-British classification, and karyotypic abnormalities. Am J Clin Pathol. 109:211–220, 1998.
69. Morgado JM, Sánchez-Muñoz L, Teodósio CG, et al: Immunophenotyping in systemic mastocytosis diagnosis: “CD25 positive” alone is more informative than the “CD25 and/or CD2” WHO criterion. Mod Pathol. 25(4):516–521, 2012. 70. Hawse WF, Champion MM, Joyce MV, et al: Cutting edge: Evidence for a dynamically driven T cell signaling mechanism. J Immunol. 188(12):5819–5823, 2012. 71. Proust R, Bertoglio J, Gesbert F: The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling. PLoS One. 7(8):e43200, 2012. 72. Oliveira JL, Grogg KL, Macon WR, et al: Clinicopathologic Features of B-Cell Lineage Neoplasms with Aberrant Expression of CD3: A Study of 21 Cases. Am J Surg Pathol. 36(9):1364– 1370, 2012. 73. Miceli MC, Parnes JR: The roles of CD4 and CD8 in T cell activation. Semin Immunol. 3(3):133–141, 1991. 74. Killeen N, Davis CB, Chu K, et al: CD4 function in thymocyte differentiation and T cell activation. Philos Trans R Soc Lond B Biol Sci.. 342(1299):25–34, 1993. 75. Bamberger M, Santos AM, Gonçalves CM, et al: A new pathway of CD5 glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase. J Biol Chem. 286(35): 30324–30336, 2011. 76. Sestero CM, McGuire DJ, De Sarno P, et al: CD5-Dependent CK2 Activation Pathway Regulates Threshold for T Cell Anergy. J Immunol. 189(6):2918–2930, 2012. 77. Kawano H, Minagawa K, Wakahashi K, et al: Diminished expression of CD5 and/or CD7 surface antigens as the first clue of diagnosis for monoclonal T lymphocytosis. Rinsho Ketsueki. 53(8):785–787, 2012. 78. Hishima T, Fukayama M, Fujisawa M, et al: CD5 expression in thymic carcinoma. Am J Pathol. 145:268–275, 1994. 79. Saati T, Alibaud L, Lamant L, et al: A New Monoclonal AntiCD7 Antibody Reactive on Paraffin Sections. Appl Immunohistochem. 9(4):289–296, 2001. 80. Rausei-Mills V, Chang KL, Gaal KK, et al: Aberrant expression of CD7 in myeloblasts is highly associated with de novo acute myeloid leukemias with FLT3/ITD mutation. Am J Clin Pathol. 129(4):624–629, 2008. 81. Chang H, Yeung J, Brandwein J, et al: CD7 expression predicts poor disease free survival and post-remission survival in patients with acute myeloid leukemia and normal karyotype. Leuk Res. 31(2):157–162, 2007. 82. Kanavaros P, Boulland ML, Petit B, et al: Expression of cytotoxic proteins in peripheral T-cell and natural killer-cell (NK) lymphomas: association with extranodal site, NK or T gamma delta phenotype, anaplastic morphology and CD30 expression. Leuk Lymphoma. 38(3–4):317–326, 2000. 83. Chan JK, Sin VC, Wong KF, et al: Nonnasal lymphoma expressing the natural killer cell marker CD56: a clinicopathologic study of 49 cases of an uncommon aggressive neoplasm. Blood. 89(12):4501–4513, 1997. 84. Pozdnyakova O, Morgan EA, Li B, et al: Patterns of expression of CD56 and CD117 on neoplastic plasma cells and association with genetically distinct subtypes of plasma cell myeloma. Leuk Lymphoma. 53(10):1905–1910, 2012. 85. Tsang WY, Chan JK, Ng CS, et al: Utility of a paraffin sectionreactive CD56 antibody (123C3) for characterization and diagnosis of lymphomas. Am J Surg Pathol. 20(2):202–210, 1996. 86. Pongpruttipan T, Sukpanichnant S, Assanasen T, et al: Extranodal NK/T-cell lymphoma, nasal type, includes cases of natural killer cell and αβ, γδ, and αβ/γδ T-cell origin: a comprehensive clinicopathologic and phenotypic study. Am J Surg Pathol. 36(4):481– 499, 2012. 87. Dukers DF, Vermeer MH, Jaspars LH, et al: Expression of killer cell inhibitory receptors is restricted to true NK cell lymphomas and a subset of intestinal enteropathy-type T cell lymphomas with a cytotoxic phenotype. J Clin Pathol. 54(3):224–228, 2001. 88. Ng CS, Chan JK, Hui PK, et al: Application of a T cell receptor antibody beta F1 for immunophenotypic analysis of malignant lymphomas. Am J Pathol. 132(2):365–371, 1988. 89. Banks PM. Microscopic mimicry of lymphomas: diagnostic pitfalls. Mod Pathol. 12:116–124, 1999. 90. Attygalle AD, Liu H, Shirali S, et al: Atypical marginal zone hyperplasia of mucosa-associated lymphoid tissue: a reactive
References condition of childhood showing immunoglobulin lambda lightchain restriction. Blood. 104:3343–3348, 2004. 91. Bryant RJ, Banks PM, O’Malley DP: Ki67 staining pattern as a diagnostic tool in the evaluation of lymphoproliferative disorders. Histopathology. 48:505–515, 2006. 92. Mason DY, Banks PM, Chan J, et al: Nodular lymphocyte predominance Hodgkin’s disease. A distinct clinicopathological entity. Am J Surg Pathol. 18:526–530, 1994. 93. Gupta D, Lim MS, Medeiros LJ, et al: Small lymphocytic lymphoma with perifollicular, marginal zone, or interfollicular distribution. Mod Pathol. 13:1161–1166, 2000. 94. Hunt JP, Chan JA, Samoszuk M, et al: Hyperplasia of mantle/ marginal zone B cells with clear cytoplasm in peripheral lymph nodes. A clinicopathologic study of 35 cases. Am J Clin Pathol. 116:550–559, 2001. 95. Reynolds DJ, Banks PM, Gulley ML: New characterization of infectious mononucleosis and a phenotypic comparison with Hodgkin’s disease. Am J Pathol. 146:379–388, 1995. 96. Maric I, Bryant R, Abu-Asab M, et al: Human herpesvirus-6associated acute lymphadenitis in immunocompetent adults. Mod Pathol. 17:1427–1433, 2004. 97. Craig F, Gulley ML, Banks PM: Post-transplantation lymphoproliferative disorders. Am J Clin Pathol. 99:265–276, 1993. 98. Thomason RW, Craig FE, Banks PM, et al: Epstein-Barr virus and lymphoproliferation in methotrexate-treated rheumatoid arthritis. Mod Pathol. 9:261–266, 1996. 99. Krishnan C, Warnke RA, Arber DA, et al: PD-1 expression in T-cell lymphomas and reactive lymphoid entities: potential overlap in staining patterns between lymphoma and viral lymphadenitis. Am J Surg Pathol. 34:178–189, 2010. 100. Lim MS, Straus SE, Dale JK, et al: Pathological findings in human autoimmune lymphoproliferative syndrome. Am J Pathol. 153:1541–1550, 1998. 101. Gascoyne RD: Hematopathology approaches to diagnosis and prognosis of indolent B-cell lymphomas. Hematology Am Soc Hematol Educ Program. 299–306, 2005. 102. Wang SA, Wang L, Hochberg EP, et al: Low histologic grade follicular lymphoma with high proliferation index: morphologic and clinical features. Am J Surg Pathol. 29(11):1490–1496, 2005. 103. Huang JC, Finn WG, Goolsby CL, et al: CD5- small B-cell leukemias are rarely classifiable as chronic lymphocytic leukemia. Am J Clin Pathol. 111(1):123–130, 1999. 104. Wierda WG, O’Brien S, Wang X, et al: Multivariable model for time to first treatment in patients with chronic lymphocytic leukemia. J Clin Oncol. 29(31):4088–4095, 2011. 105. Sellner L, Dietrich S, Dreger P, et al: Can prognostic factors be used to direct therapy in chronic lymphocytic leukemia? Curr Hematol Malig Rep. 7(1):3–12, 2012. 106. Gualco G, Weiss LM, Harrington WJ, Jr, et al: BCL6, MUM1, and CD10 expression in mantle cell lymphoma. Appl Immunohistochem Mol Morphol. 18(2):103–108, 2010. 107. Mozos A, Royo C, Hartmann E, et al: SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype. Haematologica. 94(11):1555–1562, 2009. 108. Sander B: Mantle cell lymphoma: recent insights into pathogenesis, clinical variability, and new diagnostic markers. Semin Diagn Pathol. 28(3):245–255, 2011. 109. Nygren L, Baumgartner Wennerholm S, Klimkowska M, et al: Prognostic role of SOX11 in a population-based cohort of mantle cell lymphoma. Blood. 119(18):4215–4223, 2012. 110. Adam P, Schiefer AI, Prill S, et al: Incidence of preclinical manifestations of mantle cell lymphoma and mantle cell lymphoma in situ in reactive lymphoid tissues. Mod Pathol. 2012. doi: 10.1038/modpathol.2012.117. [Epub ahead of print] 111. Gertz MA: Waldenström macroglobulinemia: 2012 update on diagnosis, risk stratification, and management. Am J Hematol. 87(5):503–510, 2012. 112. Lin P, Molina TJ, Cook JR, et al: Lymphoplasmacytic lymphoma and other non-marginal zone lymphomas with plasmacytic differentiation. Am J Clin Pathol. 136(2):195–210, 2011. 113. Swerdlow SH, Campo E, Harris NL, et al, editors: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, ed 4, (World Health Organization Classification of Tumours), Lyon, 2008, International Agency for Research on Cancer (IARC).
188.e3
114. Braggio E, Dogan A, Keats JJ, et al: Genomic analysis of marginal zone and lymphoplasmacytic lymphomas identified common and disease-specific abnormalities. Mod Pathol. 25(5):651–660, 2012. 115. Molina TJ, Lin P, Swerdlow SH, et al: Marginal zone lymphomas with plasmacytic differentiation and related disorders. Am J Clin Pathol. 136(2):211–225, 2011. 116. Traverse-Glehen A, Bertoni F, Thieblemont C, et al: Nodal marginal zone B-cell lymphoma: a diagnostic and therapeutic dilemma. Oncology (Williston Park). 26(1):92–99, 103–104, 2012. 117. Arcaini L, Lucioni M, Boveri E, et al: Nodal marginal zone lymphoma: current knowledge and future directions of an heterogeneous disease. Eur J Haematol. 83(3):165–174, 2009. 118. Swerdlow SH: Pediatric follicular lymphomas, marginal zone lymphomas, and marginal zone hyperplasia. Am J Clin Pathol. 122(Suppl):S98–S109, 2004. 119. Isaacson PG, Du MQ: MALT lymphoma: from morphology to molecules. Nat Rev Cancer. 4(8):644–653, 2004. 120. Jaso J, Chen L, Li S, et al: CD5-positive mucosa-associated lymphoid tissue (MALT) lymphoma: a clinicopathologic study of 14 cases. Hum Pathol. 43(9):1436–1443, 2012. 121. João C, Farinha P, da Silva MG, et al: Cytogenetic abnormalities in MALT lymphomas and their precursor lesions from different organs. A fluorescence in situ hybridization (FISH) study. Histopathology. 50(2):217–224, 2007. 122. Remstein ED, Dogan A, Einerson RR, et al: The incidence and anatomic site specificity of chromosomal translocations in primary extranodal marginal zone B-cell lymphoma of mucosaassociated lymphoid tissue (MALT lymphoma) in North America. Am J Surg Pathol. 30(12):1546–1553, 2006. 123. Nakamura S, Ye H, Bacon CM, et al: Clinical impact of genetic aberrations in gastric MALT lymphoma: a comprehensive analysis using interphase fluorescence in situ hybridization. Gut. 56(10):1358–1363, 2007. 124. Smith SB, Snow AN, Perry RL, et al: Helicobacter pylori: to stain or not to stain? Am J Clin Pathol. 137(5):733–738, 2012. 125. Sherman MJ, Hanson CA, Hoyer JD: An assessment of the usefulness of immunohistochemical stains in the diagnosis of hairy cell leukemia. Am J Clin Pathol. 136(3):390–399, 2011. 126. Tiacci E, Trifonov V, Schiavoni G, et al: BRAF mutations in hairy-cell leukemia. N Engl J Med. 364(24):2305–2315, 2011. 127. Watkins AJ, Huang Y, Ye H, et al: Splenic marginal zone lymphoma: characterization of 7q deletion and its value in diagnosis. J Pathol. 220(4):461–474, 2010. 128. Kanellis G: Splenic diffuse red pulp small B-cell lymphoma: revision of a series of cases reveals characteristic clinicopathologic features. Haematologica. 95:1122–1129, 2010. 129. Dunphy CH: Reaction patterns of TRAP and DBA.44 in hairy cell leukemia, hairy cell variant, and nodal and extranodal marginal zone B-cell lymphomas. Appl Immunohistochem Mol Morphol. 16:135–139, 2008. 130. Petit B, Parrens M, Soubeyran I, et al: Among 157 marginal zone lymphomas, DBA.44(CD76) expression is restricted to tumour cells infiltrating the red pulp of the spleen with a diffuse architectural pattern. Histopathology. 54:626–631, 2009. 131. Summers TA, Jaffe ES: Hairy cell leukemia diagnostic criteria and differential diagnosis. Leuk Lymphoma. 52(Suppl 2):6–10, 2011. 132. Traverse-Glehen A, Baseggio L, Bauchu EC, et al: Splenic red pulp lymphoma with numerous basophilic villous lymphocytes: a distinct clinicopathologic and molecular entity? Blood. 111:2253–2260, 2008. 133. Lorsbach RB, Hsi ED, Dogan A, et al: Plasma cell myeloma and related neoplasms. Am J Clin Pathol. 136(2):168–182, 2011. 134. Alizadeh AA, Eisen MB, Davis RE, et al: Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 403(6769):503–511, 2000. 135. Cabanillas F: Non-Hodgkin’s lymphoma: the old and the new. Clin Lymphoma Myeloma Leuk. 11(Suppl 1):S87–S90, 2011. 136. Hans CP, Weisenburger DD, Greiner TC, et al: Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 103(1): 275–282, 2004.
188.e4
Immunohistology of Non-Hodgkin Lymphoma
137. Morton LM, Cerhan JR, Hartge P, et al: Immunostaining to identify molecular subtypes of diffuse large B-cell lymphoma in a population-based epidemiologic study in the pre-rituximab era. Int J Mol Epidemiol Genet. 2(3):245–252, 2011. 138. Choi WW, Weisenburger DD, Greiner TC, et al: A new immunostain algorithm classifies diffuse large B-cell lymphoma into molecular subtypes with high accuracy. Clin Cancer Res. 15(17):5494–5502, 2009. 139. Meyer PN, Fu K, Greiner TC, et al: Immunohistochemical methods for predicting cell of origin and survival in patients with diffuse large B-cell lymphoma treated with rituximab. J Clin Oncol. 29(2):200–207, 2011. 140. Natkunam Y, Lossos IS, Taidi B, et al: Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation. Blood. 105(10):3979– 3986, 2005. 141. Gualco G, Bacchi LM, Domeny-Duarte P, et al: The contribution of HGAL/GCET2 in immunohistological algorithms: a comparative study in 424 cases of nodal diffuse large B-cell lymphoma. Mod Pathol. 2012. doi: 10.1038/modpathol.2012.119. [Epub ahead of print] 142. Seegmiller AC, Garcia R, Huang R, et al: Simple karyotype and bcl-6 expression predict a diagnosis of Burkitt lymphoma and better survival in IG-MYC rearranged high-grade B-cell lymphomas. Mod Pathol. 23(7):909–920, 2010. 143. Aukema SM, Siebert R, Schuuring E, et al: Double-hit B-cell lymphomas. Blood. 117(8):2319–2331, 2011. 144. Tapia G, Lopez R, Muñoz-Mármol AM, et al: Immunohistochemical detection of MYC protein correlates with MYC gene status in aggressive B cell lymphomas. Histopathology. 59(4):672– 678, 2011. 145. Went P, Zimpfer A, Tzankov A, et al: CD5 expression in de novo diffuse large B-cell lymphomas. Ann Oncol. 20(4):789–790, 2009. 146. Vela-Chávez T, Adam P, Kremer M, et al: Cyclin D1 positive diffuse large B-cell lymphoma is a post-germinal center-type lymphoma without alterations in the CCND1 gene locus. Leuk Lymphoma. 52(3):458–466, 2011. 147. Gibson SE, Hsi ED: Epstein-Barr virus-positive B-cell lymphoma of the elderly at a United States tertiary medical center: an uncommon aggressive lymphoma with a nongerminal center B-cell phenotype. Hum Pathol. 40(5):653–661, 2009. 148. Hoeller S, Tzankov A, Pileri SA, et al: Epstein-Barr virus-positive diffuse large B-cell lymphoma in elderly patients is rare in Western populations. Hum Pathol. 41(3):352–357, 2010. 149. Adam P, Bonzheim I, Fend F, et al: Epstein-Barr virus-positive diffuse large B-cell lymphomas of the elderly. Adv Anat Pathol. 18(5):349–355, 2011. 150. Hsi ED, Lorsbach RB, Fend F, et al: Plasmablastic lymphoma and related disorders. Am J Clin PathoL. 136(2):183–194, 2011. 151. Montes-Moreno S, Montalbán C, Piris MA: Large B-cell lymphomas with plasmablastic differentiation: a biological and therapeutic challenge. Leuk Lymphoma. 53(2):185–194, 2012. 152. Tousseyn T, De Wolf-Peeters C: T cell/histiocyte-rich large B-cell lymphoma: an update on its biology and classification. Virchows Arch. 459(6):557–563, 2011. 153. Laurent C, Do C, Gascoyne RD, et al: Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis. J Clin Oncol. 27(25):4211– 4216, 2009. 154. Morgan EA, Nascimento AF: Anaplastic lymphoma kinasepositive large B-cell lymphoma: an underrecognized aggressive lymphoma. Adv Hematol. 2012:529–572, 2012. 155. Montes-Moreno S, Montalbán C, Piris MA: Large B-cell lymphomas with plasmablastic differentiation: a biological and therapeutic challenge. Leuk Lymphoma. 53(2):185–194, 2012. 156. Zamò A, Malpeli G, Scarpa A, et al: Expression of TP73L is a helpful diagnostic marker of primary mediastinal large B-cell lymphomas. Mod Pathol. 18(11):1448–1453, 2005. 157. Hoeller S, Zihler D, Zlobec I, et al: BOB.1, CD79a and cyclin E are the most appropriate markers to discriminate classical Hodgkin’s lymphoma from primary mediastinal large B-cell lymphoma. Histopathology. 56(2):217–228, 2010. 158. Naresh KN, Ibrahim HA, Lazzi S, et al: Diagnosis of Burkitt lymphoma using an algorithmic approach—applicable in both
resource-poor and resource-rich countries. Br J Haematol. 2011. doi: 10.1111/j.1365–2141.2011.08771.x. [Epub ahead of print] 159. Seegmiller AC, Garcia R, Huang R, et al: Simple karyotype and bcl-6 expression predict a diagnosis of Burkitt lymphoma and better survival in IG-MYC rearranged high-grade B-cell lymphomas. Mod Pathol. 23(7):909–920, 2010. 160. Eberle FC, Salaverria I, Steidl C, et al: Gray zone lymphoma: chromosomal aberrations with immunophenotypic and clinical correlations. Mod Pathol. 24(12):1586–1597, 2011. 161. Salama ME, Rajan Mariappan M, Inamdar K, et al: The value of CD23 expression as an additional marker in distinguishing mediastinal (thymic) large B-cell lymphoma from Hodgkin lymphoma. Int J Surg Pathol. 18(2):121–128, 2010. 162. Vose J, Armitage J, Weisenburger D: International T-Cell Lymphoma Project. International peripheral T-cell and natural killer/ T-cell lymphoma study: pathology findings and clinical outcomes. J Clin Oncol. 26(25):4124–4130, 2008. 163. Savage KJ: Update: peripheral T-cell lymphomas. Curr Hematol Malig Rep. 6(4):222–230, 2011. 164. Chizhevsky V, O’Malley DP: Utility of Bcl2, PD1 and CD25 Expressions in the diagnosis of T-Cell lymphoma. CAP Annual Meeting. Dallas TX. 2011. 165. Weiss L: Lymph Nodes, Cambridge, UK, 2008, Cambridge University Press. 166. Jiang L, Yuan CM, Hubacheck J, et al: Variable CD52 expression in mature T cell and NK cell malignancies: implications for alemtuzumab therapy. Br J Haematol. 145(2):173–179, 2009. 167. Weisenburger DD, Savage KJ, Harris NL, et al: Peripheral T-cell lymphoma, not otherwise specified: a report of 340 cases from the International Peripheral T-cell Lymphoma Project. Blood. 117(12):3402–3408, 2011. 168. Cotta CV, Hsi ED: Pathobiology of mature T-cell lymphomas. Clin Lymphoma Myeloma. 8(Suppl 5):S168–S179, 2008. 169. Barry TS, Jaffe ES, Sorbara L, et al: Peripheral T-cell lymphomas expressing CD30 and CD15. Am J Surg Pathol. 27(12):1513– 1522, 2003. 170. Federico M, Rudiger T, Bellei M, et al: Clinicopathologic Characteristics of Angioimmunoblastic T-Cell Lymphoma: Analysis of the International Peripheral T-Cell Lymphoma Project. J Clin Oncol. 2012. [Epub ahead of print] 171. Au WY, Weisenburger DD, Intragumtornchai T, et al: Clinical differences between nasal and extranasal natural killer/T-cell lymphoma: a study of 136 cases from the International Peripheral T-Cell Lymphoma Project. Blood. 113(17):3931–3937, 2009. 172. Kim SJ, Kim BS, Choi CW, et al: Ki-67 expression is predictive of prognosis in patients with stage I/II extranodal NK/T-cell lymphoma, nasal type. Ann Oncol. 18(8):1382–1387, 2007. 173. Cao W, Liu Y, Zhang H, et al: Expression of LMP-1 and Cyclin D1 protein is correlated with an unfavorable prognosis in nasal type NK/T cell lymphoma. Mol Med Report. 1(3):363–368, 2008. 174. van de Water JM, Cillessen SA, Visser OJ, et al: Enteropathy associated T-cell lymphoma and its precursor lesions. Best Pract Res Clin Gastroenterol. 24(1):43–56, 2010. 175. Delabie J, Holte H, Vose JM, et al: Enteropathy-associated T-cell lymphoma: clinical and histological findings from the international peripheral T-cell lymphoma project. Blood. 118(1):148– 155, 2011. 176. Ensari A: Gluten-sensitive enteropathy (celiac disease): controversies in diagnosis and classification. Arch Pathol Lab Med. 134(6):826–836, 2010. 177. Wilcox RA: Cutaneous T-cell lymphoma: 2011 update on diagnosis, risk-stratification, and management. Am J Hematol. 86(11):928–948, 2011. 178. Burg G, Kempf W, Smoller B, et al: Mycosis fungoides. In LeBoit PE, Burg G, Weedon D, et al, editors: World Health Organization Calssification of Tumours. Pathology and genetic of skin tumours, Lyon, 2006, IARC Press, pp 169–174. 179. Pavlovsky L, Mimouni D, Amitay-Laish I, et al: Hyperpigmented mycosis fungoides: an unusual variant of cutaneous T-cell lymphoma with a frequent CD8+ phenotype. J Am Acad Dermatol. 67(1):69–75, 2012. 180. Nikolaou VA, Papadavid E, Katsambas A, et al: Clinical characteristics and course of CD8+ cytotoxic variant of mycosis
References fungoides: a case series of seven patients. Br J Dermatol. 161(4):826–830, 2009. 181. Fischer L, Hummel M, Burmeister T, et al: Skewed expression of natural-killer (NK)-associated antigens on lymphoproliferations of large granular lymphocytes (LGL). Hematol Oncol. 24(2):78–85, 2006. 182. Morice WG, Kurtin PJ, Leibson PJ, et al: Demonstration of aberrant T-cell and natural killer-cell antigen expression in all cases of granular lymphocytic leukaemia. Br J Haematol. 120(6):1026– 1036, 2003. 183. Matutes E: Adult T-cell leukaemia/lymphoma. J Clin Pathol. 60(12):1373–1377, 2007. 184. Suzumiya J, Ohshima K, Tamura K, et al: The International Prognostic Index predicts outcome in aggressive adult T-cell leukemia/lymphoma: analysis of 126 patients from the International Peripheral T-Cell Lymphoma Project. Ann Oncol. 20(4):715–721, 2009. 185. Kraus MD, Shenoy S, Chatila T, et al: Light microscopic, immunophenotypic, and molecular genetic study of autoimmune lymphoproliferative syndrome caused by fas mutation. Pediatr Dev Pathol. 3(1):101–109, 2000. 186. Kneile JR, Tan G, Suster S, et al: Expression of CD30 (Ber-H2) in nasopharyngeal carcinoma, undifferentiated type and lymphoepithelioma-like carcinoma. A comparison study with anaplastic large cell lymphoma. Histopathology. 48:855–861, 2006. 187. Lauwers GY, Scott GV, Karpeh MS: Immunohistochemical evaluation of bcl-2 protein expression in gastric adenocarcinomas. Cancer. 75:2209–2213, 1995. 188. Bos R, van Diest PJ, van der Groep P, et al: Protein expression of B-cell lymphoma gene 6 (BCL-6) in invasive breast cancer is associated with cyclin D1 and hypoxia-inducible factor-1alpha (HIF-1alpha). Oncogene. 22:8948–8951, 2003. 189. Chu PG, Arber DA, Weiss LM: Expression of T/NK-cell and plasma cell antigens in nonhematopoietic epithelioid neoplasms.
188.e5
An immunohistochemical study of 447 cases. Am J Clin Pathol. 120:64–70, 2003. 190. Redondo M, Villar E, Torres-Muñoz J, et al: Overexpression of clusterin in human breast carcinoma. Am J Pathol. 157:393–399, 2000. 191. Marchevsky AM: Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Arch Pathol Lab Med. 132:397–401, 2008. 192. Chilosi M, Castelli P, Martignoni G, et al: Neoplastic epithelial cells in a subset of human thymomas express the B cell-associated CD20 antigen. Am J Surg Pathol. 16:988–997, 1992. 193. Falini B, Pileri S, Stein H, et al: Variable expression of leucocytecommon (CD45) antigen in CD30 (Ki1)-positive anaplastic large-cell lymphoma: implications for the differential diagnosis between lymphoid and nonlymphoid malignancies. Hum Pathol. 21:624–629, 1990. 194. Strickler JG, Michie SA, Warnke RA, et al: The “syncytial variant” of nodular sclerosing Hodgkin’s disease. Am J Surg Pathol. 10:470–477, 1986. 195. Lai R, Medeiros LJ, Dabbagh L, et al: Sinusoidal CD30-positive large B-cell lymphoma: a morphologic mimic of anaplastic large cell lymphoma. Mod Pathol. 13:223–228, 2000. 196. Gonzalez-Crussi F, Mangkornkanok M, Hsueh W: Large-cell lymphoma. Diagnostic difficulties and case study. Am J Surg Pathol. 11:59–65, 1987. 197. Stacchini A, Barreca A, Demurtas A, et al: Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature. Histopathology. 60:452–459, 2012. 198. Moretti L, Medeiros LJ, Kunkalla K, et al: N-terminal PAX8 polyclonal antibody shows cross-reactivity with N-terminal region of PAX5 and is responsible for reports of PAX8 positivity in malignant lymphomas. Mod Pathol. 25(2):231–236, 2012.
C H A P T E R 7
IMMUNOHISTOLOGY NEOPLASMS
OF
MELANOCYTIC
VICTOR G. PRIETO
Overview 189 Biology of Antigens and Antibodies 189 Neuroendocrine Markers in Melanocytic Lesions 196 Sentinel Lymph Node Biopsies for Metastatic Melanoma 197 Application of Immunohistochemistry to Selected Differential Diagnosis 198 Prognostic Markers and Targeted Therapy for Melanoma 202 Summary 203
Overview Melanoma continues to represent one of the greatest diagnostic challenges in surgical pathology and is an important source of litigation. Both as a primary lesion in the skin and in metastatic sites, this neoplasm is capable of assuming many different macroscopic and histologic appearances that mimic other diseases, both benign and malignant. The four main microscopic phenotypes of primary malignant melanoma (MM) include 1) superficial spreading, 2) lentigo maligna, 3) acral lentiginous mucosal, and 4) nodular melanoma (Figs. 7-1 to 7-4). Other morphologic variants include nevoid, balloon (clear) cell, pleomorphic sarcomatoid, spindle cell/desmoplastic/neuroid, small cell (neuroendocrinelike), signet-ring cell, myxoid, metaplastic, and rhabdoid.1-3 All of these variants may be amelanotic in nature, which renders the morphologic differential diagnosis difficult. Accordingly, electron microscopy, immunohistology, and cytogenetic analysis have become exceedingly important in the accurate recognition of melanoma. This discussion focuses on the second of those investigative modalities and is directed principally at diagnostic questions concerning melanocytic tumors in general.4 In addition, we will discuss the use of immunohistochemistry (IHC) to help determine the
prognosis and, possibly, aid in screening for targeted therapies.
Biology of Antigens and Antibodies Filamentous Proteins in Melanocytic Neoplasms Intermediate filament protein (IFP) analysis has been an important cornerstone of IHC evaluation for nearly 30 years. Immunoanalysis for keratins, vimentin, desmin, neurofilament proteins, and glial fibrillary acidic protein (GFAP) are broadly capable of distinguishing between histologically similar classes of neoplasms with dissimilar lineages.5,6 Regarding melanocytic neoplasms, nevi and MMs typically are labeled only for vimentin, and only rarely do they express keratins or the other IFPs (Fig. 7-5).6-10 Moreover, the density of vimentin in melanogenic tumors is high, and yields intense immunoreactivity for that marker in most instances. Similarly, GFAP and desmin have been reported in a small minority (<1%) of MMs,2 usually tumors that demonstrate “metaplastic” sarcomatoid microscopic features or, conversely, desmoplastic and neuroid characteristics. For practical purposes, and in specific reference to studies on paraffin sections for these IFPs, more than 95% of melanocytic neoplasms are labeled solely for vimentin, even after application of techniques such as heat-mediated epitope retrieval. Muscle-specific actin, recognized by monoclonal antibody HHF-35; α-isoform, or smooth muscle actin (SMA), recognized by antibody 1A4; and caldesmon are also preferentially seen in nonepithelial, nonmelanocytic, nonglial tissues. They are rarely detected in melanocytic lesions, especially in spindle cell melanomas that show myofibroblastic differentiation.2
Cell Membrane Proteins A diversity of proteins associated with cell membranes come into play in relationship to the differential 189
190
Immunohistology of Melanocytic Neoplasms
Figure 7-1 Lentigo maligna–type melanoma, characterized by atypical melanocytes arranged in a confluent pattern in sundamaged skin. Note the flat pattern of the epidermis with effacement of the rete ridges (arrow).
Figure 7-3 Acral lentiginous melanoma. Large, dendritic, pigmented melanocytes arranged in single cells and nests at all levels of the epidermis.
diagnosis of melanoma. Nonetheless, these fall into two broad categories: those associated with epithelial cells and those that relate to hematopoietic elements.
plasmacellular tumors.11,12 Melanocytic proliferations are rarely reactive for this marker (Fig. 7-6).11-13 It may be observed within foci of melanomas that border zones of geographic necrosis, but those areas should be considered as likely to be artifactual.
EPITHELIAL DETERMINANTS Epithelial Membrane Antigen
Carcinoembryonic Antigen
Epithelial membrane antigen (EMA), a family of glycoproteins related to the milk fat globule proteins, is expressed by a variety of epithelia and their neoplasms.11 The principal exceptions in the latter group include germ cell tumors, adrenocortical proliferations, and hepatocellular neoplasms.12,13 An EMA-like moiety also may be observed in selected lymphoid and
Carcinoembryonic antigen (CEA) characterizes a family of glycoproteinaceous cell membrane constituents present mainly in tissues and neoplasms with endodermal differentiation. In the past, it has been contended that CEA may be observed in melanomas,14 but this observation is considered to be of an artifactual nature.15 Such reagents recognize several proteins other than CEA (e.g., nonspecific cross-reacting antigen, biliary
Figure 7-2 Superficial spreading melanoma. Proliferation of large melanocytes as nests and single cells with pagetoid upward migration.
Figure 7-4 Nodular melanoma. In situ and invasive melanoma. Notice the lack of intraepidermal extension beyond the dermal component.
Biology of Antigens and Antibodies
191
group of adjunctive epithelial markers are useful supplements to others for keratin, EMA, and CEA in helping to exclude epithelial neoplasms when considering the differential diagnosis of melanoma. It can safely be stated that virtually all somatic carcinomas should be reactive for at least one of the membrane determinants cited earlier in this discussion, in addition to keratin, in contrast to those rare melanomas that express keratin but lack the other markers. Placental-like Alkaline Phosphatase
Figure 7-5 Keratin is only rarely expressed in melanoma paraffin sections, usually low-molecular-weight keratins, and in a patchy manner (antikeratin “cocktail” AE1/AE3, CAM5.2, Zym5.2, and MNF116 and diaminobenzidine).
Placental-like alkaline phosphatase (PLAP) is an isozyme commonly synthesized by neoplastic germ cells and certain somatic epithelial malignancies.18 As such, it is a useful screening marker for gonadal tumors such as seminoma, embryonal carcinoma, and yolk sac carcinoma, all of which may enter the differential diagnosis of melanoma. In contrast, melanocytic proliferations are consistently negative for PLAP, although we have recently encountered a melanoma lesion with focal PLAP expression.19 HEMATOPOIETIC MARKERS
glycoprotein), many of which are not restricted to epithelial cells. If monoclonal antibodies are used with suitable specificity to restricted CEA epitopes, melanocytic neoplasms should be negative.16 Tumor-associated Glycoprotein-72 and BER-EP4 and MOC-31 Antigens
Tumor-associated glycoprotein-72 (TAG-72/CA72-4, recognized by monoclonal antibody B72.3), BER-EP4, and MOC-31 are all cell-membrane glycoproteins most consistently synthesized by epithelial cells.17 Rare exceptions to this do exist, however, such as TAG-72 presence in some epithelioid vascular tumors; but melanocytic neoplasms should be negative for these three glycoproteins.16 Therefore, IHC studies against this
Figure 7-6 Poorly differentiated, amelanotic melanoma focally expresses epithelial membrane antigen (EMA; arrows), anti-EMA, and diaminobenzidine.
Selected cell-surface antigens typically associated with hematopoietic cells and neoplasms also are potentially seen in melanocytic cells and neoplasms. Among others, these include CD10, CD44, CD56, CD57, CD59, CD68, CD74, CD99, CD117 (c-kit protein), CD146, class II major histocompatibility antigens (MHC2As; human leukocyte antigen [HLA]-DR, HLADP, and HLA-DQ), β-2-microglobulin (B2M), and Bcl-2 protein.20-28 These markers may be seen in some examples of Spitz nevus (epithelioid and spindle cell), architecturally disordered (dysplastic) nevus, and melanoma. Conversely, melanocytes uniformly lack other hematopoietic determinants that may enter into differential diagnostic evaluations of melanoma, such as terminal deoxynucleotidyl transferase (TdT); factor XIIIa; myeloperoxidase; and CD15, CD20, CD21, CD23, CD30, CD35, CD43, CD45, and CD138.29,30 The expression of MHC2A proteins and B2M by melanocytic proliferations appears to be a property confined to inflamed intradermal or architecturally disordered nevi and occasional melanomas.31 Interestingly, MMs that escape immune surveillance have been noted to downregulate their expression of B2M and histocompatibility antigens over time27 through mutations in the corresponding gene complexes and other mechanisms. Because of the immunophenotypic heterogeneity seen in melanocytic lesions, as well as other tumors, for MHC2A and B2M, such determinants should not be used to distinguish or rule out a melanocytic lesion. The ability of melanomas to express Bcl-2 protein and CD10, CD68, CD56, CD57, CD99, and CD117 creates a possible diagnostic pitfall in that such markers may lead the clinician to interpret these as lymphomas, histiocytic lesions, primitive neuroectodermal or neuroendocrine neoplasms, and gastrointestinal stromal tumors. As usual, the application of carefully constructed panels of antibody reagents, tailored to specific diagnostic scenarios, should preclude those mistakes.
192
Immunohistology of Melanocytic Neoplasms
NB84
NB84 is a monoclonal antibody raised against a cellmembrane determinant found in neuroblastomas. It is active in paraffin sections and labels not only neuroblastic neoplasms but also a subset of primitive neuroectodermal tumors (PNETs).32 Melanocytic proliferations are nonreactive with this reagent, a fact helpful when the differential diagnosis centers around small cell melanoma versus a neuroblastic or neuroectodermal tumor arising in a large congenital nevus or neurocristic hamartoma.33
Calcium-Binding Proteins Several proteins that affect intracellular calcium metabolism can be seen in melanocytic proliferations, including Annexins V and VI, Cap G, calmodulin, calretinin, and S-100 protein.34 The most important ones for the differential diagnosis are discussed further here. S-100 PROTEIN
One of the first and most important markers for melanoma is S-100 protein. This 21-kD moiety was first detected in glial cells of the central nervous system (CNS), and it was given its name because of solubility in 100% saturated ammonium sulfate solution.35 In 1981, Gaynor and colleagues36 recognized the fact that S-100 protein was present in human melanoma cells also, leading to its widespread application as a diagnostic indicator (Fig. 7-7).
S
The function of S-100 has not been determined with precision; however, it is thought to function in intracellular calcium trafficking or microtubular assembly or both.37,38 It has a loose physiologic relationship to calmodulin, another calcium flux protein.34 Two subunits to S-100, α and β, yield three possible dimeric forms: α-α, α-β, and β-β.39 Melanocytes synthesize only the first of those combinations. Immunoreactivity for S-100 protein is both nuclear and cytoplasmic. Immunoelectron microscopic studies have confirmed that this marker is present in both intracellular compartments in normal and neoplastic melanocytes.40 There are many antibodies against S-100, some of which are dimer-specific monoclonal products.41 In general practice, most clinical laboratories still use heteroantisera that recognize all three isotypes of the protein against this marker, yielding a high-sensitivity screening tool. In this context, the author’s experience has been that more than 98% of MM cases can be labeled for S-100 at least focally, regardless of histologic subtype. Smoller42 also reported a similar experience with S-100 protein (97.4%). Other S-100 protein–positive tumor types that enter into differential diagnosis include various carcinomas (e.g., breast carcinomas), selected histiocytic proliferations, gliomas, peripheral nerve sheath tumors (PNSTs), and Langerhans histiocytosis.40,42-46 It should be obvious, then, that S-100 is most valuable in this setting as an initial screening reagent for melanocytic tumors rather than as a specific marker for such neoplasms. Various isoforms of S-100 protein (A2, A6, A8/A9, and A12) have also been considered diagnostically in regard to various melanocytic lesions. Ribe and McNutt47 suggested that S-100A6 was differentially expressed by Spitz nevi and melanomas. All Spitz tumors were A6 reactive, whereas only 33% of melanomas showed positivity. Moreover, differences in the scope of labeling were observed, with Spitz nevi being globally A6 reactive; in contrast, melanomas showed weak and patchy staining. Another possible application for this monoclonal marker (S-100A6) is secondary to its expression in cellular neurothekeomas.48 Possible pitfalls are related to S-100 protein expression. The antibody S-100 protein in formalin-fixed tissue may not work on frozen sections, alcohol-fixed tissue, or on formalin-fixed, paraffin-embedded (FFPE) sections of previously frozen tissue. Furthermore, if the tissue is overfixed or underfixed, S-100 expression may be impaired. Also, it has been suggested that S-100 expression may be decreased in sun-damaged melanocytes.49 CALRETININ
Figure 7-7 Intense nuclear and cytoplasmic immunoreactivity for S-100 protein in primary cutaneous melanoma.
Calretinin is a cytoplasmic 31-kD protein first isolated from CNS tissues.50 It is also seen in peripheral nerves in the skin and elsewhere.51 Otherwise, this polypeptide is rather restricted in distribution, having been detected thus far only in mesothelium,50 germinal-surface epithelium of the ovary,50 and certain adenocarcinomas, most notably a subset in the colon,
Biology of Antigens and Antibodies
Figure 7-8 A, Metastatic melanoma with severe cytologic atypia. B, Very weak expression of S-100 protein in rare melanocytes. C, Compare the S-100 protein immunoresult with that of anti–MART-1, shown here, which highlights the majority of melanoma cells (anti–S-100 protein and anti–MART-1, diaminobenzidine, and hematoxylin).
A
rectum, and skin.52-54 Melanocytic tumors are not included in the list of neoplasms that show potential calretinin immunoreactivity.
Melanocyte-Specific Monoclonal Antibodies Beginning in the early 1980s, monoclonal antibody technology was applied to a quest for a melanomaspecific reagent, not only for diagnostic but also for therapeutic utility.55 A variety of antimelanocyte hybridoma products have been described, some of which are applicable to routinely processed tissue specimens.42 Products with activity restricted to frozen tissue substrates include PAL-M1 and PAL-M2, 691-13-17 and 691-15-Nu4B, and MEL (melanoma) series antibodies 1 through 4. In contrast, human melanosome-associated antigen (HMSA)-2, 2-139-1, 6-26-3, KBA62, 1C11, 7H11, MEL-CAM, MEL-5, and SM5-1 do have immunoreactivity with FFPE specimens and are therefore more useful in routine specimens.42,56,57 Products that have been used in a clinical context are considered in the following sections.
B
193
C
the inner membranes of types 1, 2, and 3 premelanosomes; they similarly serve as potential targets for cytotoxic T lymphocytes, probably in concert with MHC2A.61 Indeed, one marker in this group—MART-1, melanoma antigen recognized by T-cells 1—was named specifically for that property; it is recognized by two monoclonal antibodies, A103 and M2-7C10, and is also known as melan-A (Figs. 7-8 and 7-9).62-73 Nucleotide sequence analysis performed by Adema and colleagues59 has shown by alternative splicing that the cDNAs for gp100 and PMel-17 emanate from a single gene. This conclusion gained further support by their observation that gp100 and PMel-17 are consistently expressed concomitantly by both nonneoplastic and neoplastic melanocytic populations.74 The protein
gp100 AND PMEL-17–RELATED MONOCLONAL ANTIBODIES
Several monoclonal antibodies were raised against a glycoprotein antigenic group restricted to cells of melanocytic lineage. This group is designated “gp100,” and its corresponding cDNA has been cloned.58,59 Two proteins are encoded by that nucleic acid sequence, gp100 itself, with a molecular weight of 100 kD, and gp10, with a molecular weight of 10 kD.59 The translational product related to gp100-C1 complementary DNA (cDNA) is closely homologous, but not identical, to yet another melanocytic protein, PMel-17.60 Both are localized to
Figure 7-9 Diffuse cytoplasmic immunoreactivity for MART-1/ melan-A protein in this metastatic melanoma.
194
Immunohistology of Melanocytic Neoplasms
A
B
target of HMB-45 is probably a unique, premelanosomerelated polypeptide.75,76 Interestingly, gp100 transcripts are not specific for melanocytes and have been observed in other tissue types.58 Nonetheless, translation of the mRNA in question apparently occurs only in melanocytic elements. This finding strongly suggests that immunohistology is the most practical technologic method for assessment of gp100 as a melanocyte marker (Figs. 7-10 and 7-11) and that nucleic acid–based procedures, such as in situ hybridization (ISH) and polymerase chain reaction (PCR), are likely to be positive in other, nonmelanocytic pigment-producing cells. Antibodies in the gp100/PMel-17 group include NKI-beteb and NKI/C3, HMB-45 and HMB-50, and MART-1/melan-A.59,60,62-74,76-94 These demonstrate variable levels of specificity and sensitivity for
HMB Figure 7-11 Intense cytoplasmic positivity with HMB-45 in epithelioid melanoma metastatic to a lymph node (HMB-45 and diaminobenzidine).
Figure 7-10 Labeling with HMB-45 in a nevus (A) and in a primary melanoma (B). The deep portion of the nevus has fewer positive cells than the upper portion (maturation). In contrast, both superficial and deep areas in a melanoma show similar numbers of positive cells. (HMB-45, diaminobenzidine, and hematoxylin).
melanocytes, nevi, and melanomas vis-à-vis other cell lineages and tumor types. In practice, HMB-45 and MART-1 have thus far enjoyed the greatest use as agents to confirm the identity of S-100 protein–positive neoplasms as melanocytic in nature. Unfortunate peculiarities exist that relate to the commercial distribution of HMB-45. Dr. Mark Wick evaluated the original supernatant product from the HMB-45 clone, received as a gift from Dr. Allen Gown, one of the developers of the antibody, in the mid-1980s. He then found HMB-45 to be more than 95% sensitive for melanoma and essentially 100% specific for that diagnosis among non–spindle-cell malignancies.94 Afterward, HMB-45 was sold to a commercial firm that marketed an impure form of the hybridoma product. It showed a much lower degree of specificity and was seen to label a variety of tumors other than melanomas.40,95-98 When other firms subsequently assumed distributorship of HMB-45, its specificity improved. HMB-45, HMB-50, MART-1, and other gp100related reagents can also label angiomyolipomas, lymphangioleiomyomatosis, clear-cell myomelanocytic “sugar” tumors of the lung, and perivascular epithelioid cell tumors (PEComas).99-105 These lesions manifest ultrastructural evidence of premelanosome synthesis and therefore have at least partial melanocytic differentiation.100,101 Hence, gp100-related antibodies are not manifesting cross-reactivity in labeling such pathologic entities; that affinity is merely a biologic extension of their specificity for premelanosomeassociated proteins. One true exception to the last statement is represented by the ability of MART-1/melan-A (but not HMB-45, NKI-beteb, or HMB-50) to label the tumor cells of a subset of adrenocortical carcinomas and sex-cord tumors of the gonads.68,106 No evidence of true melanocytic differentiation has been seen in such neoplasms, and MART-1 antibodies are presumably
Biology of Antigens and Antibodies
195
recognizing an antigenic epitope in those steroidogenic proliferations, which is shared with the gp100/PMel-17 molecules. One drawback related to this group of antibodies is the low level of positivity (<10%) seen in spindle-cell/ desmoplastic/neuroid melanomas.69,84,85 This observation is likely a reflection of the fact that such neoplasms typically manifest clonal evolution from a melanocytic phenotype to a more fibroblastic or schwannian pattern, losing, in the process, their ability to synthesize premelanosomes and proteins related to those organelles.107 On the other hand, anti–MART-1 may label macrophages, possibly because they have phagocytized fragments of melanocytes (see also below).107 KEY DIAGNOSTIC POINTS
Figure 7-12 Multifocal, intense positivity for tyrosinase in epithelioid melanoma metastatic to the liver (antityrosinase with diaminobenzidine and hematoxylin).
gp100 Antibody Group • HMB-45 and melan-A, both highly specific for melanocytic cell types, have sensitivities in the 60% to 80% range. • The gp100 antibody group regularly reacts with cells of angiomyolipoma and lesions of the PEComa group of neoplasms. • Melan-A decorates adrenocortical carcinomas and sex-cord tumors of the ovary. • A minority of desmoplastic/spindle cell melanomas show reactivity with the gp100 antibody group.
TYROSINASE-RELATED ANTIBODIES
In normal melaninogenesis, the amino acid tyrosine is hydroxylated to form 3,4-dihydroxyphenylalanine (“dopa”), which is then oxidized to dopa-quinone. The latter moiety is polymerized to form melanin, thereafter combining with melanoprotein to form a stable complex within premelanosomes and melanosomes.108 Tyrosinase plays a central role in this process by catalyzing the first step in the stated sequence.109 As such, it is a specific marker for melanocytic differentiation. This premise has been affirmed by studies that show tyrosinase gene transcripts are confined to melanin-producing cells and therefore might be used to detect isolated tumor cells by PCR.110 T311 and monoclonal anti-tyrosinase (MAT-1) are the two antityrosinase monoclonal antibodies that have been best analyzed in surgical pathology.111-118 The second of them is an immunoglobulin G (IgG) reagent raised by using a synthetic peptide, corresponding to the carboxy terminus of human tyrosinase as an immunogen.117 Both antibodies show a high level (>80%) of sensitivity and virtually absolute specificity for melanoma of the non–spindle-cell type among all malignant tumors (Fig. 7-12). Interestingly, nevi are said to be nonreactive with MAT-1 in many instances, as are nonneoplastic melanocytes.116 Hence, the epitope it recognizes is presumably related to melanocytic maturation and differentiation. In light of that finding, and because of the excellent specificity of antityrosinase antibodies, it would seem logical and permissible to use them diagnostically as mixtures, so-called cocktails.
MICROPHTHALMIA TRANSCRIPTION FACTOR PROTEIN
The microphthalmia gene encodes a transcription factor that is necessary to the survival and development of melanocytes during embryogenesis. Microphthalmia transcription-factor protein (MiTF) is a nuclear, basic helix-loop-helix leucine zipper moiety that, with the PAX3 and MSG1 gene products, plays a role in controlling the activity of melanogenic enzymes by upregulating the cyclic adenosine monophosphate pathway.119 (Incidentally, another related gene, TFE, has similar properties, and its transcription factor may emerge in the future as an additional melanocyte marker.)120 These reagents are fairly sensitive and specific for the identification of melanocytic differentiation (Fig. 7-13). As is true of most other markers in this general category, except for S-100 protein, MiTF typically labels epithelioid melanocytic lesions and, to a lesser degree, spindle-cell/desmoplastic melanomas.121-123 Because of
Figure 7-13 Invasive melanoma. Numerous cells are highlighted by the nuclear expression of microphthalmia transcription factor (MiTF; anti-MiTF with diaminobenzidine and hematoxylin).
196
Immunohistology of Melanocytic Neoplasms
its nuclear localization, one situation in which we favor the use of MiTF is in the analysis of intraepidermal melanocytic proliferations on sun-damaged skin. The nuclear pattern is relatively easy to distinguish from the melanin contained in the atypical melanocytes of pigmented actinic keratosis. Similar to other melanocytic markers, MiTF is also positive in angiomyolipoma, lymphangioleiomyomatosis, and so-called sugar tumors.124,125 Interestingly, cellular neurothekeoma, a nonmelanocytic lesion that may enter the differential diagnosis of melanoma, also expresses MiTF.126 p16
Upregulation of p16INK4a inhibits melanocyte growth in culture, and it inhibits loss of replicative potential in melanocytic nevi.127 Oncogene-induced senescence seems to inhibit further nevus growth and thereby inhibits formation of cutaneous melanoma. Expression of p16 is preserved in most nevi, both standard and Spitz, and may be lost in melanomas.128,129 PNL2
In 2003, Rochaix and colleagues130 described the clinical use of a new monoclonal antibody, PNL2, which had been raised against a fixative-resistant melanocyte antigen. In that study, which encompassed a spectrum of benign and malignant melanocytic lesions, the authors observed little if any labeling of banal melanocytes in the dermis, whereas junctional nevus cells were PNL2 positive. Melanomas were also immunoreactive in all cases, except for tumors with a desmoplastic appearance. PNL2 has also been tested against nonmelanocytic neoplasms,131-132 and it appears to have adequate specificity to justify its use in clinical settings. SOX9
SOX9 is a transcription factor that participates in chondrocyte differentiation, originally described in chondrocytes.133 This marker is routinely expressed in cartilaginous lesions. However, a possible pitfall is its expression in a number of melanomas (Fig. 7-14).134 SOX10
SOX10 is a transcription factor seen in cells from the neural crest. It is involved in maturation and maintenance of Schwann cells and melanocytes. SOX10 nuclear expression has been found in a majority of melanomas and nevi, in benign neural lesions, in most schwannomas and neurofibromas, and in almost half of malignant PNSTS.135 SOX10 appears to be strongly and diffusely expressed in spindle-cell/desmoplastic melanoma and is therefore helpful in distinguishing melanoma from scar.136 SOLUBLE ADENYLYL CYCLASE
Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a molecule
Figure 7-14 Nuclear expression of SOX9 in a case of metastatic melanoma (anti-SOX9 with diaminobenzidine and hematoxylin).
involved in regulating melanocyte function.137 R21, a mouse monoclonal antibody against sAC, shows nuclear expression in melanoma in situ, lentigo maligna type, but is mostly negative in benign melanocytes. Thus it may be helpful in distinguishing melanoma in situ from melanocytic hyperplasia in sun-damaged skin. Ki-67
Ki-67 is a nuclear marker expressed in proliferating cells. Its pattern of expression, similar to gp100, highlights the presence or absence of maturation. Common nevi and dysplastic nevi exhibit reactivity in less than 1% of cells, generally disposed at the dermal-epidermal junction or in the more superficial dermal compartment. In contrast, melanomas do not show this “maturation” pattern, but rather contain positive cells throughout the dermal component, with a mean proliferative fraction of more than 10%, particularly at the deep edge of the lesion (Fig. 7-15).138 Similarly, desmoplastic melanomas have a much higher proliferation rate, as detected with Ki-67 antibody, than do desmoplastic nevi.139
Neuroendocrine Markers in Melanocytic Lesions The association of both melanocytic and neuroendocrine proliferations with the neuroectoderm suggests that these neoplasms may demonstrate immunohistologic homologies. Proteins that are restricted anatomically to neurosecretory granules and neurosynaptic vesicles, such as chromogranins and synaptophysin, are occasionally present in melanocytic lesions.140-146 In addition, it is apparent that some lesions that are basically neuroendocrine or neuroectodermal neoplasms (e.g., neuroendocrine carcinomas, paragangliomas, PNETs, PNSTS) may exhibit a variable degree of melanocytic differentiation. These lesions include
Sentinel Lymph Node Biopsies for Metastatic Melanoma
Figure 7-15 Differential expression of Ki-67 in a nevus (A) and a primary melanoma (B). The deep portion of the nevus has few positive cells compared with melanoma (B; Ki-67, diaminobenzidine, and hematoxylin).
A
pigmented carcinoid,147,148 pigmented paraganglioma,149 melanotic neuroectodermal tumor,150,151 and melanotic schwannoma,152 both benign and malignant (Fig. 7-16). In these lesions, some of the cells have the same immunophenotype as cutaneous nevi or MMs (i.e., S-100P+, HMB-45+, MART-1+, tyrosinase+) in addition to having monodifferentiated epithelial, neural, or schwannian cells. The latter cells lack melanocytic markers altogether and produce a mostly dimorphic and mutually exclusive immunophenotype. Other neuroendocrine determinants that may be seen in melanoma are, in fact, synthesized by a broad repertoire of cell types. They include, but are not limited to, melanocytes, neuroendocrine epithelial cells, neuroblasts, primitive mesenchymal neuroectodermal cells, and Schwann cells. The markers in question are
Figure 7-16 Melanotic schwannoma, in which a high proportion of the tumor cells demonstrate divergent melanocytic differentiation.
197
B
principally represented by neuron-specific (γ dimer) enolase, neural-cell adhesion molecule (CD56), CD57, and CD99 (MIC2 protein).153-158 With the exception of CD99, these markers tend to be observed preferentially in melanocytic proliferations with neuroid features, such as neurotized intradermal nevi and desmoplastic/ neurotropic melanomas.156 Because of these similarities, such markers cannot be used to distinguish between truly neuroendocrine and melanocytic neoplasms.
Sentinel Lymph Node Biopsies for Metastatic Melanoma During the last two decades, analysis of sentinel lymph nodes (SLNs) has become increasingly widespread in assessing patients with melanoma for possible metastasis.159 The basic precept underlying this technique is that the absence of melanoma in an SLN biopsy obviates the need for regional lymphadenectomy and is associated with a better prognosis. This evaluation includes the use of IHC techniques to increase the sensitivity.160,161 Indeed, multiantibody “cocktails” have been devised for application to lymph nodes in that specific setting.162 Several authors have expressed points of view against the performance of this technique,163,164 These authors question the utility of SLN, because it has not been yet proved that removal of SLN improves survival. These authors also indicate a paucity of treatments for patients with metastatic melanoma. However, it is my opinion that SLN should be considered a procedure to help determine the prognosis of patients with cutaneous melanoma, and therefore analysis of SLNs is important in the clinical management of patients with melanoma.165
198
Immunohistology of Melanocytic Neoplasms
At our institution, we “bread-loaf” the SLN without performing frozen sections166 and then examine one hematoxylin and eosin (H&E) section.160 If that slide is negative, we cut three sections in the block (after ~200 μm), and then we stain one with H&E and another with a cocktail that includes four antibodies: anti– MART-1, A-103, HMB-45, and antityrosinase. We examine any possible positive cells in the immunoslides and determine whether they have morphology consistent with metastatic melanoma, possibly by comparing them with the cells from the primary lesion. The most important differential diagnosis is with isolated macrophages and capsular nevi. Some macrophages can be labeled with the antibodies used in the cocktail167 and therefore may constitute a diagnostic pitfall. In addition, as many as 20% of lymphadenectomies may contain benign melanocytes (nodal nevus). These cells are mostly located in the capsule but may also involve the parenchyma.168 If the morphology is still unclear between melanoma cells and nevus cells, IHC may be helpful, because most nodal nevi are negative for HMB-45 and have negligible proliferation rates with anti–Ki-67. Most clinical studies confirm that examination of SLN provides significant prognostic information. Our study on 237 positive SLNs out of 1417 patients169 suggests a stratification in three groups with progressively worse prognoses: 1) involvement of one or two SLN and metastasis size of 2 mm or less in the largest nest, and no ulceration in the primary lesion; 2) ulceration in the primary lesion or any metastatic nest greater than 2 mm; and 3) involvement of three or more SLNs or ulceration in the primary lesion and any metastatic nest greater than 2 mm. This stratification provides prognostic classification similar to that seen by using the American Joint Committee on Cancer (AJCC) staging.
Application of Immunohistochemistry to Selected Differential Diagnoses Melanoma Versus Melanocytic Nevus Variants Several papers have reported on the use of immunohistology for the separation of benign and malignant melanocytic lesions.4,47,138,170-177 Because no IHC markers are exclusively expressed in either nevus or melanoma, these markers are used in a scaled manner rather than for a positive/negative result. For example, HMB-45 labels melanocytic lesions in patterns different for benign and malignant melanocytic lesions. In nevi, HMB-45 shows two patterns: it most commonly labels the intraepithelial and superficial cells, with decreased labeling with depth in the dermis; cells located in the deep reticular dermis are either negative or only weakly positive. This pattern recapitulates the pattern of maturation of nevi (see Fig 7-10). A second, less frequent pattern is that of diffuse and strong labeling in blue nevi, cellular blue nevi, deep penetrating nevi, and some
Spitz/Reed nevi; most Spitz and Reed nevi show patterns of maturation. In nevi, Ki-67 is expressed in patterns similar to HMB-45 and thus shows maturation. Intraepithelial and superficial melanocytes express it, whereas deeply located melanocytes are negative for Ki-67. In contrast, melanomas express Ki-67 in a patchy manner throughout the lesion, without showing the zonal distribution of nevi maturation (see Fig 7-15).
Proliferative Dermal Nodules in Congenital Nevi Versus Melanoma In large congenital compound nevi, dermal proliferative melanocytic nodules (PMNs) that assume a “clonal” appearance and occasional mitotic figures may raise the possibility of melanoma originating in a nevus. The delimitation between the area of putative PMN and the surrounding congenital nevus is very important for the diagnosis. Even though at low power it seems that PMNs are well demarcated from the adjacent, benignappearing, small melanocytes, an intimate relationship exists between the two types of cells. In contrast, melanomas that arise in congenital nevi are sharply delimited from the adjacent nevus cells and often have a different stroma, with more fibrosis and lymphocytic infiltrates.178 Herron and colleagues179 have systematically examined a series of congenital nevi that contain PMNs, finding that the tumor cells in those foci paradoxically expressed putatively mutant p53 protein and Bcl-2 protein, both of which are antiapoptotic, with Bax protein, a proapoptotic mediator. CD117 was also usually retained in PMNs, as opposed to its relatively common absence in melanomas. The specified immunoprofile may therefore prove to be helpful in distinguishing PMNs in congenital nevi from malignant melanocytic lesions.
Primary Versus Secondary Intracutaneous Melanoma Cutaneous melanoma has a well-known ability to metastasize back to the skin; when it does, that tumor may even demonstrate apparent intraepidermal growth and pagetoid spread.180 Although most melanoma metastases to the skin present as circumscribed dermal or subcutaneous nodules, unfortunately, no reliable morphologic features can distinguish primary melanoma from secondary so-called epidermotropic melanoma. Guerriere-Kovach and colleagues181 assessed a group of cases in which both of the latter possibilities were represented, by using antibodies to Bcl-2 protein, mutant p53 protein, Ki-67, proliferating-cell nuclear antigen (PCNA), SMA, and CD117 (c-kit protein). Although some trends were observed toward greater labeling of metastatic melanomas for mutant p53 protein and Ki-67, but with diminution of CD117 reactivity, that pattern was not consistent. In summary, at the present time, there is no absolute immunohistologic solution to this specific problem. It is thus necessary to establish clinical-pathologic correlation that should include determination as to whether single or multiple lesions are present, whether the lesion
Application of Immunohistochemistry to Selected Differential Diagnoses
Melanocytic Neoplasms Versus Histiocytic Proliferations
appeared recently, or whether it is located close to a previous melanoma. Then the clinician should look for possible vascular invasion, which is much more common in metastatic than in primary melanoma; obvious mitotic figures; and an irregular (patchy) pattern of HMB-45 and high proliferation with anti–Ki-67.180
In the skin and elsewhere, it may be histologically difficult to separate amelanotic melanocytic lesions from histiocytic proliferations such as epithelioid histiocytoma, foam-cell–poor xanthogranuloma, atypical fibroxanthoma, and reticulohistiocytoma.184-186 Even though occasional histiocytic tumors may label for S-100 protein, they are consistently nonreactive with HMB-45, HMB-50, MART-1, antityrosinase, and anti-MiTF. In contrast, both histiocytic lesions and melanocytic neoplasms may be reactive for factor XIIIa and CD68, so-called histiocytic markers.24,187-189 We prefer CD163190 as a marker of monocyte-macrophage differentiation, because it is lacking in melanocytic proliferations.191 A possible pitfall is the interpretation of normal dendritic dermal cells that express S-100 protein as part of the neoplasm, rather than stromal cells, and thus consider the lesion to be melanocytic. We have seen at least one case of an epithelioid fibrous histiocytoma diagnosed as melanoma based upon its epithelioid morphology and the presence of dermal mitotic figures (Fig. 7-18). Another lesion of putative “fibrohistiocytic” differentiation is cellular neurothekeoma. These tumors usually are found on the head, neck, and upper extremities, more commonly in children and young women. Cellular neurothekeomas usually behave in a benign fashion regardless of the degree of cytologic atypia. These lesions are negative for S-100 protein but are strongly positive
Melanoma In Situ Versus Pigmented Actinic Keratosis A common problem for dermatopathologists is the separation of melanoma in situ (MIS) with relatively bland cytologic features from pigmented actinic keratosis (PAK) or actinically damaged skin with occasional atypical melanocytes. Shabrawi-Caelen and colleagues182 have addressed this differential diagnosis specifically, by using antibodies to S-100 protein, HMB-45, MART-1, and tyrosinase. The results of that study suggested that immunohistology was indeed useful in making the specified distinction. However, it was also implied that MART-1 was overly sensitive in the context under discussion, yielding unwanted labeling of PAK cases. Our opinion is that anti–MART-1 is very helpful in this differential diagnosis,183 because this potential pitfall may be avoided by counting the bodies of the melanocytes regardless of the area immunolabeled. Another marker that may be helpful in this diagnosis is MiTF. Because of its nuclear pattern of expression, it is relatively easy to distinguish positive cells (melanocytes) from adjacent, pigmented keratinocytes (Fig. 7-17).
A
199
B
Figure 7-17 Nuclear expression of microphthalmia transcription factor (MiTF). Compare the rare, scattered melanocytes in a case of actinic keratosis (A) and melanoma in situ (B; anti-MiTF with diaminobenzidine and hematoxylin).
200
Immunohistology of Melanocytic Neoplasms
A
B
C
D
Figure 7-18 Epithelioid fibrous histiocytoma resembling melanoma. A and B, Hematoxylin and eosin images of epithelioid cells with irregular nuclei. C, Expression of factor XIIIa. D, Expression of CD68 by many tumor cells. (C and D, anti-factor XIIIa and anti-CD68 with diaminobenzidine and hematoxylin).
for S-100A6, NKI-C3, neuron-specific enolase (NSE), SMA, protein gene product 9.5 (PGP9.5), and MiTF (Fig. 7-19).48,192
Recognition of Rhabdoid and Sarcomatoid Malignant Melanomas Rhabdoid and sarcomatoid melanomas include desmoplastic, myxoid, neurotropic, and osteochondroid subtypes, and these may demonstrate antigenic deletion or aberrancy. We and others have seen rhabdoid melanomas that lack melanocytic markers while expressing keratin or desmin.193-197 Obviously, an extended panel of reagents is needed to support the diagnosis of these melanomas. Sarcomatoid melanomas are consistently S-100– protein positive, but only 3% to 10% express other melanocytic markers (Fig. 7-20).84,125,126,128,198 Furthermore, such lesions may express CD56, CD57, nerve growth factor receptor, desmin, and actin isoforms (Figs. 7-21 and 7-22).2,153-158,193
Amelanotic Melanoma Versus Other Epithelioid Malignancies In reference to the expression of melanocytic markers in metastatic tumors, Plaza and colleagues199 have demonstrated that there is little if any antigenic “drift” compared with primary melanoma. A classical differential diagnostic question posed by surgical pathologists is that of melanoma versus poorly differentiated carcinoma versus large cell non-Hodgkin lymphoma or syncytial Hodgkin disease.198,200 This is particularly true of tumors found in lymph nodes with no known history of a prior malignancy. Under such circumstances, applicable antibody panels should include reagents to keratins, EMA, vimentin, S-100 protein, HMB-45, tyrosinase, MART-1, CD15, CD30, and CD45. Expected results in the specified classes of tumor under consideration are depicted in Figure 7-23. The same basic approach may be used when the differential diagnosis is that of metastatic melanoma
Application of Immunohistochemistry to Selected Differential Diagnoses
201
B
A
C
Figure 7-19 Cellular neurothekeoma. A-B, Nests of epithelioid cells. C, Expression of S-100 protein A6 (S-100A6) by most tumor cells (anti–S-100A6 protein with diaminobenzidine and hematoxylin).
involving serosae versus malignant mesothelioma, except that antibodies to calretinin, thrombomodulin, and HBME-1 should be included. The great majority of mesotheliomas, if not all of them, will express at least one of the latter three determinants, whereas MM is nonreactive for all of them.201-203 Neuroendocrine carcinomas may be separated from MM by attention to keratin reactivity patterns and positivity for chromogranin and synaptophysin.144 Keratin often assumes a dotlike, perinuclear, globular intracytoplasmic pattern of labeling in neuroendocrine
carcinomas,204 but in the small minority of MMs that show keratin expression, that configuration is not seen. Rare melanoma cases may express synaptophysin or chromogranin.
Metastatic Melanoma Versus Malignant Glioma In the CNS, metastatic amelanotic melanoma may closely imitate the microscopic appearance of a highgrade malignant glioma. This resemblance is further
L7
Figure 7-20 Immunolabeling for tyrosinase in sarcomatoid melanoma. Less than 10% of examples of this tumor variant express melanocytic markers other than S-100 protein.
Figure 7-21 CD57 immunolabeling in a malignant peripheral nerve sheath tumor.
202
Immunohistology of Melanocytic Neoplasms
Figure 7-22 CD57/Leu 7 (L7) in sarcomatoid/desmoplastic malignant melanoma likely reflects neuroid differentiation in that tumor.
complicated by the common reactivity seen in both lesions for S-100 protein,98,153,205,206 and the potential for a small minority of melanomas to express GFAP as well.22 Hence, other melanocytic markers such as HMB-45, anti-tyrosinase, and MART-1 are crucial to making this diagnostic distinction. All of those markers should be absent in pure glial neoplasms.98,206
Melanoma Versus Soft Tissue Sarcomas The ability of melanoma to simulate the appearance of various soft tissue sarcomas is also well documented.207 Among others, these include gastrointestinal stromal tumors (GISTs), epithelioid angiosarcoma, rhabdoid tumors, osteosarcomas, and PNETs.208 The detailed immunophenotypic properties of those lesions are provided elsewhere in this book. However, most sarcomas do not react for gp100-related melanocytic markers or tyrosinase. A notable exception to that statement is represented by clear cell sarcoma of soft tissue (“melanoma of soft parts”), which clearly does exhibit true melanocytic 100%
EMM LCNHL LCC SHD
90% 80% 70% 60%
differentiation.209-211 Although the immunohistologic profile of that tumor and MM are superimposable,211 clear cell sarcoma regularly shows a t(12;22) chromosomal translocation, apposing the EWS and ATF1, or a t(2;22) translocation that apposes the EWS and CREB genes.212-214 The most difficult diagnostic distinction to make in this context is that of sarcomatoid MM versus superficial malignant peripheral nerve sheath tumor (SMPNST). Indeed, in many ways, the two are nearly identical.215 Obviously, a history of previous melanoma would be important information to have in such a case, but if that is unavailable, a diagnosis of SMPNST would be favored in lesions associated with a large nerve or preexisting neurofibroma, or when they occur in patients with neurofibromatosis.216 As mentioned earlier in reference to small cell lesions, a useful ICH clue in the distinction between spindle cell melanoma and SMPNST concerns the intensity and level of cellular labeling for S-100 protein. If the neoplastic cells are only focally positive or negative for that marker, a diagnosis of SMPNST is favored. Both of these neoplastic categories share potential reactivity for nestin, p16 (cINK4a protein), CD10, CD99, Bcl-2 protein, and Wilms tumor 1 (WT-1) protein.217-222 Furthermore, to illustrate the close relationship between these two lesions of neuroectodermal derivation, we have observed tumors with all the histologic features of MPNSTs—dense fascicles of spindle cells with numerous mitotic figures, hemangiopericytoid vascular pattern, and prominent endothelial cells—but arising in congenital nevi and showing strong and diffuse S-100 expression; as mentioned before, these two features are associated with melanoma.
Melanoma Versus Cutaneous Granular Cell Tumor Melanoma is one of the lesions in the skin that may assume a granular cell composition, along with cutaneous granular cell tumor (CGCT), basal cell carcinoma, angiosarcoma, and leiomyosarcoma. Gleason and Nascimento223 have compared S-100 protein–positive CGCTs and melanomas with regard to their immunoreactivity for S-100 protein, MART-1, HMB-45, and MiTF. Of these markers, HMB-45 was the best discriminator, followed closely by MART-1. On the other hand, MiTF was commonly seen in both CGCT and melanoma.223 Calretinin and inhibin represent additional determinants that are selective for CGCT in this specific setting.224
50% 40%
Prognostic Markers and Targeted Therapy for Melanoma
30% 20% 10% 0% KER
VIM
HMB45
MART-1
CD30
Figure 7-23 Immunoreactivity patterns in large cell undifferentiated malignancies. EMM, Epithelioid melanoma; KER, keratin; LCC, large cell carcinoma; LCNHL, large cell non-Hodgkin lymphoma; SHD, syncytial Hodgkin disease; VIM, vimentin.
IHC has been proposed as an adjunct technique for evaluating primary and metastatic melanomas regarding prognosis. Among the markers studied are p53 protein, a promoter of programmed cell death; Ki-67, proliferating cell nuclear antigen, and Ki-S5, cell-cycle–related indicators of proliferation; heat shock proteins, markers of
Summary
replicating or “activated” cells; Bcl-2 protein, an inhibitor of apoptosis; VLA-4 and α-v/β-3 integrins, intercellular adhesion molecules that correlate with entrance into the vertical-growth phase of melanoma; CD26/dipeptidylaminopeptidase IV, a membrane-bound protease that assists tumor cell invasion; osteopontin, an adhesive matricellular glycoprotein that upregulates metalloproteinase production; NM23, a metastasis-suppressor gene product; Cdc42 and CXCR4 proteins, moieties that influence cellular motility; E-cadherin, a protein concerned with cell-matrix interaction; and cyclin-D1, cyclin-D3, Trk-A, and p16INK4-α (CDKN2A), gene products (cell-cycle regulators).225-235 These markers have been reported to be associated with decreased survival: decreased p16, increased p21; increased p27, predominantly when located in the cytoplasm; decreased growth-arrest DNA damage (GADD); increased human double minute 2 (HDM-2); increased activating transcription factor 1 (ATF1); increased galectin 3, predominantly when located in the nucleus; increased inducible nitric oxide synthase; and altered retinoid receptors. Another method proposed for prognostic purposes uses immunostains for endothelial and lymphatic markers (e.g., von Willebrand factor [vWF], CD31, CD34, D2-40/podoplanin)236-238 to assess stromal vascularity in vertical-growth–phase melanomas, on the premise that increased angiogenesis is correlated with an adverse prognosis.239,240 Furthermore, anti–D2-40 has been used to detect lymphatic space invasion, and it correlates with positivity in the SLN and with decreased disease-free survival.241 Further analysis is necessary to determine whether any of these markers should be used along with the current histopathologic features included in the AJCC: Breslow thickness, ulceration, and mitotic count.
203
Probably the most exciting application of IHC is to help detect mutations suitable for targeted therapy. The first target was c-kit, a member of the protein tyrosine kinases (PTKs). This protein, essential also in acute myelogenous leukemia and GISTs, is mutated or overexpressed in a number of melanomas, particularly in acral lentiginous and mucosal melanomas.242 Thus some of these patients have responded to anti-kit therapies such as imatinib (Gleevec). IHC may be used to try to detect those melanoma cases that overexpress c-kit but are lacking the mutations commonly analyzed.243 Other targets currently being studied are AKT, MEK/MAPKK, MAPK, BRAF, and mTOR. Of these, BRAF inhibitors have already demonstrated therapeutic effectiveness. Recently, an antibody that detects the BRAF V600E mutation may help in the selection of patients eligible for targeted therapy.244
Summary The many faces of malignant melanoma pose a serious diagnostic dilemma to the pathologist, especially when presenting as an undifferentiated tumor. IHC can be used as a diagnostic aid in primary melanoma, versus benign nevocytic lesions, to report prognostic factors and, in the near future, to report potential theranostic parameters.
REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Nakhleh RE, Wick MR, Rocamora A, et al: Morphologic diversity in malignant melanomas. Am J Clin Pathol. 93:731–740, 1990. 2. Banerjee SS, Harris M: Morphologic and immunophenotypic variation in malignant melanoma. Histopathology. 36:387–402, 2000. 3. Reed RJ, Martin P: Variants of melanoma. Semin Cutan Med Surg. 16:137–158, 1997. 4. Prieto VG, Shea CR: Immunohistochemistry of Melanocytic Proliferations. Arch Pathol Lab Med. 135:853–859, 2011. 5. Battifora H: Clinical application of the immunohistochemistry of filamentous proteins. Am J Surg Pathol. 12(Suppl):24–42, 1988. 6. Osborn M: Intermediate filaments as histologic markers: an overview. J Invest Dermatol. 81(Suppl 1):s104–s109, 1983. 7. Ramaekers FCS, Puts JJ, Moesker O, et al: Intermediate filaments in malignant melanomas: identification and use as a marker in surgical pathology. J Clin Invest. 71:635–643, 1983. 8. Caselitz J, Janner M, Breitbart E, et al: Malignant melanomas contain only the vimentin type of intermediate filaments. Virchows Arch A. 400:43–51, 1983. 9. Zarbo RJ, Gown AM, Nagle RB, et al: Anomalous cytokeratin expression in malignant melanoma: one- and two-dimensional Western blot analysis and immunohistochemical survey of 100 melanomas. Mod Pathol. 3:494–502, 1990. 10. Miettinen M, Franssila K: Immunohistochemical spectrum of malignant melanoma: the common presence of keratins. Lab Invest. 61:623–628, 1989. 11. Swanson PE: Monoclonal antibodies to human milk fat globule proteins. In Wick MR, Siegal GP, editors. Monoclonal antibodies in diagnostic immunohistochemistry, New York, 1988, Dekker, pp 227–284. 12. Pinkus GS, Kurtin PJ: Epithelial membrane antigen—a diagnostic discriminant in surgical pathology. Hum Pathol. 16:929–940, 1985. 13. Sloane JP, Ormerod MG: Distribution of epithelial membrane antigen in normal and neoplastic tissues and its value in diagnostic tumor pathology. Cancer. 47:1786–1795, 1981. 14. Selby WL, Nance KV, Park KH: Carcinoembryonic antigen— immunoreactivity in metastatic malignant melanoma. Mod Pathol. 5:415–419, 1992. 15. Ravindranath MH, Shen P, Habal N, et al: Does human melanoma express carcinoembryonic antigen? Anticancer Res. 20:3083–3092, 2000. 16. Ben-Izhak B, Levy R, Weill S, et al: Anorectal malignant melanoma: a clinicopathology study, including immunohistochemistry and DNA flow cytometry. Cancer. 79:18–25, 1997. 17. Muraro R, Kuroki M, Wunderlich D, et al: Generation and characterization of B72.3 second generation monoclonal antibodies reactive with the tumor associated glycoprotein72 antigen. Cancer Res. 48:4588–4596, 1988. 18. Wick MR, Swanson PE, Manivel JC: Placental alkaline phosphatase-like reactivity in human tumors: an immunohistochemical study of 520 cases. Hum Pathol. 18:946–954, 1987. 19. Frieling G, Al-Zaid T, Prieto V: Placental Alkaline Phosphatase Positivity in Metastatic Melanoma: Report of a Case and Review of the Literature. Annual Meeting of the American Society of Dermatopathology Chicago. 2012. 20. Chorvath B, Hunakova L, Turzova M, et al: Monoclonal antibodies to two adhesive cell surface antigens (CD43 and CD59) with different distribution on hematopoietic and non-hematopoietic tumor cell lines. Neoplasma. 39:325–329, 1992. 21. Carrel S, Schmidt-Kessen A, Mach JP, et al: Expression of common acute lymphoblastic leukemia antigen (CALLA) on human malignant melanoma cell lines. J Immunol. 130:2456– 2460, 1983. 22. Herbold KW, Zhou J, Haggerty JG, et al: CD44 expression on epidermal melanocytes. J Invest Dermatol. 106:1230–1235, 1996. 23. Sarlomo-Rikala M, Kovatich AJ, Barusevicius A, et al: CD117: a sensitive marker for gastrointestinal stromal tumors that is more specific than CD34. Mod Pathol. 11:728–734, 1998. 24. Pernick NL, DaSilva M, Gangi MD, et al: “Histiocytic” markers in melanoma. Mod Pathol. 12:1072–1077, 1999.
203.e1
25. Tang NE, Luyten GP, Mooy CM, et al: HNK-1 antigens on uveal and cutaneous melanoma cell lines. Melanoma Res. 6:411–418, 1996. 26. Weidner N, Tjoe J: Immunohistochemical profile of monoclonal antibody O13. Am J Surg Pathol. 18:486–494, 1994. 27. Tron VA, Krajewski S, Klein-Parker H, et al: Immunohistochemical analysis of bcl-2 protein regulation in cutaneous melanoma. Am J Pathol. 146:643–650, 1995. 28. Wilkerson AE, Glasgow MA, Hiatt KM: Immunoreactivity for CD99 in invasive malignant melanoma. J Cutan Pathol. 33:663– 666, 2006. 29. Chu PG, Arber DA, Weiss LM: Expression of T/NK-cell and plasma-cell antigens in nonhematopoietic epithelioid neoplasms: an immunohistochemical study of 447 cases. Am J Clin Pathol. 120:64–70, 2003. 30. Sagi A, Yun SS, Yu GH, et al: Utility of CD138 (syndecan-1) in distinguishing carcinomas from mesotheliomas. Diagn Cytopathol. 33:65–70, 2005. 31. Ruiter DJ, Bhan AK, Harrist TJ, et al: Major histocompatibility antigens and mononuclear inflammatory infiltrate in benign nevomelanocytic proliferations and malignant melanoma. J Immunol. 129:2808–2815, 1982. 32. Miettinen M, Chatten J, Paetan A, et al: Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors. Am J Surg Pathol. 22:327–332, 1998. 33. Mezebish D, Smith K, Williams J, et al: Neurocristic cutaneous hamartoma: a distinctive dermal melanocytosis with an unknown malignant potential. Mod Pathol. 11:573–578, 1998. 34. Van Ginkel PR, Gee RL, Walker TM, et al: The identification and differential expression of calcium-binding proteins associated with ocular melanoma. Biochim Biophys Acta. 1448:290– 297, 1998. 35. Ludwin SK, Kosek JC, Eng LF: The topographical distribution of S100 protein and GFA protein in the adult rat brain: an immunohistochemical study using horseradish peroxidaselabeled antibodies. J Comp Neurol. 165:197–208, 1976. 36. Gaynor R, Irie R, Morton D, et al: S100 protein: a marker for human malignant melanomas? Lancet. 1:869–871, 1981. 37. Baudier J, Briving C, Deinum J, et al: Effect of S100 protein and calmodulin on calcium-induced disassembly of brain microtubule proteins in vitro. FEBS Lett. 147:165–168, 1982. 38. Stefansson K, Wollmann R, Jerkovic M: S100 protein in soft tissue tumors derived from Schwann cells and melanocytes. Am J Pathol. 106:261–268, 1982. 39. Takahashi K, Isobe T, Ohtsuki Y, et al: Immunohistochemical study on the distribution of alpha and beta subunits of S100 protein in human normal and neoplastic tissues. Virchows Arch B. 45:385–396, 1984. 40. Herrera GA, Pena JR, Turbat-Herrera EA, et al: The diagnosis of melanoma: current approaches addressing tumor differentiation. Pathol Annu. 29(Part 1):233–260, 1994. 41. Loeffel SC, Gillespie GY, Mirmiram SA, et al: Cellular immunolocalization of S100 protein within fixed tissue sections by monoclonal antibodies. Arch Pathol Lab Med. 109:117–122, 1985. 42. Smoller BR: Immunohistochemistry in the diagnosis of melanocytic neoplasms. Pathol State Art Rev. 2:371–383, 1994. 43. Kindblom LG, Lodding P, Rosengren L, et al: S100 protein in melanocytic tumors. Acta Pathol Microbiol Scand. 92:219–230, 1984. 44. Nakajima T, Watanabe S, Sato Y, et al: An immunoperoxidase study of S100 protein distribution in normal and neoplastic tissues. Am J Surg Pathol. 7:715–727, 1982. 45. Cochran AJ, Lu HF, Li PX, et al: S100 protein remains a practical marker for melanocytic and other tumors. Melanoma Res. 3:325–330, 1993. 46. Swanson PE, Wick MR: Immunohistochemistry of cutaneous tumors. In Applied immunohistochemistry for the surgical pathologist, Cambridge, UK, 1993, Edward Arnold, pp 269– 308. 47. Ribe A, McNutt NS: S100A6 protein expression is different in Spitz nevi and melanomas. Mod Pathol. 16:505–511, 2003. 48. Plaza, JA, et al: Immunohistochemical expression of S100A6 in cellular neurothekeoma: clinicopathologic and
203.e2
Immunohistology of Melanocytic Neoplasms
immunohistochemical analysis of 31 cases. Am J Dermatopathol. 31(5):419–422, 2009. 49. Sigal AC, Keenan M, et al: P75 nerve growth factor receptor as a useful marker to distinguish spindle cell melanoma from other spindle cell neoplasms of sun-damaged skin. Am J Dermatopathol. 34(2):145–150, 2012. 50. Tos AP, Doglioni C: Calretinin: a novel tool for diagnostic immunohistochemistry. Adv Anat Pathol. 5:61–66, 1998. 51. Schulze E, Witt M, Fink T, et al: Immunohistochemical detection of human skin nerve fibers. Acta Histochem. 99:301–309, 1997. 52. Gotzos V, Wintergerst ES, Musy JP, et al: Selective distribution of calretinin in adenocarcinomas of the human colon and adjacent tissues. Am J Surg Pathol. 23:701–711, 1999. 53. Mahalingam M, Nguyen LP, Richards JE, et al: The diagnostic utility of immunohistochemistry in distinguishing primary skin adnexal carcinomas from metastatic adenocarcinoma to skin: an immunohistochemical reappraisal using cytokeratin 15, nestin, p63, D2-40, and calretinin. Mod Pathol. 23:713–719, 2010. 54. Prieto VG, Ivan D: The use of immunohistochemistry in the differential diagnosis of primary cutaneous adnexal neoplasms and metastatic adenocarcinomas to the skin. Diagnostic Histopathology. 16:409–416, 2010. 55. Nance KV, Siegal GP: The use of monoclonal antibodies in the search for tumor-specific antigens. In Wick MR, Siegal GP, editors: Monoclonal Antibodies in Diagnostic Immunohistochemistry, New York, 1988, Dekker, pp 593–622. 56. Shih IM, Nesbit M, Herlyn M, et al: A new MEL-CAM (CD146)specific monoclonal antibody, MN-4, on paraffin-embedded tissue. Mod Pathol. 11:1098–1106, 1998. 57. Trefzer U, Rietz N, Chen Y, et al: SM5-1: a new monoclonal antibody which is highly sensitive and specific for melanocytic lesions. Arch Dermatol Res. 292:583–589, 2000. 58. Brouwenstijn N, Slager EH, Bakker AB, et al: Transcription of the gene encoding melanoma-associated antigen gp10 tissues and cell lines other than those of the melanocytic lineage. Br J Cancer. 76:1562–1566, 1997. 59. Adema GJ, deBoer AJ, Vogel AM, et al: Molecular characterization of the melanocyte lineage-specific antigen gp100. J Biol Chem. 269:20126–20133, 1994. 60. Adema GJ, Bakker AB, deBoer J, et al: PMel17 is recognized by monoclonal antibodies NKI-beteb, HMB-45, HMB-50, and by anti-melanoma cytotoxic T-cells. Br J Cancer. 73:1044–1048, 1996. 61. Kawakami Y, Nishimura MI, Restifo NP, et al: T-cell recognition of human melanoma antigens. J Immunother. 14:88–93, 1993. 62. Chen YT, Stockert E, Jungbluth A, et al: Serological analysis of Melan-A (MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas. Proc Natl Acad Sci USA. 93:5916–5919, 1996. 63. Kawakami Y, Battles JK, Kobayashi T, et al: Production of recombinant MART-1 proteins and specific anti-MART-1 polyclonal and monoclonal antibodies: use in the characterization of the human melanoma antigen MART-1. J Immunol Methods. 202:13–25, 1997. 64. Kageshita T, Kawakami Y, Hirai S, et al: Differential expression of MART-1 in primary and metastatic melanoma lesions. J Immunother. 20:460–465, 1997. 65. Nicotra MR, Nistico P, Mangoni A, et al: Melan-A/MART-1 antigen expression in cutaneous and ocular melanomas. J Immunother. 20:466–469, 1997. 66. Pierard-Franchimont C, Letawe C, Nikkels AF, et al: Patterns of the immunohistochemical expression of melanoma-associated antigens and density of CD45R0+ activated T-lymphocytes and L1-protein-positive macrophages in primary cutaneous melanomas. Int J Mol Med. 2:721–724, 1998. 67. Fetsch PA, Marincola FM, Filie A, et al: Melanoma-associated antigen recognized by T-cells (MART-1): the advent of a preferred immunocytochemical antibody for the diagnosis of metastatic malignant melanoma with fine needle aspiration. Cancer. 87:37–42, 1999. 68. Busam KJ, Jungbluth AA: Melan-A, a new melanocytic differentiation marker. Adv Anat Pathol. 6:12–18, 1999. 69. Orosz Z: Melan-A/MART-1 expression in various melanocytic lesions and in non-melanocytic soft tissue tumors. Histopathology. 34:517–525, 1999.
70. Yu LL, Flotte TJ, Tanabe KK, et al: Detection of microscopic melanoma metastases in sentinel lymph nodes. Cancer. 86:617– 627, 1999. 71. Anichini A, Molla A, Mortarini R, et al: An expanded peripheral T-cell population to a cytotoxic T-lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J Exp Med. 190:651–667, 1999. 72. Bergman R, Azzam H, Sprecher E, et al: A comparative immunohistochemical study of MART-1 expression in Spitz nevi, ordinary melanocytic nevi, and malignant melanomas. J Am Acad Dermatol. 42:496–500, 2000. 73. Heegaard S, Jensen OA, Prause JU: Immunohistochemical diagnosis of malignant melanoma of the conjunctiva and uvea: comparison of the novel antibody against Melan-A with S100 protein and HMB-45. Melanoma Res. 10:350–354, 2000. 74. Adema GJ, deBoer AJ, VantHullenaar R, et al: Melanocyte lineage-specific antigens recognized by monoclonal antibodies NKI-beteb, HMB-50, and HMB-45 are encoded by a single cDNA. Am J Pathol. 143:1579–1585, 1993. 75. Chiamenti AM, Vella F, Bonetti F, et al: Anti-melanoma monoclonal antibody HMB-45 on enhanced chemi-luminescenceWestern blotting recognizes a 30-35 kDa melanosome-associated sialated glycoprotein. Melanoma Res. 6:291–298, 1996. 76. Schaumburg-Lever G, Metzler G, Kaiserling E: Ultrastructural localization of HMB-45 binding sites. J Cutan Pathol. 18:432– 435, 1991. 77. Esclamado RM, Gown AM, Vogel AM: Unique proteins defined by monoclonal antibodies specific for human melanoma. Am J Surg. 152:376–385, 1986. 78. Gown AM, Vogel AM, Hoak DH, et al: Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Pathol. 123:195–203, 1986. 79. Palazzo JP, Duray PH: Congenital agminated Spitz nevi: immunoreactivity with a melanoma-associated monoclonal antibody. J Cutan Pathol. 15:166–170, 1988. 80. Smoller BR, McNutt NS, Hsu A: HMB-45 staining of dysplastic nevi: support for a spectrum of progression toward melanoma. Am J Surg Pathol. 13:680–684, 1989. 81. Skelton HG III, Smith KJ, Barrett TL, et al: HMB-45 staining in benign and malignant melanocytic lesions: a reflection of cellular activation. Am J Dermatopathol. 131:543–550, 1991. 82. Wick MR: HMB-45: a clue to the biology of malignant melanoma? J Cutan Pathol. 18:307–308, 1991. 83. Smoller BR, Hsu A, Krueger J: HMB-45 monoclonal antibody recognizes an inducible and reversible melanocyte cytoplasmic protein. J Cutan Pathol. 18:315–322, 1991. 84. Wick MR, Swanson PE, Rocamora A: Recognition of malignant melanoma by monoclonal antibody HMB-45: an immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol. 15:201–207, 1988. 85. Blessing K, Sanders DS, Grant JJ: Comparison of immunohistochemical staining of the novel antibody melan-A with S100 protein and HMB-45 in malignant melanoma and melanoma variants. Histopathology. 32:139–146, 1998. 86. Vogel AM, Esclamado RM: Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody. Cancer Res. 48:1286–1294, 1988. 87. Kim RY, Wistow GJ: The cDNA RPE1 and monoclonal antibody HMB-50 define gene products preferentially expressed in retinal pigment epithelium. Exp Eye Res. 55:657–662, 1992. 88. Bar H, Schlote W: Malignant melanoma in the CNS: sub-typing and immunocytochemistry. Clin Neuropathol. 16:337–345, 1997. 89. Barrett AW, Bennett JH, Speight PM: A clinicopathological and immunohistochemical analysis of primary oral mucosal melanoma. Eur J Cancer B Oral Oncol. 31B:100–105, 1995. 90. Fernando SS, Johnson S, Bate J: Immunohistochemical analysis of cutaneous malignant melanoma: comparison of S100 protein, HMB-45 monoclonal antibody, and NKI/C3 monoclonal antibody. Pathology. 26:16–19, 1994.
References 91. Bishop PW, Menasce LP, Yates AJ, et al: An immunophenotypic survey of malignant melanomas. Histopathology. 23:159–166, 1993. 92. Mackie RM, Campbell I, Turbitt M: Use of NKI/C3 monoclonal antibody in the assessment of benign and malignant melanocytic lesions. J Clin Pathol. 37:367–372, 1984. 93. Yaziji H, Gown AM: Immunohistochemical markers of melanocytic tumors. Int J Surg Pathol. 11:11–15, 2003. 94. Mangini J, Li N, Bhawan J: Immunohistochemical markers of melanocytic lesions: a review of their diagnostic usefulness. Am J Dermatopathol. 24:270–281, 2002. 95. Friedman HD, Tatum AH: HMB-45 positive malignant lymphoma: a case report with literature review of aberrant HMB-45 reactivity. Arch Pathol Lab Med. 115:826–830, 1991. 96. Hancock C, Allen BC, Herrera GA: HMB-45 detection in adenocarcinomas. Arch Pathol Lab Med. 115:886–890, 1991. 97. Unger PD, Hoffman K, Thung SN, et al: HMB-45 reactivity in adrenal pheochromocytomas. Arch Pathol Lab Med. 116:151– 153, 1992. 98. Zimmer CM, Gottschalk J, Goebel S, et al: Melanoma-associated antigens in tumors of the nervous system: an immunohistochemical study with the monoclonal antibody HMB-45. Virchows Arch A. 420:121–126, 1992. 99. Eble JN, Amin MB, Young RH: Epithelioid angiomyolipoma of the kidney: a report of five cases with prominent and diagnostically confusing epithelioid smooth muscle component. Am J Surg Pathol. 21:1123–1130, 1997. 100. Fetsch PA, Fetsch JF, Marincola FM, et al: Comparison of melanoma antigen recognized by T-cells (MART-1) to HMB-45: additional evidence to support a common lineage for angiomyolipoma, lymphangiomyomatosis, and clear cell sugar tumor. Mod Pathol. 11:699–703, 1998. 101. Ribalta T, Lloreta J, Munne A, et al: Malignant pigmented clear cell epithelioid tumor of the kidney: clear cell (“sugar”) tumor versus malignant melanoma. Hum Pathol. 31:516–519, 2000. 102. Zamboni G, Pea M, Martignoni G, et al: Clear cell “sugar” tumor of the pancreas: a novel member of the family of lesions characterized by the presence of perivascular epithelioid cells. Am J Surg Pathol. 20:722–730, 1996. 103. Bonetti F, Pea M, Martignoni G, et al: Clear cell (“sugar”) tumor of the lung is a lesion strictly related to angiomyolipoma—the concept of a family of lesions characterized by the presence of the perivascular epithelioid cells (PEC). Pathology. 26:230–236, 1994. 104. Tanaka Y, Ijiri R, Kato K, et al: HMB-45/Melan-A and smooth muscle actin-positive clear-cell epithelioid tumor arising in the ligamentum teres hepatis: additional example of clear-cell “sugar” tumors. Am J Surg Pathol. 24:1295–1299, 2000. 105. Gaffey MJ, Mills SE, Zarbo RJ, et al: Clear cell tumor of the lung: immunohistochemical and ultrastructural evidence of melanogenesis. Am J Surg Pathol. 15:644–653, 1991. 106. Busam KJ, Iversen K, Coplan KA, et al: Immunoreactivity for A103, an antibody to melan-A (MART-1) in adrenocortical and other steroid tumors. Am J Surg Pathol. 22:57–63, 1998. 107. Huttenbach Y, Prieto VG, Reed JA: Desmoplastic and spindle cell melanomas express protein markers of the neural crest but not of later committed stages of Schwann cell differentiation. J Cutan Pathol. 29:562–568, 2002. 108. Lerner AB, Fitzpatrick TB: Biochemistry of melanin formation. Physiol Rev. 30:91–126, 1950. 109. Fitzpatrick TB, Miyomato M, Iskikawa K: The evolution of concepts of melanin biology. Arch Dermatol. 96:305–323, 1967. 110. Pellegrino D, Bellina CR, Manca G, et al: Detection of melanoma cells in peripheral blood and sentinel lymph nodes by RT-PCR analysis: a comparative study with immunohistochemistry. Tumori. 86:336–338, 2000. 111. Orchard GE: Comparison of immunohistochemical labeling of melanocyte differentiation antibodies melan-A, tyrosinase, and HMB-45 with NKI/C3 and S100 protein in the evaluation of benign nevi and malignant melanoma. Histochem J. 32:475–481, 2000. 112. Jungbluth AA, Iversen K, Coplan K, et al: T311—an antityrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues. Pathol Res Pract. 196:235– 242, 2000.
203.e3
113. Kaufmann O, Koch S, Burghardt J, et al: Tyrosinase, melan-A, and KBA62 as markers for the immunohistochemical identification of metastatic amelanotic melanomas on paraffin sections. Mod Pathol. 11:740–746, 1998. 114. Blaheta HJ, Schittek B, Breuninger H, et al: Lymph node micrometastases of cutaneous melanoma: increased sensitivity of molecular diagnosis in comparison to immunohistochemistry. Int J Cancer. 79:318–323, 1998. 115. Cormier JN, Abati A, Fetsch P, et al: Comparative analysis of the in vivo expression of tyrosinase, MART-1/Melan-A, and gp100 in metastatic melanoma lesions: implications for immunotherapy. J Immunother. 21:27–31, 1998. 116. Sato N, Suzuki S, Takimoto H, et al: Monoclonal antibody MAT-1 against human tyrosinase can detect melanogenic cells on formalin-fixed paraffin-embedded sections. Pigment Cell Res. 9:72–76, 1996. 117. Chen YT, Stockert E, Tsang S, et al: Immunophenotyping of melanomas for tyrosinase: implications for vaccine development. Proc Natl Acad Sci U S A. 92:8125–8129, 1995. 118. Clarkson KS, Sturdgess IC, Molyneux AJ: The usefulness of tyrosinase in the immunohistochemical assessment of melanocytic lesions: comparison of the novel T311 antibody (antityrosinase) with S100, HMB-45, and A103 (MART-1; Me). 119. Vachtenheim J, Novotna H: Expression of genes for microphthalmia isoforms, Pax3 and MSG1, in human melanomas. Cell Mol Biol. 45:1075–1082, 1999. 120. Verastegui C, Bertolotto C, Bille K, et al: TFE3, a transcription factor homologous to microphthalmia, is a potential transcriptional activator of tyrosinase and TyrpI genes. Mol Endocrinol. 14:449–456, 2000. 121. Koch MB, Shih IM, Weiss SW, et al: Microphthalmia transcription factor and melanoma cell adhesion molecule expression distinguish desmoplastic/spindle-cell melanoma from morphologic mimics. Am J Surg Pathol. 25:58–64, 2001. 122. King R, Googe PB, Weilbaecher KN, et al: Microphthalmia transcription factor expression in cutaneous benign and malignant melanocytic, and nonmelanocytic tumors. Am J Surg Pathol. 25:51–57, 2001. 123. Miettinen M, Fernandez M, Franssila K, et al: Microphthalmia transcription factor in the immunohistochemical diagnosis of metastatic melanoma: comparison with four other melanoma markers. Am J Surg Pathol. 25:205–211, 2001. 124. Zavala-Pompa A, Folpe AL, Jimenez RE, et al: Immunohistochemical study of microphthalmia transcription factor and tyrosinase in angiomyolipoma of the kidney, renal cell carcinoma, and retroperitoneal sarcomas: comparative evaluation with traditional diagnostic markers. Am J Surg Pathol. 25:65–70, 2001. 125. Folpe AL, Goodman ZD, Ishak KG, et al: Clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres: a novel member of the perivascular epithelioid cell family of tumors with a predilection for children and young adults. Am J Surg Pathol. 24:1239–1246, 2000. 126. Page RN, King R, Mihm MC Jr, et al: Microphthalmia transcription factor and NKI/C3 expression in cellular neurothekeoma. Mod Pathol. 17:230–234, 2004. 127. Michaloglou C, Vredeveld LC, Soengas MS, et al: BRAFE600associated senescence-like cell cycle arrest of human naevi. Nature. 436(7051):720–724, 2005. 128. Alonso SR, Ortiz P, Pollan M, et al: Progression in Cutaneous Malignant Melanoma Is Associated with Distinct Expression Profiles: A Tissue Microarray-Based Study. Am J Pathol. 164(1):193–203, 2004. 129. Reed JA, Loganzo F Jr, Shea CR, et al: Loss of expression of the p16/cyclin-dependent kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with invasive stage of tumor progression. Cancer Res. 55(13):2713–2718, 1995. 130. Rochaix P, Lacroix-Triki M, Lamant L, et al: PNL2, a new monoclonal antibody directed against a fixative-resistant melanocyte antigen. Mod Pathol. 16:481–490, 2003. 131. Busam KJ, Kucukgol D, Sato E, et al: Immunohistochemical analysis of novel monoclonal antibody PNL2 and comparison with other melanocyte differentiation markers. Am J Surg Pathol. 29:400–406, 2005.
203.e4
Immunohistology of Melanocytic Neoplasms
132. Ohsie SJ, Sarantopoulos GP, Cochran AJ, et al: Immunohistochemical characteristics of melanoma. J Cutan Pathol. 35:433– 444, 2008. 133. Akiyama H, Chaboissier MC, Martin JF, et al: The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 16:2813–2828, 2002. 134. Rao P, Fuller GN, Prieto VG: Expression of Sox-9 in Metastatic Melanoma-A Potential Diagnostic Pitfall. Am J Dermatopathol. 32(3):262–266, 2010, 5/2010. e-Pub 1/2010. 135. Nonaka D, Chiriboga L, Rubin BP: Sox10: a pan-schwannian and melanocytic marker. Am J Surg Pathol. 32(9):1291–1298, 2008. 136. Ramos-Herberth FI, Karamchandani J, Kim J, et al: SOX10 immunostaining distinguishes desmoplastic melanoma from excision scar. J Cutan Pathol. 37(9):944–952, 2010. 137. Magro CM, Yang SE, Zippin JH, et al: Expression of Soluble Adenylyl Cyclase in Lentigo Maligna. Arch Pathol Lab Med. 2012. [Epub ahead of print]. 138. Rudolph P, Schubert C, Schubert B, et al: Proliferation marker Ki-S5 as a diagnostic tool in melanocytic lesions. J Am Acad Dermatol. 37(2 Pt 1):169–178, 1997. 139. Harris GR, Shea CR, Horenstein MG, et al: Desmoplastic (sclerotic) nevus: an underrecognized entity that resembles dermatofibroma and desmoplastic melanoma. Am J Surg Pathol. 23(7):786–794, 1999. 140. Wick MR, Stanley SJ, Swanson PE: Immunohistochemical diagnosis of sinonasal melanoma, carcinoma, and neuroblastoma with monoclonal antibodies HMB-45 and antisynaptophysin. Arch Pathol Lab Med. 112:616–620, 1988. 141. Franquemont DW, Mills SE: Sinonasal malignant melanoma: a clinicopathologic and immunohistochemical study of 14 cases. Am J Clin Pathol. 96:689–697, 1991. 142. Wiedenmann B, Franke WW, Kuhn C, et al: Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci U S A. 83:3500–3504, 1986. 143. Lloyd RV, Wilson BS: Specific endocrine tissue marker defined by a monoclonal antibody. Science. 222:628–630, 1983. 144. Kontochristopoulos GJ, Stavropoulos PG, Krasagakis K, et al: Differentiation between Merkel cell carcinoma and malignant melanoma: an immunohistochemical study. Dermatology. 201:123–126, 2000. 145. Lee H, Torres FX, McLean SA, et al: Immunophenotypic heterogeneity of primary sinonasal melanoma with aberrant expression of neuroendocrine markers and calponin. Appl Immunohistochem. Mol Morphol. 19:48–53, 2011. 146. Eyden B, Pandit D, Banerjee SS: Malignant melanoma with neuroendocrine differentiation: clinical, histological, immunohistochemical and ultrastructural features of three cases. Histopathology. 47:402–409, 2005. 147. Klemm KM, Moran CA, Suster S: Pigmented thymic carcinoids: a clinicopathological and immunohistochemical study of two cases. Mod Pathol. 12:946–948, 1999. 148. Gal AA, Koss MN, Hochholzer L, et al: Pigmented pulmonary carcinoid tumor: an immunohistochemical and ultrastructural study. Arch Pathol Lab Med. 117:832–836, 1993. 149. Moran CA, Albores-Saavedra J, Wenig BM, et al: Pigmented extraadrenal paragangliomas: a clinicopathologic and immunohistochemical study of five cases. Cancer. 79:398–402, 1997. 150. Pettinato G, Manivel JC, d’Amore ESG, et al: Melanotic neuroectodermal tumor of infancy: a reexamination of a histogenetic problem based on immunohistochemical, flow cytometric, and ultrastructural study of 10 cases. Am J Surg Pathol. 15:233–245, 1991. 151. Kapadia SB, Frisman DM, Hitchcock CL, et al: Melanotic neuroectodermal tumor of infancy: clinicopathological, immunohistochemical, and flow cytometric study. Am J Surg Pathol. 17:566–573, 1993. 152. Mennemeyer RP, Hallman KO, Hammar SP, et al: Melanotic schwannoma: clinical and ultrastructural studies of three cases with evidence of intracellular melanin synthesis. Am J Surg Pathol. 3:3–10, 1979. 153. Orchard GE, Wilson-Jones E: Immunocytochemistry in the diagnosis of malignant melanoma. Br J Biomed Sci. 51:44–56, 1994.
154. Springall DR, Gu J, Cocchia D, et al: The value of S100 immunostaining as a diagnostic tool in human malignant melanomas: a comparison using S100 and neuron-specific enolase antibodies. Virchows Arch A. 400:331–343, 1983. 155. Dhillon AP, Rode J, Leathem A: Neuron-specific enolase: an aid to the diagnosis of melanoma and neuroblastoma. Histopathology. 6:81–92, 1982. 156. Reed JA, Finnerty B, Albino AP: Divergent cellular differentiation pathways during the invasive stage of cutaneous malignant melanoma progression. Am J Pathol. 155:549–555, 1999. 157. Sangueza OP, Requena L: Neoplasms with neural differentiation: a review. Part II: Malignant neoplasms. Am J Dermatopathol. 20:89–102, 1998. 158. Mooy CM, Luyten GP, DeJong PT, et al: Neural cell adhesion molecule distribution in primary and metastatic uveal melanomas. Hum Pathol. 26:1185–1190, 1995. 159. Cochran AJ, Roberts A, Wen DR, et al: Update on lymphatic mapping and sentinel node biopsy in the management of patients with melanocytic tumors. Pathology. 36:478–484, 2004. 160. Prieto VG, Clark SH: Processing of sentinel lymph nodes for detection of metastatic melanoma. Ann Diagn Pathol. 6:257– 264, 2002. 161. Spanknebel K, Coit DG, Bieligk SC, et al: Characterization of micrometastatic disease in melanoma sentinel lymph nodes by enhanced pathology: recommendations for standardizing pathologic analysis. Am J Surg Pathol. 29:305–317, 2005. 162. Shidham VB, Komorowski R, Macias V, et al: Optimization of an immunostaining protocol for the rapid intraoperative evaluation of melanoma sentinel lymph node imprint smears with the “MCW melanoma cocktail”. Cytojournal. 1:2, 2004. 163. Wick MR: Principles of evidence-based medicine as applied to “sentinel” lymph node biopsies. Pathol Case Rev. 13:102–108, 2008. 164. Medalie N, Ackerman AB: Sentinel lymph node biopsy has no benefit for patients with primary cutaneous melanoma: an assertion based on comprehensive, critical analysis, ed 2, New York, 2004, Ardor Scribendi Press, pp 1–95. 165. Prieto VG: Sentinel lymph nodes in cutaneous melanoma: handling, examination, and clinical repercussion. Arch Pathol Lab Med. 134(12):1764–1769, 2010. 166. Prieto VG: Use of frozen sections in the examination of sentinel lymph nodes in patients with melanoma. Semin Diagn Pathol. (25):112–115, 2008. 167. Trejo O, Reed JA, Prieto VG: Atypical cells in human cutaneous re-excision scars for melanoma express p75NGFR, C56/NCAM and GAP-43: evidence of early Schwann cell differentiation. J Cutan Pathol. 29(7):397–406, 2002. 168. Biddle DA, Evans HL, Kemp BL, et al: Intraparenchymal nevus cell aggregates in lymph nodes: a possible diagnostic pitfall with malignant melanoma and carcinoma. Am J Surg Pathol. 27(5):673–681, 2003. 169. Prieto VG, Diwan AD, Lazar AFJ, et al: Histologic Quantification of Tumor Size in Sentinel Lymph Node Metastases Correlates with Prognosis in Patients with Cutaneous Malignant Melanoma. Modern Pathology. 19:87A, 2006. 170. Nagasaka T, Lai R, Medeiros LJ, et al: Cyclin D1 over-expression in Spitz nevi: an immunohistochemical study. Am J Dermatopathol. 21:115–120, 1999. 171. Kanter-Lewensohn L, Hedblad MA, Wedje J, et al: Immunohistochemical markers for distinguishing Spitz nevi from malignant melanomas. Mod Pathol. 10:917–920, 1997. 172. Bergman R, Shemer A, Levy R, et al: Immunohistochemical study of p53 protein expression in Spitz nevi as compared with other melanocytic tumors. Am J Dermatopathol. 17:547–550, 1995. 173. Penneys NS, Seigfried E, Nahass G, et al: Expression of proliferating cell nuclear antigen in Spitz nevus. J Am Acad Dermatol. 32:964–967, 1995. 174. Takahashi H, Maeda K, Maeda K, et al: Immunohistochemical characterization of Spitz’s nevus: differentiation from common melanocytic nevi, dysplastic melanocytic nevus, and malignant melanoma. J Dermatol. 14:533–541, 1987. 175. Nasr MR, El-Zammar O: Comparison of pHH3, Ki-67, and survivin immunoreactivity in benign and malignant melanocytic lesions. Am J Dermatopathol. 30:117–122, 2008.
References 176. Prieto VG, Shea CR: Use of immunohistochemistry in melanocytic lesions. J Cutan Pathol. (35):1–10, 2008. 177. Ivan D, Prieto VG: Use of immunohistochemistry in the diagnosis of melanocytic lesions: applications and pitfalls. Future Oncol. 6(7):1163–1175, 2010. 178. Paradela S, Fonseca E, Prieto VG: Melanoma in children. Arch Pathol Lab Med. 135:307–316, 2011. 179. Herron MD, Vanderhoof SL, Smock K, et al: Proliferative nodules in congenital melanocytic nevi: a clinicopathologic and immunohistochemical analysis. Am J Surg Pathol. 28:1017–1025, 2004. 180. Plaza JA, Torres-Cabala C, Evans H, et al: Cutaneous metastases of malignant melanoma: a clinicopathologic study of 192 cases with emphasis on the morphologic spectrum. Am J Dermatopathol. 32(2):129–136, 2010. 181. Guerriere-Kovach PM, Hunt EL, Patterson JW, et al: Primary melanoma of the skin and cutaneous melanomatous metastases: comparative histologic features and immunophenotypes. Am J Clin Pathol. 122:70–77, 2004. 182. Shabrawi-Caelen LE, Kerl H, Cerroni L: Melan-A: not a helpful marker in distinction between melanoma in-situ on sun-damaged skin and pigmented actinic keratosis. Am J Dermatopathol. 26:364–366, 2004. 183. Wiltz KL, Qureshi H, Patterson JW, et al: Immunostaining for MART-1 in the interpretation of problematic intraepidermal pigmented lesions. J Cutan Pathol. 34:601–605, 2007. 184. Glusac EJ, McNiff JM: Epithelioid cell histiocytoma: a simulant of vascular and melanocytic neoplasms. Am J Dermatopathol. 21:1–7, 1999. 185. Busam KJ, Granter SR, Iversen K, et al: Immunohistochemical distinction of epithelioid histiocytic proliferations from epithelioid melanocytic nevi. Am J Dermatopathol. 22:237–241, 2000. 186. Busam KJ, Rosai J, Iversen K, et al: Xanthogranulomas with inconspicuous foam cells and giant cells mimicking malignant melanoma: a clinical, histologic, and immunohistochemical study of three cases. Am J Surg Pathol. 24:864–869, 2000. 187. Ma CK, Zarbo RJ, Gown AM: Immunohistochemical characterization of atypical fibroxanthoma and dermatofibrosarcoma protuberans. Am J Clin Pathol. 97:478–483, 1992. 188. Diaz-Cascajo C, Borghi S, Bonczkowitz M: Pigmented atypical fibroxanthoma. Histopathology. 33:537–541, 1998. 189. Gloghini A, Rizzo A, Zanette I, et al: KP1/CD68 expression in malignant neoplasms including lymphomas, sarcomas, and carcinomas. Am J Clin Pathol. 103:425–431, 1995. 190. Miettinen M, Fetsch JF: Reticulohistiocytoma (solitary epithelioid histiocytoma): a clinicopathologic and immunohistochemical study of 44 cases. Am J Surg Pathol. 30:521–528, 2006. 191. Lau SK, Chu PG, Weiss LM: CD163: a specific marker of macrophages in paraffin-embedded tissue samples. Am J Clin Pathol. 122:794–801, 2004. 192. Fetsch JF, Laskin WB, Hallman JR, et al: Neurothekeoma: an analysis of 178 tumors with detailed immunohistochemical data and long-term patient follow-up information. Am J Surg Pathol. 31(7):1103–1114, 2007. 193. Banerjee SS, Eyden B: Divergent differentiation in malignant melanomas: a review. Histopathology. 52:119–129, 2008. 194. Suster S: Tumors of the skin composed of large cells with abundant eosinophilic cytoplasm. Semin Diagn Pathol. 16:162–177, 1999. 195. Borek BT, McKee PH, Freeman JA, et al: Primary malignant melanoma with rhabdoid features: a histologic and immunocytochemical study of two cases. Am J Dermatopathol. 20:123– 127, 1998. 196. Laskin WB, Knittel DR, Frame JN: S100 protein and HMB-45negative “rhabdoid” malignant melanoma: a totally dedifferentiated malignant melanoma? Am J Clin Pathol. 103:772–773, 1995. 197. Anstey A, Cerio R, Ramnarain N, et al: Desmoplastic malignant melanoma: an immunocytochemical study of 25 cases. Am J Dermatopathol. 16:14–22, 1994. 198. Gatter KC, Alcock K, Heryet A, et al: The differential diagnosis of routinely-processed anaplastic tumors using monoclonal antibodies. Am J Clin Pathol. 82:33–43, 1984. 199. Plaza JA, Suster S, Perez-Montiel D: Expression of immunohistochemical markers in primary and metastatic malignant
203.e5
melanoma: a comparative study in 70 patients using a tissue microarray technique. Appl Immunohistochem Mol Morphol. 15:421–425, 2007. 200. Strickler JG, Michie SA, Warnke RA, et al: The “syncytial” variant of nodular sclerosing Hodgkin’s disease. Am J Surg Pathol. 10:470–477, 1986. 201. Ritter JH, Mills SE, Gaffey MJ, et al: Clear cell tumors of the alimentary tract and abdominal cavity. Semin Diagn Pathol. 14:213–219, 1997. 202. Ordonez NG: Role of immunohistochemistry in differentiating epithelial mesothelioma from adenocarcinoma. Am J Clin Pathol. 112:75–89, 1999. 203. Mizutani H, Ohyanagi S, Hayashi T, et al: Functional thrombomodulin expression on epithelial skin tumors as a differentiation marker for suprabasal keratinocytes. Br J Dermatol. 135:187– 193, 1996. 204. Battifora H, Silva EG: The use of antikeratin antibodies in the immunohistochemical distinction between neuroendocrine (Merkel cell) carcinoma of the skin, lymphoma, and oat cell carcinoma. Cancer. 58:1040–1046, 1986. 205. Clark HB: Immunohistochemistry of nervous system antigens: diagnostic applications in surgical neuropathology. Semin Diagn Pathol. 1:309–316, 1984. 206. Gottschalk J, Jautzke G, Schreiner C: Epithelial and melanoma antigens in gliosarcoma: an immunohistochemical study. Pathol Res Pract. 188:182–190, 1992. 207. Lodding P, Kindblom LG, Angervall L: Metastases of malignant melanoma simulating soft tissue sarcoma: a clinicopathological, light- and electron microscopic and immunohistochemical study of 21 cases. Virchows Arch A. 417:377–388, 1990. 208. Banerjee SS, Coyne JD, Menasce LP, et al: Diagnostic lessons of mucosal melanoma with osteocartilaginous differentiation. Histopathology. 33:255–260, 1998. 209. Swanson PE, Wick MR: Clear cell sarcoma: an immunohistochemical analysis of six cases and comparison with other epithelioid neoplasms of soft tissue. Arch Pathol Lab Med. 113:55–60, 1989. 210. Mechtersheimer G, Tilgen W, Klar E, et al: Clear cell sarcoma of tendons and aponeuroses: case presentation with special reference to immunohistochemical findings. Hum Pathol. 20:914– 917, 1989. 211. Almeida MM, Nunes AM, Frable WJ: Malignant melanoma of soft tissue: a report of three cases with diagnosis by fine needle aspiration cytology. Acta Cytol. 38:241–246, 1994. 212. Stenman G, Kindblom LG, Angervall L: Reciprocal translocation t(12;22)(q13;q13) in clear-cell sarcoma of tendons and aponeuroses. Genes Chromosomes Cancer. 4:122–127, 1992. 213. Langezaal SM, Graadt van Roggen JF, et al: Malignant melanoma is genetically distinct from clear cell sarcoma of tendons and aponeuroses (malignant melanoma of soft parts). Br J Cancer. 84:535–538, 2001. 214. Curry CV, Dishop MK, Hicks MJ, et al: Clear cell sarcoma of soft tissue: diagnostic utility of fluorescence in-situ hybridization and reverse transcriptase polymerase chain reaction. J Cutan Pathol. 35:411–417, 2008. 215. Swanson PE, Scheithauer BW, Wick MR: Peripheral nerve sheath neoplasms: clinicopathologic and immunochemical observations. Pathol Annu. 30(Pt, 2):1–82, 1995. 216. Wick MR: Malignant peripheral nerve sheath tumors of the skin. Mayo Clin Proc. 65:279–282, 1990. 217. Suster S, Fisher C, Moran CA: Expression of bcl-2 oncoprotein in benign and malignant spindle-cell tumors of soft tissue, skin, serosal surfaces, and gastrointestinal tract. Am J Surg Pathol. 22:863–872, 1998. 218. Kanitakis J, Bourchany D, Claudy A: Expression of the CD10 antigen (neutral endopeptidase) by mesenchymal tumors of the skin. Anticancer Res. 20:3539–3544, 2000. 219. Olsen SH, Thomas DG, Lucas DR: Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol. 19:659– 668, 2006. 220. Shimada S, Tsuzuki T, Kuroda M, et al: Nestin expression as a new marker in malignant peripheral nerve sheath tumors. Pathol Int. 57:60–67, 2007.
203.e6
Immunohistology of Melanocytic Neoplasms
221. Yoo J, Park SY, Kang SJ, et al: Altered expression of G1 regulatory proteins in human soft tissue sarcomas. Arch Pathol Lab Med. 126:567–573, 2002. 222. Brychtova S, Fluraskova M, Hlovilkova A, et al: Nestin expression in cutaneous melanomas and melanocytic nevi. J Cutan Pathol. 34:370–375, 2007. 223. Gleason BC, Nascimento AF: HMB-45 and Melan-A are useful in the differential diagnosis between granular cell tumor and malignant melanoma. Am J Dermatopathol. 29:22–27, 2007. 224. Ray S, Jukic DM: Cutaneous granular cell tumor with epidermal involvement: a potential mimic of melanocytic neoplasia. J Cutan Pathol. 34:188–194, 2007. 225. Wick MR: Prognostic factors for cutaneous melanoma. Am J Clin Pathol. 110:713–718, 1998. 226. Moretti S, Spallanzani A, Chiarugi A, et al: Correlation of Ki-67 expression in cutaneous primary melanoma with prognosis in a prospective study: different correlation according to thickness. J Am Acad Dermatol. 44:188–192, 2001. 227. Florenes VA, Faye RS, Maelandsmo GM, et al: Levels of cyclin D1 and D3 in malignant melanoma: deregulated cyclin expression is associated with poor clinical outcome in superficial melanomas. Clin Cancer Res. 6:3614–3620, 2000. 228. Straume O, Sviland L, Akslen LA: Loss of nuclear p16 protein expression correlates with increased tumor proliferation (Ki-67) and poor prognosis in patients with vertical growth phase melanoma. Clin Cancer Res. 6:1845–1853, 2000. 229. Kaleem Z, Lind AC, Humphrey PA, et al: Concurrent Ki-67 and p53 immunolabeling in cutaneous melanocytic neoplasms: an adjunct for recognition of the vertical growth phase in malignant melanomas? Mod Pathol. 13:217–222, 2000. 230. Henrique R, Azevedo R, Bento MJ, et al: Prognostic value of Ki-67 expression in localized cutaneous malignant melanoma. J Am Acad Dermatol. 43:991–1000, 2000. 231. Florenes VA, Maelandsmo GM, Holm R, et al: Expression of activated Trk-A protein in melanocytic tumors: relationship to cell proliferation and clinical outcome. Am J Clin Pathol. 122:412–420, 2004. 232. Rangaswami H, Kundu GC: Osteopontin stimulates melanoma growth and lung metastasis through NIK-MeKK1-dependent MMP-9 activation pathways. Oncol Rep. 18:909–915, 2007. 233. Rangel J, Nosrati M, Torabian S, et al: Osteopontin as a molecular prognostic marker for melanoma. Cancer. 112:144–150, 2008.
234. Ferrari D, Lombardi M, Ricci R, et al: Dermatopathological indicators of poor melanoma prognosis are significantly inversely correlated with the expression of NM23 protein in primary cutaneous melanoma. J Cutan Pathol. 34:705–712, 2007. 235. Tucci MG, Lucarini G, Brancorsini D, et al: Involvement of E-cadherin, beta-catenin, Cdc42, and CXCR4 in the progression and prognosis of cutaneous melanoma. Br J Dermatol. 157:1212– 1216, 2007. 236. Xu X, Gimotty PA, Guerry D, et al: Lymphatic invasion revealed by multispectral imaging is common in primary melanomas and associates with prognosis. Hum Pathol. 39:901–909, 2008. 237. Straume O, Jackson DG, Akslen LA: Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma. Clin Cancer Res. 9:250–256, 2003. 238. Cassarino DS, Cabral ES, Kartha RV, et al: Primary dermal melanoma: distinct immunohistochemical findings and clinical outcome compared with nodular and metastatic melanoma. Arch Dermatol. 144:49–56, 2008. 239. Graham CH, Rivers J, Kerbel RS, et al: Extent of vascularization as a prognostic indicator in thin (<0.76 mm) malignant melanomas. Am J Pathol. 145:510–514, 1994. 240. Vlaykova T, Talve L, Hahka-Kemppinen M, et al: MIB-1 immunoreactivity correlates with blood vessel density and survival in disseminated malignant melanoma. Oncology. 57:242–252, 1999. 241. Petersson F, Diwan AH, Ivan D, et al: Immunohistochemical detection of lymphovascular invasion with D2-40 in melanoma correlates with sentinel lymph node status, metastasis and survival. J Cutan Pathol. 36(11):1157–1163, 2009. 242. Shen SS, Zhang PS, Eton O, et al: Analysis of protein tyrosine kinase expression in melanocytic lesions by tissue array. J Cutan Pathol. 30(9):539–547, 2003. 243. Kim KB, Eton O, Davis DW, et al: Phase II trial of imatinib mesylate in patients with metastatic melanoma. Br J Cancer. (99):734–740, 2008. 244. Capper D, Preusser M, Habel A, et al: Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol. 122(1):11–19, 2011.
C H A P T E R 8
IMMUNOHISTOLOGY OF METASTATIC CARCINOMA OF UNKNOWN PRIMARY SITE ROHIT BHARGAVA, DAVID J. DABBS
Overview 204 Cancer of Unknown Primary Site: Clinical Aspects and Economic Considerations 204 The Diagnostic Approach to the Study of CUPS: Specimen Preparation 206 Determining Site of Origin: Stepwise Approach 207 Combined Antibody (Panel) Approach to Solving Diagnostic Problems 237 Special Clinical Presentations 237 Molecular Approach in Determining Site of Origin 242 Summary 244
Overview The impact of diagnostic immunohistochemistry (IHC) for the surgical pathologist is legendary, and it is best appreciated when studying malignancies of unknown primary site. A cost-effective tool, IHC is performed in most hospital laboratories, is often automated, and provides for a rapid turnaround time—all desirable qualities to the pathologist. The number of antibodies available for diagnostic use rises exponentially each year, a testament to the importance of ongoing research in this field. Since the first edition of this book, there has been a substantial addition of important antibodies that are especially useful in the workup for patients with metastatic malignancy of unknown primary site. Even with the larger armamentarium of antibodies, a paucity of specific antibodies remains that allow unequivocal and definitive diagnosis in every case. Indeed, it has been said that “it may be dangerous to base any distinction in tumor pathology primarily on the basis of the pattern of immunoreactivity of a given marker, no matter how 204
specific it is purported to be.”1 This statement echoes the importance of histopathologic morphology that is the basis of diagnosis in surgical pathology. The standard tissue section is the starting point for raising questions that need to be answered for the patient when morphology alone is not enough, and IHC is perhaps the best method to obtain more information from the paraffin section. Even the most specific antibodies—such as thyroid transcription factor 1 (TTF-1), paired box gene 8 (Pax8), Wilms tumor 1 (WT1), or prostate-specific antigen (PSA)—are not entirely site specific; therefore we resort to panels of antibodies that give statistical power to our morphologic diagnoses. Relevant diagnostic panels of antibodies change rapidly, based on information from IHC studies, and we can expect this constant infusion of new data on antibody sensitivity and specificity to impart an uncomfortable state of chronic flux on the discipline of IHC. Nevertheless, change is often incremental, and the basics of separating the category of metastatic malignancy of unknown primary into the categories of carcinoma, melanoma, lymphoma, germ cell neoplasia, and sarcoma have stood the test of time. Although the term cancer of unknown primary site (CUPS) is sometimes used interchangeably with the term carcinoma of unknown primary site—that is, cancers of epithelial differentiation—not all CUPS are epithelial in origin. This chapter will review the triage and evaluation of all types of cancers, but the main focus will remain on carcinoma, which forms the predominant category (~90% to 95% cases) of CUPS.2-4
Cancer of Unknown Primary Site: Clinical Aspects and Economic Considerations By definition, patients with CUPS have no obvious, identifiable primary site despite a careful clinical history, physical examination, radiologic imaging, and biochemical or histologic investigations. Studies of patients with
Cancer of Unknown Primary Site: Clinical Aspects and Economic Considerations
malignancies have shown that CUPS accounts for 5% to 15% of all patients who come to medical attention with a malignancy.3,5-7 The impact of recent improvements in radiologic imaging has reduced this percentage of patients to 3% to 7% of those who receive a CUPS diagnosis.2,8,9 The clinical presentation in a CUPS case depends on a number of factors that include age, gender, sites of involvement, and line of differentiation (epithelial, mesenchymal, lymphoid, germ cell, melanocytic). It appears that the tumors that present as CUPS are biologically and clinically different from the known primary tumors that metastasize several years after diagnosis. CUPS patients fail to show any symptoms related to the primary tumor and demonstrate an unpredictable pattern of spread; that is, frequency of involvement of a particular site is different than would be expected for a known primary tumor. The majority of CUPS cases have multiple sites of involvement, although a few present with only one or two sites. Based on the sites of involvement, several clinicopathologic entities have been characterized that are helpful in identifying the primary site. The liver is one of the single largest repositories for metastatic malignancies of all types, especially for carcinomas. The most common malignancies metastatic to the liver are from the gastrointestinal (GI) tract, with colorectal carcinomas leading this group. Lung and breast carcinomas also commonly metastasize to the liver, as do pancreaticobiliary carcinomas. This entire group of adenocarcinomas may appear similar to primary cholangiocarcinoma of the liver and may simulate some hepatocellular carcinomas, particularly the less-differentiated hepatocellular carcinomas. Prostate carcinoma, although unusual, does metastasize to the liver and can be confused with cholangiocarcinoma. Thus for hepatic metastases of unknown primary in women, colorectal, breast, and lung carcinomas are of primary consideration; whereas in men, colorectal, lung, and prostate carcinomas top the list. Malignant melanoma metastatic to the liver is not uncommon, and the highest frequency of liver metastases is seen with primary eye melanomas.5 In a fine needle biopsy study of 200 malignant aspirates of the liver, Pisharodi and associates10 found that 32% were hepatocellular carcinomas, 49.5% were readily diagnosed as metastatic carcinomas, and 18.5% were problematic. Of this latter group, IHC contributed to definitive diagnosis in half of the cases. Along with the liver, the lung is a major repository for metastatic carcinomas, especially adenocarcinomas. Identification of the origin of an adenocarcinoma in the lung is a frequent, difficult, and challenging process for the surgical pathologist, because adenocarcinomas not only are the most frequent primary lung tumor, they are also the most common metastatic tumor found in the lung. Distinction among these tumor types can be especially challenging on scant biopsy materials, such as transbronchial biopsy or fine needle aspiration biopsy (FNAB). Clinical information in regard to circumscription and number of lesions is quite useful. Metastatic tumors often present as multiple circumscribed nodules, whereas primary lung tumors often show a dominant
205
infiltrative lesion. It is important to identify those carcinomas that can be treated by chemotherapy or hormonal manipulation or both, especially metastatic breast or prostate carcinomas. In the brain, distinguishing metastatic adenocarcinoma or poorly differentiated carcinoma from a glial tumor is straightforward, although determining the source of the metastasis may be problematic, especially when the occult primary is unknown.11,12 Lung carcinomas are the most likely primary to be discovered subsequent to central nervous system (CNS) presentation; other common primaries include breast, kidney, thyroid, and GI tract.11-13 Patients with other adenocarcinomas—such as ovarian, prostatic, and pancreaticobiliary carcinomas—rarely come to medical attention with brain metastases, because there is almost always evidence of widespread dissemination of these tumors before the occurrence of cerebral metastases. The site of origin of carcinoma remains unknown in as many as 5% of patients.14 The survival of most patients with carcinomatous brain metastases is in the range of 3 to 11 months.15-17 Patients who are seen initially with skeletal metastases often have primary carcinomas in the lung, breast, kidney, or urogenital region, and imaging studies have been particularly useful in elucidating the primary tumor.18 For patients who come to medical attention with pleural effusions, the breast is the most common primary site for women; the lung is the most common site for a primary tumor in men.19 Malignant lymphomas are seen in both sexes. In women who are seen initially with malignant abdominal effusions (malignant ascites), common abdominal sites include a müllerian primary, often of the fallopian tubes and ovaries; whereas men with malignant ascites typically have primary tumor sites in the GI tract, predominantly in the colon, rectum, pancreas, or stomach.20 Patients with peritoneal carcinomatosis of nongynecologic origin most often have origins in the stomach, colon, or pancreas and have a median survival of 3 months.21 For patients who are seen initially with primary lymph node metastases, clues to the primary site of the tumor may be based on tumor morphology (adenocarcinoma, squamous cell carcinoma, or undifferentiated carcinoma) and the anatomic site of lymph node involvement. In women with adenocarcinoma in the axilla, the primary tumor is most often found in the ipsilateral breast. For the patient with metastatic adenocarcinoma in the neck, the metastatic workup will begin in the lung for males or in the breast for females, although GI and prostate adenocarcinomas both show a predilection for the left side of the neck.22 Undifferentiated carcinomas of the head and neck are the most common primary source for metastatic tumors in head and neck lymph nodes,6 and the majority of these are of squamous mucosal derivation. The prognosis for this group of patients rests largely on the nodal status; patients with stage N3 lesions have a poor prognosis.23 For a squamous cell carcinoma that involves the mid and upper cervical lymph nodes, a thorough examination of the oropharynx, hypopharynx,
206
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
nasopharynx, larynx, and upper esophagus by direct vision and by fiberoptic nasopharyngolaryngoscopy, with biopsy of any suspicious areas, is more valuable than further pathologic examination. Advanced diagnostic techniques such as computerized tomography (CT) and positron emission tomography (PET) scans are helpful in determining the primary site.24-26 Occasionally, systematic random biopsies of mucosal sites such as nasopharynx, base of tongue, pyriform sinus, and tonsils may reveal an occult tumor.27 Metastatic squamous cell carcinoma that involves the lower cervical lymph nodes, or those at any other site except inguinal nodes, is highly suspicious for a lung primary.28 Occasionally, esophageal squamous cell carcinomas may preferentially involve the lower cervical lymph nodes.29 The vast majority of patients with squamous cell carcinoma that involves the inguinal nodes generally have detectable primary tumor in the anogenital area.30 Therefore all women must undergo a thorough evaluation of the vulva, vagina, and cervix, and all men should undergo careful inspection of the penis; both sexes must also have examination of the anorectal region. The current clinical approach is an attempt to identify favorable prognostic groups in patients with unknown primary tumors so that they can be managed appropriately.31-35 This group of tumors includes leukemia/lymphoma, germ cell tumors (GCTs), small cell carcinoma of the lung, and carcinomas of the breast, ovary, endometrium, adrenal gland, thyroid, and prostate.5,36,37 When possible, it is useful to separate regional from distant metastases, because localized disease is more amenable to treatment.5,38 Other favorable clinical features that have been described include location of tumor in the retroperitoneum or peripheral lymph nodes, tumor limited to one or two metastatic sites, a negative smoking history, and youth.5 Kirsten and colleagues36 studied 286 patients with CUPS and concluded that the factors that predicted survival were lymph node presentation, good performance status, and body weight loss of less than 10%. Using a panel of antibodies to determine differentiation of tumors in 41 patients with CUPS, Van der Gaast and associates concluded that the IHC panel approach to uncover tumor origin is useful for selecting appropriate treatment of patients, especially those who may benefit from combination chemotherapy.32,39 Other IHC studies of CUPS have elucidated the origin of tumors in as few as 5% to as many as 70% of patients.40 However, most investigators have arrived at the same conclusion: for individual patient therapy, knowledge of site of origin improves patient survival.31,41 Furthermore, with the advent of targeted therapy, although still in its infancy, the benefit of identifying the site of origin may be manifold.42-44 With the incorporation of next-generation sequencing in diagnostic testing, it may become less important to identify the site of origin and more useful to find actionable targets for an individual patient. However, finding the site of origin may still have genetic implications, and both patient and family need to know where the tumor came from. The appropriate workup for identifying a primary tumor depends on the patient’s clinical symptoms, age,
history, gender, and the likelihood of finding the primary tumor. Patients with CUPS do poorly as a group, with a median survival of 6 to 11 months, and the importance of establishing the origin of the primary site guides therapeutic interventions of hormonal manipulation, chemotherapy, and radiation.45-47 The clinician must also take into account the economics of an extensive clinical workup in addition to the inconvenience and discomfort to which the patient is subjected.48 The economic considerations of clinical workup in these patients have not been extensively studied, and few data are available on the cost-effectiveness of IHC in surgical pathology. Schapira and Jerrett7 analyzed the clinical workups in a group of 199 patients and concluded that the search for a primary neoplasm incurred an average cost of $17,973 and only 19.6% of patients survived for more than 1 year. As a matter of fact, IHC is probably undervalued and is likely a cost-effective maneuver in the study of CUPS. Radiologic studies have limited value in the management of these patients, and prognosis is not affected.49,50 Even autopsies on some of these patients may not detect the primary site of tumor because of small size, extensive dissemination, or regression as a result of therapy.51 In 1988, Le Chevalier and coworkers52 studied 302 autopsy specimens from patients who came to medical attention with CUPS. The primary tumor site was located premortem in 27% of patients, at autopsy in 57%, and remained unidentified in 16%.52 The most common primary tumor sites in this study included pancreas, lung, kidney, and colon/rectum, a list that includes the two malignancies with the highest incidence in both men and women.
Diagnostic Approach to the Study of CUPS: Specimen Preparation The goal of the surgical pathologist is to identify the line of differentiation of the tumor and identify those tumors that are within the “treatable” group of tumors, namely, carcinomas of breast, prostate, ovary, endometrium, and thyroid and adrenal glands in addition to GCTs and neuroendocrine carcinomas (NECs).32 Hormonal and antihormonal therapies are useful for patients with breast, prostate, and adrenal carcinomas; neuroendocrine, thyroid, and GCTs may be responsive to suppression by chemical agents. The therapeutic response of other carcinomas is less certain,53 but the identity of the carcinoma, if available, is useful to determine more useful therapeutic regimens for these patients prospectively.32,47,52,54 Some studies on patients with CUPS demonstrate that as many as one third respond to taxane-based therapies.55,56 Tissue procurement is the first step in the workup for tumors of unknown primary origin. It is a common practice to obtain tissue by FNAB or by obtaining a core tissue biopsy. The sensitivity of FNAB for metastatic carcinoma in a series of 266 superficial lymph nodes was 96.5%, with no false-positive results and nine falsenegative results.57 Tissue from both FNAB and core
Determining Site of Origin: Stepwise Approach
biopsies can be triaged for ancillary studies in the same manner. Immunocytochemical study of malignant effusions is also of great value, and often these are the first samples available by virtue of therapeutic evacuation.58-64 Whatever the method of obtaining tissue, it is ideal to monitor the process so that adequate tissue may be obtained to triage the patient’s problem appropriately; namely, to triage the specimen for immunohistology, electron microscopy, flow cytometry, and molecularcytogenetic studies. If not enough tissue is available, our recommendation is to freeze at least some tumor sample after tissue has been collected for morphologic analysis, because fresh-frozen tissue is the best sample for molecular analysis.65 Monitoring of the tissue-procurement process can be performed with frozen sections, immediate interpretation of FNAB, or tissue imprints. In addition to tissue procurement, the pathologist must define the problem by taking into account the patient’s age, gender, known risk factors, duration of symptoms, and clinical and radiologic findings. Based on this information and the morphologic appearance of the tumor, the quest for the study of tumor origin begins. In surgical pathology and cytopathology, poorly differentiated carcinomas can be broadly classified as large cell undifferentiated, small cell undifferentiated, and spindle cell. The starting point for diagnostic interpretation is the standard hematoxylin and eosin (H&E) or Papanicolaou-stained slide. The importance of the histologic morphology should not be underestimated in arriving at a definitive diagnosis; indeed, morphology is the foundation upon which the interpretation of all IHC studies rests. In this chapter, the role of diagnostic IHC in diagnosing CUPS is emphasized, especially as it relates to adenocarcinoma and poorly differentiated carcinoma of unknown primary site and GCTs, because these account for more than 90% cases of CUPS. Specific tables are presented that aid in the differential diagnosis of tumors in specific anatomic sites. The role of molecular studies in combination with IHC for patients with CUPS is also discussed.
Determining Site of Origin: Stepwise Approach Carcinomas form the predominant category (~90% to 95% cases) of CUPS2-4 and will therefore remain the main focus of this chapter. Because most carcinomas show significant positivity for cytokeratins (CKs), carcinomatous differentiation becomes readily apparent when the tumor is diffusely CK positive. The simple and broad-spectrum CKs are the initial antibodies of choice for detecting carcinomatous differentiation. More specific subcategorization of the tumor origin is then possible by using a variety of site-specific CKs and antibodies directed against various cellular products. A combination of these cellular antigens may yield a costeffective approach to tumor categorization.
207
The approach to definitive diagnosis of the patient with CUPS effectively follows five sequential steps: 1. Determine the cell line of differentiation by using major lineage markers that include keratins, lymphoid, melanoma, germ cell, and sarcoma markers. 2. Determine the CK type or types of distribution in the tumor cells, because some subsets are unique to certain tumor types. 3. Determine whether vimentin is coexpressed. 4. Determine whether expression of supplemental antigens of epithelial or germ cell derivation is present; that is, whether carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), or placental alkaline phosphatase (PLAP) are evident. 5. Determine whether expression of cell-specific products, cell-specific structures, transcription factors, or receptors that are unique identifiers of cell types is present; for example, neuroendocrine granules, peptide hormones, thyroglobulin, PSA, prostate-specific membrane antigen, NKX3-1, inhibin, gross cystic disease fluid protein (GCDFP), villin, uroplakin (URO), thyroid transcription factor-1 (TTF-1), transcription factor CDX-2, or Pax-8.
Step One: Screening Immunohistochemistry An abbreviated first-line panel to determine the line of differentiation should be composed of epithelial markers (pankeratin AE1/AE3 and CAM5.2, both used together), mesenchymal marker (vimentin), lymphoid marker (leukocyte common antigen [LCA]), and melanocytic marker (S-100 protein). Although vimentin is included in the above panel, it is generally the least helpful and should therefore be interpreted with caution; vimentin is considered a mesenchymal marker,66 but it may be expressed quite diffusely in many poorly differentiated carcinomas.67 As a matter of fact, vimentin coexpression in a carcinoma may point to a specific primary site. Except for vimentin, a diffuse strong expression of any of the markers above is generally suggestive of a particular line of differentiation. LINE OF DIFFERENTIATION: LYMPHOID
The above first-line panel generally leads to a more extensive workup. At this point, if the tumor is strongly positive for LCA and negative for keratins, further workup is directed toward classifying the lymphoma by using pan–B-cell and pan–T-cell antigens (CD20, CD79a, and CD3) and other markers.68,69 If LCA is equivocal, pan–B-cell and pan–T-cell markers are negative, and the morphology is still suggestive of lymphoid neoplasm, it is not unreasonable to think about a myeloid neoplasm and to perform a test by using myeloid markers for granulocytic sarcoma. This diagnosis may be missed even after an expensive and extended immunohistologic evaluation.70-72 Stains that are helpful in demonstrating myeloid lineage are myeloperoxidase, chloracetate esterase, lysozyme stains, and CD117 (also known as C-KIT or stem cell
208
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
marker).73-75 CD43 and CD68 stains are also positive in granulocytic sarcomas.71,76,77 LINE OF DIFFERENTIATION: MELANOCYTIC
A diffuse strong staining with S-100 in a tumor of unknown origin that is negative for cytokeratins is good evidence that it may be a melanoma.78-80 However, this diagnosis still must be confirmed by additional melanoma markers such as human melanoma black 45 (HMB-45), melan-A, tyrosinase, or anti-melanoma antibody clone NKI/C3. Additional markers are needed because, although S-100 is a very sensitive marker, it is not a specific marker for melanoma. S-100 is also expressed by some carcinomas81,82 and sarcomas (liposarcoma, chondrosarcoma, neural tumors).83,84 Although, the typical variants of these sarcomas are easy to diagnose on H&E stain alone, the unusual variants—such as dedifferentiated liposarcoma, mesenchymal chondrosarcoma, or a malignant peripheral nerve sheath tumor (MPNST)—may pose a challenge to distinguish from melanomas, therefore additional melanoma markers should be used for definitive diagnosis. Similarly, a pathologist should also be careful in distinguishing melanoma from carcinoma based on only a limited number of immunostains. As mentioned earlier, some carcinomas may show strong S-100 expression; an additional pitfall is that some melanomas may show polyclonal CEA (pCEA) and/or focal CAM5.2 keratin immunoreactivity,85-87 therefore caution is advised when the diagnosis is heavily based on IHC expression of markers. LINE OF DIFFERENTIATION: MESENCHYMAL
Strong vimentin expression in a nonmelanocytic, nonlymphoid neoplasm is generally an indication of a sarcoma. Most sarcomas, but not all, are negative for epithelial markers. Epithelioid sarcoma, as the name suggests, shows some epithelial differentiation, and synovial sarcoma may have a well-defined biphasic pattern that shows strong staining for epithelial markers in the glandlike component.88-91 The sarcomas that need to be considered in a CUPS case are the ones that do not demonstrate a particular line of differentiation on morphology alone. These sarcomas may have small round blue cell tumor morphology (Ewing sarcoma, desmoplastic small round cell tumor, rhabdomyosarcoma [RMS]), spindle and epithelioid cells (synovial sarcoma, clear cell sarcoma, angiosarcoma), or pure epithelioid cells (epithelioid sarcoma). Although a number of IHC stains are available to further classify a sarcoma into a defined category, many stains are not specific enough to provide a definitive diagnosis.92 Occasionally, a high index of suspicion is required to make the correct diagnosis (Fig. 8-1). However, IHC may be performed to narrow down the differential diagnosis and streamline the molecular tests that need to be ordered.93,94 For example, a small round blue cell tumor positive for CD99 and periodic acid–Schiff (PAS) in a child or young adult should be evaluated for an EWS-FLI1 fusion transcript (Fig. 8-2). As mentioned earlier, freshfrozen tissue is the best sample for molecular testing;
reverse transcription polymerase chain reaction (RTPCR) assays can also be performed on fresh-frozen paraffin-embedded (FFPE) tissue.95,96 Triage for suspected sarcoma cases can be performed as shown in Table 8-1. LINE OF DIFFERENTIATION: EPITHELIAL
Carcinoma comprises approximately 90% of CUPS cases. Within the carcinoma category, the overwhelming majority of tumors are adenocarcinomas (~70%).97 The poorly differentiated carcinoma group comprises approximately 15% to 20%, and the remainder represent either squamous cell carcinoma (5%) or NEC (5%). CK stains are an excellent marker of epithelial differentiation and are strongly and diffusely expressed in carcinomas.98 However, examples of keratin posi tivity have been described in almost all tumor types including sarcomas, melanomas, and even lymphomas.99-106 In spite of these disturbing reports, when an epithelioid tumor is overwhelmingly positive for pankeratin stains, a diagnosis of carcinoma must be seriously evaluated.
Step Two: The Cytokeratins— An Overview The soft epithelial keratin intermediate filaments comprise approximately 20 different keratin polypeptides.107-109 The polypeptides, numbered 1 through 20, comprise the type II (basic) and type I (acidic) keratins (Table 8-2). This family of intermediate filaments is crucial in diagnostic IHC for the identification of carcinomatous differentiation and for identification of specific carcinoma subtypes. Keratin filaments are formed by tetrameric heteropolymers of two different keratins, two from type I and two from type II, to maintain cellular electrical neutrality. The vast majority of keratins are paired together as acidic and basic types, with rare exception. The classification and numbering system of the keratins is based on the catalog of Moll and associates.110 Twelve keratins with more acidic isoelectric points form the type I (acidic) keratins, and eight keratins with more basic isoelectric points form the type II (basicneutral) keratins.111 The keratins are products of two gene families: most genes for type II keratins are localized on chromosome 12, and the genes for type I keratins are localized on chromosome 17.112-114 Within each group, the CKs are numbered consecutively, from highest to lowest molecular weight in each group. Most low-molecular-weight (LMW) keratins are typically found in all epithelia except the squamous variety, whereas high-molecular-weight (HMW) keratins are typically of squamous epithelium.110 The original methods for identification of the different keratin types in tissues relied on tedious biochemical methods, chiefly those first performed by Moll and Franke and associates.110,115 More recently, the problem of keratin subtyping has been expedited by the development of numerous monoclonal keratin-specific antibodies.108-110,116 This development was crucial for
Determining Site of Origin: Stepwise Approach
A
B
C
D
E
the ease of keratin subtyping now indispensable to the surgical pathologist. The detection of keratin, and therefore carcinomatous differentiation, is possible in tumors with extensive necrosis. Judkins and colleagues117 studied a small number of tumors with necrotic areas—including carcinomas, melanomas, and sarcomas—with a panel of antibodies and found that 78% of carcinomas stained with at least one antikeratin antibody in necrotic areas with 100% specificity.
209
Figure 8-1 This high-grade tumor (A) located in the vulvar area showed patchy staining for CK7 (B) and CAM5.2 (not shown). The tumor was negative for all other epithelial markers. Diffuse strong reactivity for CD31 (C), CD34 (D), and vimentin (E) supported the correct diagnosis of postradiation angiosarcoma. The patient was treated with radiation therapy for vulvar squamous cell carcinoma several years earlier.
DISTRIBUTION OF KERATIN ANTIGENS IN TISSUES Simple Epithelial Keratins
Simple epithelial keratins are the first keratins to appear in embryonic development, because they are expressed in virtually all simple (nonstratified), ductal, and pseudostratified epithelial tissues.109,110,115 Because these keratins are widespread, they may be useful for the identification of epithelial differentiation. Almost all mesotheliomas and carcinomas,109,110,115 except
210
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
B Split
EWS DNA Probe
Fusion Split Split
Figure 8-2 Undifferentiated tumor (A) in the pelvis of a 40-yearold woman was positive for CD99 (B) but negative for all other stains. Fluorescence in situ hybridization with EWS break-apart probe demonstrates one red-green fusion signal and one split signal (C) consistent with EWS gene rearrangement. This supports a diagnosis of extraskeletal Ewing sarcoma/primitive neuroectodermal tumor. (Courtesy Dr. Esther Elishaev.)
squamous cell carcinomas, contain the simple keratins 8 and 18. A few visceral organs, such as liver, contain only keratins 8 and 18. Although identified by many keratin antibodies that recognize a cocktail of keratin peptides (e.g., pankeratin antibodies AE1 and AE3 antibodies), CAM5.2 and 35βH11 recognize keratins 8 and 18 almost exclusively (Fig. 8-3). This group of antibodies is perhaps the most commonly used to demonstrate the simple keratins in surgical pathology. Because simple keratins are widely distributed in most carcinomas, these antibodies are particularly useful in the initial approach to investigation for carcinomatous differentiation (Table 8-3; see also Table 8-2). Cytokeratin 19 (CK19). The cytokeratin with the lowest molecular weight of the group, CK19 is a simple keratin that has a distribution similar to keratins 8 and 18, is also present in the basal layer of the squamous epithelium of mucosal surfaces, and may be seen in epidermal basal cells.118 CK19 is a good screening marker for epithelial neoplasms because of its wide distribution in simple epithelia and in many squamous tissues. The monoclonal antibody AE1 reacts with CK19 as does the AE1/AE3 cocktail. Also reacting in
C
Split Fusion
Split Split
EWS/22q12
Fusion
Positive for the EWS Gene Rearrangement
formalin-fixed tissues is a monoclonal antibody to CK19-RCK108.119 CK19 is mostly negative, or it is rarely seen focally in hepatocellular carcinoma.119 Cytokeratin 7 (CK7). CK7 is a 54-kD type II simple keratin that has a restricted distribution compared with keratins 8 and 18. Its presence in many simple, pseudostratified, and ductal epithelia and mesothelia is similar in distribution to that of keratins 8 and 18. Much of the data in the literature on CK7 is based on the reactivity patterns of antibody OV-TL 12/30 in FFPE tissues. The OV-TL 12/30 antibody parallels the CK7 immunoreactivity with RCK105, an antibody for use on frozen sections.120-122 Predigestion with protease or heat-induced epitope retrieval (HIER) is required for OV-TL 12/30. The lack or extreme paucity of CK7 distribution in tissues such as colonic epithelium, hepatocytes, and prostatic acinar tissue is used to diagnostic advantage.120-124 This antibody identifies transitional cell epithelium (Fig. 8-4, A) but is predominantly negative in most squamous epithelia. The restricted topography of CK7 makes it especially useful in evaluating the origin of adenocarcinomas, because this keratin is present in most breast, lung, ovarian, pancreatobiliary, and transitional cell carcinomas, but it is either
Determining Site of Origin: Stepwise Approach
211
TABLE 8-1 Sarcomas That Can Present as Cancer of Unknown Primary Source Ancillary Techniques to Confirm Diagnosis
Sarcoma Type
Age/Site
Morphology
Special Stains/IHC
Ewing sarcoma/ PNET
Usually <30 years Occurs in chest wall, extremities, retroperitoneum, pelvis Metastases to lungs and bone
Small round blue cell tumor
PAS+, CD99+, FLI1+
RT-PCR for EWS/FLI1, EWS/ERG, EWS/ETV1, EWS/E1AF, EWS/ FEV; EWS translocations can also be shown by FISH with EWS break-apart probe.
RMS Alveolar (A-RMS), embryonal (E-RMS), and pleomorphic (P-RMS)
A-RMS: 10 to 20 years; Occurs in extremities and perineum E-RMS: 3 to 10 years Occurs in prostate, paratesticular region, orbit, nasal cavity P-RMS: 50+ years Occurs in abdomen, retroperitoneum, chest wall, testes, extremities
Small round blue cells with alveolar growth pattern in A-RMS; round and spindle cells in E-RMS; round, spindle and pleomorphic cells in P-RMS
MSA+, desmin+, myoglobin+, myogenin (most specific)+, MyoD1+
RT-PCR for PAX3FOXO1 (FKHR) and PAX7-FOXO1 in alveolar RMS only
Desmoplastic small round cell tumor
Young adults, often adolescent boys Occurs in abdomen and pelvis, peritoneal implants
Round/oval cells in desmoplastic stroma in classic cases, variable morphology in other cases
Vimentin+, cytokeratin+, EMA+, desmin+, WT1+
RT-PCR for EWS/WT1
Synovial sarcoma
Young adults Occurs in extremities around large joints; now described in various locations, including lung and pleura
Spindle cell or biphasic glandular and spindle cell pattern; small round cells in poorly differentiated tumor
EMA+, keratin+ (biphasic tumors), CD99+, Bcl-2+, and recently TLE1+
RT-PCR for SYT/SSX1 and SYT/SSX2
Clear cell sarcoma (melanoma of soft parts)
Young adults Occurs in deep soft tissue with nodal and lung metastases
Mixed epithelioid and spindle cells in nested growth pattern
S-100+, HMB-45+, melan-A+
RT-PCR for EWS/ATF1 (not seen in cutaneous melanoma)
Alveolar soft-part sarcoma
Young adults, most often females Occurs in deep soft tissue; lung metastases common
Large polygonal cells, granular cytoplasm, prominent nucleoli, rare mitoses
PASD+, TFE3+
Membrane-bound rhomboidal crystals by EM; RT-PCR for ASPSCR1(ASPL)/ TFE3
PEComas
40 to 50 years, usually affects women Occurs in various visceral organs and soft tissue
Epithelioid and spindle cells with perivascular arrangement, clear to granular cytoplasm
HMB-45+, melanA+, but S-100 negative; musclemarker positive
EM: glycogen, premelanosomes, occasional dense bodies
Epithelioid sarcoma
Young adults Occurs in deep soft tissue of extremities; metastases to lung, lymph node, and skin
Epithelioid tumor cells, granuloma-like growth pattern
Keratin+, EMA+, vimentin+, CD34+, CK5/6−, p63−
Nothing specific; EM may be helpful
Vascular tumors
Adults Occurs in soft tissue and various visceral organs
Angiosarcoma: epithelioid and spindle cell tumor, vasoformative areas; epithelioid in hemangioendothelioma
Factor VIII+, CD31+, CD34+, FLI1+, thrombomodulin+, patchy keratin+
EM to identify endothelial cells is rarely required.
Leiomyosarcoma
Adults Occurs in abdomen, pelvis, and various other locations
Spindle or epithelioid cells with areas of smooth muscle differentiation
SMA+, HHF-35+, desmin+, caldesmon+, patchy keratin+
EM: smooth muscle differentiation
Continued
212
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
TABLE 8-1 Sarcomas That Can Present as Cancer of Unknown Primary Source—cont’d Ancillary Techniques to Confirm Diagnosis
Sarcoma Type
Age/Site
Morphology
Special Stains/IHC
Malignant peripheral nerve sheath tumor
Adults, NF1 patients (50%) Occurs in deep soft tissue in association with major nerve
Spindle cells with neural differentiation, abundant mitosis, ± necrosis; rarely epithelioid
S-100+ (weak, patchy), CD56+, CD57+, PGP9.5+, CD99+; negative for melanoma and vascular markers
Negative for SYT/SSX1 and SYT/SSX2; EM: neural differentiation
Chordoma
Adults, usually men Occurs in sacrococcygeal, thoracolumbar spine
Physaliferous cells, vacuolated cytoplasm, mucoid stroma
Brachyury+ (nuclear), S-100+, keratin+, CK7−/ CK20−, EMA+
EM rarely required
Extraskeletal myxoid chondrosarcoma
Adults Occurs in deep soft tissues of extremities; metastases easily confused with myoepithelial type carcinomas
Cords of spindle and epithelioid cells in myxoid stroma
S-100+, NSE+, synaptophysin+/−, keratin−, chromogranin−
RT-PCR for EWS/ CHN1 and TAF15/ CHN1
Endometrial stromal sarcoma
Adult women Found in abdominopelvic region; distant metastases to lungs
Oval/round to spindle cells; vague resemblance to proliferative pattern endometrial stroma
CD10+, ER+, Bcl-2−, CD34−; SMA and desmin positivity with smooth muscle differentiation
FISH for 7p15 translocation better than RT-PCR for JAZF1/SUZ12 (JJAZI) fusion
Gastrointestinal stromal tumor (GIST)
Adults Found in GI tract; abdominopelvic region; metastases often to the liver
Spindle or epithelioid cells
DOG1+, CD117+, CD34+; often negative for S-100, actin, and desmin
KIT-activating mutations
CK, Cytokeratin; EM, electron microscopy; EMA, epithelial membrane antigen; ER, estrogen receptor; EWS, Ewing sarcoma; FISH, fluorescence in situ hybridization; GI, gastrointestinal; HMB, human melanoma black; IHC, immunohistochemistry; MSA, muscle-specific actin; NF1, neurofibromatosis 1; NSE, neuron-specific enolase; PAS, periodic acid–Schiff; PGP9.5, protein gene product 9.5; PNET, primitive neuroectodermal tumor; RMS, rhabodmyosarcoma; RT-PCR, reverse transcription polymerase chain reaction; SMA, smooth muscle actin; WT1, Wilms tumor 1.
TABLE 8-2 Most Common Soft Keratins and Their Distribution Type II (Basic) Keratin
Molecular Weight (kD)
Typical Distribution in Normal Tissue
Type I (Acidic) Keratin
Molecular Weight (kD)
CK1
67
Epidermis of palms and soles
CK9 CK10
64 56.5
CK2
65
Epithelia, all locations
CK11
56
CK3
63
Cornea
CK12
55
CK4
59
Nonkeratinizing squamous epithelia
CK13
51
CK5
58
Basal cells of squamous and glandular epithelia, myoepithelia, mesothelium
CK14 CK15
50 50
CK6
56
Squamous epithelia, especially hyperproliferative
CK16
48
CK7
54
Simple epithelia
CK17
46
CK8
52
Basal cells of glandular epithelia, myoepithelia Simple epithelia, most glandular and squamous epithelia (basal) Simple epithelia of intestines and stomach, Merkel cells
CK18 CK19
45 40
CK20
46
From Quinlan RA, Schiller DL, Hatzfeld M, et al: Patterns of expression and organization of cytokeratin intermediate filaments. Ann NY Acad Sci. 1985;455:282-306.
Determining Site of Origin: Stepwise Approach
A
B
C
D
213
Figure 8-3 CAM5.2 stains liver and adjacent bile ducts (A), whereas keratin 34βE12 (K903) stains bile ducts (B) and stratum corneum of skin (C). CAM5.2 and 35βH11 stain only eccrine coils and not epidermis (D).
absent or present in only rare cells in colorectal, renal, and prostatic carcinomas (Table 8-4 and Box 8-1).116,120,121,125-132 CK7 stains squamous cell carcinoma and squamous dysplasias of the cervix.116 Although hepatocellular carcinomas are negative for CK7, it is expressed in the fibrolamellar variant.133 CK7 is also
typically expressed in mammary and extramammary Paget disease.134 A diagnostic pitfall in the interpretation of CK7 is that it stains subsets of endothelial cells of normal soft tissues in addition to endothelial cells in venules and lymphatics in intestinal mucosa, uterine exocervix, and lymphoid tissue.
TABLE 8-3 Keratin Antigens and Antibodies Cytokeratin Antigen
Antibody
Notes
CK8
35βH11
Carcinomas of simple epithelium
CK8
CAM5.2
Carcinomas of simple epithelium
Pankeratin
AE1/AE3
Carcinomas of simple and complex epithelium
CK1/10
34B4
Squamous cell carcinomas
CK7
OV-TL 12/30
Non–gastrointestinally derived carcinomas
CK20
K20
Most gastrointestinal carcinomas; mucinous ovarian, biliary, transitional, and Merkel cell carcinomas
CK19
RCK108
Most carcinomas; many carcinomas with squamous component; myoepithelial cells
CK1/5/10/14
34βE12
Basal cells of prostate; most duct-derived carcinomas
CK18/19
PKK1
Most carcinomas
CK10/11/13/14/15/16/19
AE1
Most squamous lesions and many carcinomas
CK8/14/15/16/18/19
MAK-6
Most carcinomas
214
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
Figure 8-4 Transitional cell carcinoma of the kidney is immunostained by CK7 (A) and CK20 (B). Metastatic gastrointestinalderived carcinoma demonstrates strong CK20 reactivity, but hepatic parenchyma is CK20 negative (C).
Diagnostic Utility of CK7. The specific diagnostic utility of CK7 lies in the fact that there are three dominant patterns of immunostaining: 1. Tumors that are characteristically strongly and diffusely positive include those of the salivary glands, lung, breast, ovary, endometrium, and bladder in addition to mesotheliomas, neuroendocrine tumors, pancreatobiliary adenocarcinomas, and the fibrolamellar variant of hepatocellular carcinoma. CK7 is also typically expressed in tumor cells of mammary and extramammary Paget disease. 2. CK7 variably stains the tumor cells in biliary and gastric tumors. 3. Carcinomas almost invariably negative but that may occasionally show rare CK7-positive cells include hepatocellular carcinomas, duodenal ampullary carcinomas, colon carcinomas, and renal, prostate, and adrenal cortical tumors. Strong diffuse CK7 immunostaining is a valuable marker in the diagnostic workup of a carcinoma and may be used as a starting point for further IHC study. Metastatic carcinomas in lung that are CK7 positive must be differentiated from a primary lung carcinoma with a panel of antibodies, and the IHC workup will be dependent on the patient’s age, gender, and findings at presentation. It is important to remember that CK7 may be expressed infrequently in certain tumors (see Table 8-4).135 In general, a high fidelity of CK7 expression is seen among primary and metastatic carcinomas.135
B
C Cytokeratin 20 (CK20). CK20 is a 46-kD LMW keratin that was discovered by Moll and associates.136 The tissue distribution is limited predominantly to GI epithelium and tumors thereof, mucinous tumors of the ovary, and Merkel cell neoplasms.127,130,131,137-139 The limited distribution of CK20 in colorectal, pancreatic, and gallbladder carcinomas; Merkel cell carcinomas; and transitional cell carcinomas (see Fig. 8-4, B) is useful in the identification of this group of tumors in primary or even metastatic sites.63,138,140 When combined with the specific tissue distribution of other keratins, such as CK7, it is possible to identify colon cancer metastases in the lung, distinguish pulmonary small cell carcinoma from Merkel cell carcinoma,141,142 and distinguish transitional cell carcinoma from other squamous cell carcinomas and poorly differentiated carcinomas. It is of importance to recognize that CK20 in this subgroup of tumors is most often distributed strongly and diffusely. Rare CK20-positive cells may be seen in some other neoplasms. As many as 10% of primary pulmonary adenocarcinomas not otherwise specified (NOS) and as many as 25% of mucinous bronchioloalveolar types may show CK20-positive cells.143,144 In addition, the controversial primary mucinous carcinoma of the lung—the so-called colloid carcinoma, goblet cell variant—shows CK20 immunostaining in approximately 50% of cases, along with nuclear positivity for CDX-2, a gut-specific marker.145 A very small percentage of müllerian and breast carcinomas may also show CK20 positivity.146 Cholangiocarcinomas of liver are also positive; the central (large duct) carcinomas are more likely to have
Determining Site of Origin: Stepwise Approach
TABLE 8-4 Cytokeratin 7: Percentage of Tumors with Expression Tumor Lung, adenocarcinoma Lung, small cell carcinoma
Percentage Expression 100 43
Ovary adenocarcinoma
100
Salivary gland, all tumors
100
Uterus, endometrium
100
Thyroid, all tumors
98
Breast, adenocarcinoma
96
Liver cholangiocarcinoma
93
Pancreas adenocarcinoma
92
Bladder, transitional cell
88
Cervix, squamous cell
87
Mesothelioma
65
Neuroendocrine carcinoma
56
Stomach adenocarcinoma
38
Head and neck, squamous cell
27
Esophagus, squamous cell
21
Kidney adenocarcinoma
11
Germ cell carcinoma
7
Colon adenocarcinoma
5
Adrenal carcinoma
0
Prostate carcinoma
0
Data from Chu P, Wu E, Weiss L: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13:962-972.
a high labeling index for CK20 in addition to CK7.147 The positive predictive value with the combination of CK7 and CK20 to predict the presence of metastatic carcinomas of colorectal or pancreatobiliary origins in the liver, based on clinical outcomes, is close to 0.9.148 It is important to remember that CK20 may be expressed infrequently in certain tumors (Table 8-5).135,149 In general, high fidelity of CK20 expression is observed among primary and metastatic carcinomas (see Fig. 8-4, C).135 Although the prominent expression of cytokeratins is the essential element of epithelial differentiation, on occasion expression of other lineage-specific markers may cloud the issue. Such is the case of finding keratins in nonepithelial tissues, the rare observation of LCA (CD45) in some undifferentiated or NECs,150 and CD30 in embryonal carcinomas. The use of a panel of antibodies and the pattern and intensity of immunostaining is critically important in these confounding situations.
215
KEY DIAGNOSTIC POINTS Simple Cytokeratins • CAM5.2 and AE1/AE3: Broad coverage for detection of carcinomatous differentiation; should be used together for screening • CK7 (positive): Adenocarcinomas of breast, lung, ovary, endometrium, and pancreas; mesothelioma; urothelial and thymic carcinomas; cervical squamous cell carcinoma; and fibrolamellar variant of hepatocellular carcinomas • CK7 (negative/rare positive): Renal, prostate, adrenocortical, squamous (except uterine cervix), small cell carcinomas, and hepatocellular carcinomas • CK20 (positive): Colorectal, pancreas (60%), and gastric carcinomas (50%); cholangiocarcinoma (40%); and mucinous ovarian, Merkel cell, and urothelial carcinomas (30%) • CK20 (negative/rare positive): Most breast, lung, and salivary gland carcinomas and hepatocellular, renal, prostate, adrenocortical, squamous, and small cell carcinomas • See Table 8-4 for CK7/CK20 immunoprofile of various carcinomas
Box 8-1 CYTOKERATINS 7 AND 20: DOMINANT IMMUNOPROFILES IN SELECT NEOPLASMS CK7+/CK20+ Transitional cell carcinoma Pancreatic carcinoma Ovarian mucinous carcinoma Gastric adenocarcinoma CK7+/CK20− Non–small cell carcinoma of lung Small cell carcinoma of lung Breast carcinoma, ductal and lobular Nonmucinous ovarian carcinoma Endometrial adenocarcinoma Gastric adenocarcinoma Pancreatic ductal adenocarcinoma Cholangiocarcinoma Mesothelioma Squamous cell carcinoma of cervix CK7−/CK20+ Colorectal adenocarcinoma Merkel cell carcinoma CK7−/CK20− Squamous cell carcinoma, lung Prostate adenocarcinoma Renal cell carcinoma Hepatocellular carcinoma Adrenocortical carcinoma Some thymic carcinoma Modified from Wang MP, Zee S, Zarbo RJ, et al: Coordinate expression of cytokeratin 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem. 1995;3:99-107; and Chu P, Wu E, Weiss L: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms. A survey of 435 cases. Mod Pathol. 2000; 13:962-972.
216
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
TABLE 8-5 Cytokeratin 20: Percentage of Tumors with Expression Site, Tumor Colon, adenocarcinoma
Percentage Expression 100
Skin, Merkel cell carcinoma
78
Pancreas, adenocarcinoma
62
Stomach, adenocarcinoma
50
Liver, cholangiocarcinoma
43
Bladder, transitional cell carcinoma
29
Lung, adenocarcinoma
10
Liver, hepatoma
9
Gut, carcinoid
6
Lung, adenocarcinoma
10
Head and neck, squamous cell carcinoma
6
Ovary, adenocarcinoma
4
Adrenal, carcinoma
0
Breast, ductal/lobular carcinoma
0
Cervix, squamous cell carcinoma
0
Esophagus, squamous cell carcinoma
0
Germ cell, carcinoma
0
Kidney, carcinoma
0
Mesothelioma
0
Prostate, adenocarcinoma
0
Salivary gland, all tumors
0
Thyroid, all tumors
0
Thymus, thymoma
0
Endometrial adenocarcinoma
0
Lung, carcinoid
0
Lung, small cell carcinoma
0
Lung, squamous cell carcinoma
0
This keratin of stratified type is also typically present in squamous epithelium, and antibody K903 is good for detecting squamous differentiation in an otherwise poorly differentiated carcinoma. These HMW structural keratins are also commonly seen in duct-derived epithelium (breast, pancreas, biliary tract, lung) and in transitional, ovarian, and mesothelial tissues.151-153 The degree of immunostaining of these tissues with HMW keratin antibodies is typically strong and diffuse, a feature that is helpful diagnostically, because HMW keratin immunostaining is seen only focally in visceral epithelial tissues such as colon, stomach, kidney, and liver. KEY DIAGNOSTIC POINTS Antibodies to Complex Keratins • Confirms the presence of basal cells of prostate (see Fig. 8-5) • Confirms the presence of myoepithelial cells in breast • Present in basal cell layer of stratified and squamous epithelium • Strong and diffuse in tumors of squamous epithelial differentiation • Present in a wide variety of duct-derived carcinomas and mesotheliomas and in most neoplasms that demonstrate tonofilaments ultrastructurally
Cytokeratin 5 (CK5) and Cytokeratin 5/6 (Ck5/6). CK5 and CK6 are basic (type II) polypeptides with molecular weights of 58 kD and 56 kD respectively. Most studies have been performed with antibodies to CK5/6, which have been found useful in the differential diagnosis of metastatic carcinoma in the pleura versus epithelial mesothelioma. Epithelial mesotheliomas are strongly positive in all cases (Fig. 8-6), but as many as 30% of pulmonary adenocarcinomas will show focal variable immunostaining.154 Almost all squamous cell carcinomas, half of transitional cell carcinomas, and many undifferentiated large cell carcinomas immunostain with CK5/6
Data from Chu P, Wu E, Weiss L: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13:962-972.
Keratins of Stratified Epithelia: Complex Keratins
Keratins of high molecular weight are observed in stratified epithelia and generally are not present in the simple visceral-type epithelia. Basal cells of prostate and myoepithelial cell populations of ducts and glandular tissue also contain an abundance of HMW type II keratins and LMW type I keratins. The antibody 34βE12 or keratin 903 (K903)151-153 can identify a cocktail of keratins that includes Moll types I, II, V, X, XI, and XIV/XV. The practical diagnostic use of this pattern of expression is to identify basal and myoepithelial cells in their respective organs (Fig. 8-5). For example, the staining of myoepithelial cells around ductal carcinoma in situ or sclerosing adenosis can confirm a noninvasive lesion.
Figure 8-5 Basal cells in this prostate section are seen with K903. Myoepithelial cells in the breast can also be seen with K903.
Determining Site of Origin: Stepwise Approach
A
217
B
Figure 8-6 Antibodies to CK5/CK6 strongly stain reactive mesothelial cells in a pleural biopsy (A) and in epithelial mesothelioma cells (B).
(Table 8-6).155-157 CK5/6 has excellent sensitivity and specificity for the detection of squamous differentiation in poorly differentiated carcinomas,155,158 and p63 is also seen with high frequency in squamous and transitional carcinomas; when used with the CK5/6 antibody, p63
TABLE 8-6 Cytokeratin 5/6: Percentage of Tumors with Expression Tumor
Percentage Expression
Skin, squamous cell
100
Skin, basal cell
100
Thymus, thymoma
100
Salivary gland, all tumors
93
Mesothelioma
76
Bladder, transitional cell
62
Endometrial adenocarcinoma
50
Pancreas, carcinoma
38
Breast, carcinoma
31
Ovary, carcinoma
25
Liver, cholangiocarcinoma
14
Gut, carcinoid
10
affords high sensitivity and specificity for squamous differentiation.155,158 Myoepithelial cells of the breast, glandular epithelium, and basal cells of the prostate express CK5/6.157 Some carcinomas of ovarian origin may also display CK5/6.157 Hyperplastic mesothelial cells can be seen on occasion in the sinuses of lymph nodes from the chest or cervical chain.159 The differential diagnosis in this instance is metastatic carcinoma. The presence of strong diffuse CK5/6 in the cells of these nests should aid in identifying them as mesothelial in origin, but this should be confirmed by using more specific markers for mesothelium, such as calretinin and WT1. Interest in these antibodies has been somewhat renewed, because both CK5 and CK5/6 are also used to identify the basal-like molecular class of breast cancer. We have recently shown that CK5 (clone XM26) is superior to CK5/6 antibody (clone D5/16B4) in identifying the basal-like phenotype of breast carcinoma with high sensitivity and specificity.160 Whether CK5 is superior to CK5/6 in other distinctions must be investigated, such as to detect mesothelioma versus carcinoma or to identify squamous differentiation. KEY DIAGNOSTIC POINTS CK5 and CK5/6
Lung, adenocarcinoma
5
Liver, hepatoma
4
Adrenal, carcinoma
0
Colon, adenocarcinoma
0
Germ cell, carcinoma
0
Kidney, carcinoma
0
Prostate, carcinoma
0
Stomach, adenocarcinoma
0
Keratins in Nonepithelial Cells
Thyroid, all tumors
0
Keratins have been documented by IHC,107,161-164 dot immunoblot,87 and PCR105 in several types of tumors in which there is no morphologic evidence of epithelial differentiation. This type of keratin immunostaining has
Data from Chu P, Weiss LM: Expression of CK 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod Pathol. 2002;6:6-10.
• Good indicators of squamous and transitional cell differentiation • Good discriminators of mesothelial differentiation versus adenocarcinoma in lung • Positive in myoepithelial cells of breast and basal cells of prostate • Sensitive and specific markers of basal-like phenotype of breast carcinoma
218
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
been variably referred to as anomalous, aberrant, spurious, and unexpected.165 The keratins most often found in these nonepithelial mesenchymal tissues or melanocytic lesions are keratins 8 and 18 and, less commonly, keratin 19. Antibodies that detect these LMW keratins have demonstrated positive immunostaining in a variety of FFPE mesenchymal tumors, including leiomyosarcomas, fibrosarcoma, liposarcoma, RMS, MPNSTs, some malignant fibrous histiocytomas, gastrointestinal stromal tumors (GISTS), rare solitary fibrous tumors of pleura, angiosarcoma, endometrial stromal sarcoma, and primitive neuroectodermal tumors (PNETs).100,166-180 Keratin usually stains scattered cells in this group of tumors in traditional FFPE tissue, whereas carcinomas and sarcomatoid carcinomas are heavily and diffusely stained (Fig. 8-7).181 In addition, keratin-positive soft tissue and bone tumors with partial epithelial differentiation are variably stained with keratin in the epithelial areas as expected. This group includes synovial sarcomas, epithelioid sarcoma, chordoma, MPNST, and adamantinoma of long bones.182-187 Although some of the soft tissue tumors may mimic metastatic carcinoma morphologically, the finding of sporadic cell immunostaining is unlike the strong, diffuse immunostaining seen in carcinomas, especially with the broad-coverage antibodies. Frozen tissues fixed in acetone or alcohol, including alcohol-fixed cytologic specimens, yield far more keratin-positive cells. This result can be confusing diagnostically, especially with cytologic specimens for which alcohol is a standard fixative for needle aspiration specimens. Malignant melanoma also demonstrates immunostaining for keratins 8 and 18, but in FFPE tissues, the prevalence is approximately 1% of cases, with focal tumor cell staining.85,188-190 Frozen sections and alcoholfixed melanomas show substantially more positive tumor cells than do formalin-fixed specimens, and it is important to recognize this fact to avoid misdiagnosing melanoma as a carcinoma, especially in alcohol-fixed cytologic preparations. The consensus regarding keratin immunostaining of nonepithelioid sarcomas and melanomas is that although the presence of keratin is real as measured by molecular techniques and more sensitive
A
immunohistologic methods (frozen sections, alcohol fixation), the observed nonexpression of keratin staining in these tumors in formalin-fixed tissue is desirable because of its diagnostic usefulness. Truly spurious keratin immunoreactivity has been described in human glial tissue and in some human astrocytomas, especially with antibodies AE1 and 34βE12.191 In addition, the cocktail AE1/AE3 may cross-react with both normal and neoplastic astrocytes.192 The spurious keratin immunoreactivity is probably due to cross-reaction with glial cells that contain glial fibrillary acidic proteins (GFAPs).191 This is an obvious pitfall for the misdiagnosis of metastatic carcinoma in the brain. The antibody CAM5.2 does not react with astroglial cells and thus is best used to detect carcinomatous differentiation in the CNS. Meningiomas, especially the secretory variant, may express also keratin in as many as one third of cases.193-196 Epithelial differentiation is simulated in lymph nodes with the LMW keratin–positive fibroblastic reticulum cells of the paracortex (Fig. 8-8).197-205 These dendritic cells immunostain with CAM5.2, and rarely with AE1/ AE3, to reveal an extensive network of extrafollicular dendritic processes in lymph nodes, tonsils, and spleen.206 These keratin-positive cells are a pitfall for the diagnosis of metastatic carcinoma, because the conventional wisdom had been that keratin-positive cells in a lymph node equated with metastatic carcinoma. The pitfall is twofold: When searching for keratin-positive micrometastases in patients with breast carcinoma, the pathologist must distinguish the dendritic processes from carcinoma cells that cluster in the subcapsular sinus. Also, needle aspirates and touch imprints of lymph nodes may contain keratin-positive cells without containing metastatic carcinoma, so the pathologist must be aware of the morphologic features of the keratinpositive cells. Keratin positivity has been described in plasma cells, plasmacytoma, and anaplastic large cell lymphoma (ALCL).206-211 For ALCL, keratins may be detected in as many as 30% of cases, and in the presence of some EMA-positive anaplastic lymphoma cells, the definitive diagnosis can be confusing. However, adherence to a
B
Figure 8-7 Smooth muscle neoplasm (A) shows scattered CAM5.2-positive cells (B), a typical focal pattern of immunostaining for keratin seen in a variety of mesenchymal tumors.
Determining Site of Origin: Stepwise Approach
Figure 8-8 In this normal lymph node, interfollicular dendritic cells are CAM5.2 positive.
broad-spectrum antibody for keratin immunoreactivity will show only focal rare staining at most in these lymphomas. Plasmacytomas likewise should be studied with broad-coverage antibodies in a panel that includes antibodies to CD138 and κ and λ light chains. The majority of keratin immunostaining is performed on FFPE tissues, and the duration of formalin fixation is a key factor when trying to optimize the technical performance of keratin immunoperoxidase stains. Fixation time is closely related to the time required for enzymatic predigestion.212 Generally, tissue fixed in 10% formalin for more than 2 days requires greater antigen retrieval; less time is required for tissues fixed briefly (hours) in 10% formalin. Depending on antibody and fixation duration, most if not all keratin antibodies require epitope retrieval for optimal keratin antibody performance. KEY DIAGNOSTIC POINTS Keratin in Nonepithelial Tumors • Focal presence is found in many sarcomas. • Focal rare presence is seen in melanoma, mainly with CAM5.2. • Occasional weak reactivity is seen in lymphoid neoplasms. • Keratin is commonly found in dendritic cells of lymph nodes, mainly with CAM5.2. • Antibody AE1/AE3 may give a spurious positive keratin result in astrocytic neoplasms.
Step Three: Carcinoma Subsets with Frequent Vimentin Coexpression Mesenchymal and endothelial cells regularly immunostain with vimentin, and this immunostaining generally provides a measure of internal quality control for the quality of immunoreactivity.213 If there is no immunostaining of blood vessels or stromal cells by vimentin, it denotes significant damage to tissue antigens or other failure of the staining procedure. Carcinomas in effusion
219
specimens are universally positive for vimentin, presumably an in vivo fluid effect, and thus have no diagnostic utility.214 Initially thought to be an intermediate filament restricted to mesenchymal cells, vimentin has been found in a diverse number of neoplasms, including a variety of carcinomas (Box 8-2). Vimentin stains virtually all spindle cell neoplasms, mesenchymal spindle cell neoplasms and sarcomatoid carcinomas included. However, vimentin stains a subset of carcinomas regularly and to a significant degree, and this may be useful in the context of a panel of antibodies to narrow a differential diagnosis. The cellular vimentin immunostaining pattern is often a perinuclear band of reactivity, particularly for endometrioid adenocarcinomas. Carcinomas with frequent (>50% to 60% of cells) and strong (>25% of cells) vimentin coexpression include spindle cell carcinomas, renal cell carcinomas (except the chromophobe variant), müllerian endometrioid adenocarcinomas, and malignant mixed müllerian tumors, serous ovarian carcinomas, pleomorphic salivary gland tumors, basal-like breast carcinomas, and follicular thyroid carcinomas.215-217 Epithelial and sarcomatoid mesotheliomas also regularly demonstrate vimentin. Certain carcinomas may immunostain with vimentin but with lesser frequency (10% to 20%) and with far less intensity (<10% of cells). This group includes adenocarcinomas of colorectum, lung, breast, and prostate and nonserous ovarian carcinomas. Therefore the finding of substantial coexpression of vimentin in a metastatic carcinoma may aid in narrowing the differential diagnosis, and it adds value to the rest of the antibody panel (Fig. 8-9). Vimentin coexpression is especially useful in differentiating endometrial endometrioid carcinomas in uterine curettage specimens from endocervical adenocarcinomas, including the endometrioid variant of endocervical adenocarcinoma. Endometrial endometrioid carcinomas immunostain strongly for vimentin, but endocervical carcinomas rarely stain; weak focal staining is seen in as many as 13% of endocervical carcinomas.218,219 However, with the current antigen retrieval techniques, moderate to occasionally strong vimentin expression may be seen in endocervical carcinomas, and a panel approach is more useful in this distinction.220 Box 8-2 MAJOR PATTERNS OF COEXPRESSION OF CYTOKERATIN/ VIMENTIN IN CARCINOMAS Coexpression Common (>50%) Endometrial adenocarcinoma Renal cell carcinoma Salivary gland carcinoma Spindle cell carcinoma Thyroid follicular carcinoma Coexpression Uncommon (<10%) Endocervical adenocarcinoma Colorectal adenocarcinoma Breast ductal-lobular carcinoma Lung non–small cell carcinoma Prostate adenocarcinoma
220
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
Figure 8-9 This sarcomatoid carcinoma of lung (A) richly coexpresses CAM5.2 (B) and vimentin (C). Most carcinomas of this type in lung and upper aerodigestive tract are sarcomatoid squamous cell carcinomas.
KEY DIAGNOSTIC POINTS Vimentin Coexpression in Carcinomas • Vimentin is common in renal, endometrioid endometrial, salivary gland, follicular thyroid, and sarcomatoid (spindle cell) carcinomas, basal-like breast carcinomas, and in the stromal components of malignant mixed müllerian tumors. • Vimentin may be seen in a few cells in 10% to 20% of colorectal, lung, breast, prostate, and ovarian adenocarcinomas. • Vimentin expression is not diagnostically useful in body cavity effusion specimens. • Epithelial and sarcomatoid mesotheliomas are usually vimentin positive. • Vimentin coexpression provides an important internal quality measure for antigen assessment in any tissue.
Step Four: Supplemental Epithelial Markers Although not specific for tissue lineage, these epithelial markers demonstrate characteristic immunostaining patterns for certain tissue types and therefore are useful to corroborate a diagnosis when used as part of a panel of antibodies. With the availability of more
B
C
tissue-specific markers and transcription factors, the usage of these supplemental markers has decreased over time. Only the more commonly used markers are discussed here. CARCINOEMBRYONIC ANTIGEN
Carcinoembryonic antigen (CEA) is a 180-kD glycoprotein that is 50% carbohydrate. Many CEA antibodies to a variety of CEA epitopes are available. The polyclonal antibodies commonly cross-react with tissue nonspecific cross-reacting antigens and biliary glycoprotein 1 (Bg1).221-223 The polyclonal (p) antibody is therefore used for demonstrating a canalicular pattern in hepatocellular carcinoma, and monoclonal (m) antibodies are used for everything else. Although CEA is a sensitive marker, adenocarcinomas of colorectal origin cannot be distinguished from lung adenocarcinomas or ductal carcinomas of the breast because of the low specificity of CEA. Primary adenocarcinomas of the lung are typically negative for CK20 and positive for CK7 and CEA, whereas colorectal carcinomas are CK7 negative but positive for CK20 and CEA; ductal and lobular breast carcinomas are negative for CK20, positive for CK7, and are often CEA positive; and ovarian carcinomas are CK7 positive, CEA negative, and may be positive or negative for CK20.121,125-129,224-227 Neoplasms that
Determining Site of Origin: Stepwise Approach
Box 8-3 CARCINOEMBRYONIC ANTIGEN (CEA) IMMUNOSTAINING OF ADENOCARCINOMA CEA Positive Paranasal sinuses Lung Colon Stomach Biliary system* Pancreas Sweat glands Breast CEA Negative Prostate Kidney Adrenal glands Endometrium Ovarian (serous) *Pericanalicular pattern with polyclonal antibody.
typically are strongly positive for most CEA antibodies include adenocarcinomas of the lung, colon, stomach, biliary tree, pancreas, urinary bladder, endocervix, paranasal sinuses, sweat glands, and breast (Box 8-3).228 The usefulness of CEA when used with keratins is to
221
corroborate expected staining for CEA, whether positive or negative. Neoplasms that are essentially negative with most CEA antibodies include adenocarcinomas of prostate, kidney, adrenal gland, and endometrium220 along with serous ovarian tumors, thyroid tumors (except the medullary type), and mesotheliomas. Liver cell–derived tumors are nonreactive with the monoclonal CEA (mCEA) antibodies but do react with the polyclonal antibodies in a distinct pattern of pericanalicular staining (Fig. 8-10), because the polyclonal antibodies cross-react with hepatic bile canalicular Bg1.229,230 Adenocarcinomas of pulmonary, GI, thymic, endocervical, and pancreaticobiliary origin typically show strong, although variable, cytoplasmic immunostaining for CEA antibodies. Immunostaining patterns of CEA in the liver are particularly useful. Epithelioid hemangioendothelioma (EH) of liver can mimic carcinoma to perfection. Demonstration of positive CD31/CD34 and factor VIII with variable (usually focal, sometimes diffuse) keratin and lack of CEA will separate this entity from hepatocellular carcinoma.231 However, it is important to remember that unlike normal liver, neoplastic liver sinusoids demonstrate the presence of immunoreactive CD34. This may be confused with EH, but it is useful in the differential diagnosis of primary liver neoplasm versus metastatic carcinoma and nonneoplastic liver, especially on small biopsy samples (Fig. 8-11).232
A
B
C
Figure 8-10 Hepatocellular carcinoma (A) may show a canalicular polyclonal carcinoembryonic antigen pattern (B) and CAM5.2 immunostaining (C).
222
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
Figure 8-11 Normal liver sinusoids (A) do not express CD34, whereas hepatic adenomas (B) and hepatocellular carcinomas (C) often show sinusoidal CD34. This characteristic is especially helpful on needle aspirates or biopsies to identify primary liver neoplasia.
KEY DIAGNOSTIC POINTS Carcinogenic Embryonic Antigen • Polyclonal CEA–positive tumor (pericanalicular pattern): hepatocellular carcinoma • Other monoclonal CEA–positive tumors: gastrointestinal, lung, breast, thymus, endocervical, primary cholangiocarcinoma • CEA-negative tumors: prostate, renal, endometrial, adrenal, and serous ovarian tumors and mesothelioma • Epithelioid hemangioendothelioma of liver (mimicker of hepatocellular carcinoma): CEA negative but positive for vascular markers
EPITHELIAL MEMBRANE ANTIGEN
Encoded by the MUC1 gene on chromosome 1 and a derivative human antigen, epithelial membrane antigen (EMA) is a transmembrane glycoprotein of the breast mucin complex, and its expression is increased in carcinomas.233,234 Unlike the normal breast, in which EMA is present on the apical cell membrane, neoplasms demonstrate EMA on the entire circumference of the cell membrane.234 Increased amounts of the large glycoprotein interfere with cell-to-cell and cell-to-matrix adhesion in neoplastic cells.235 The utility of EMA antibody is in the detection of epithelial differentiation, as a supplement to the cytokeratins. Spindle cell, small cell, and large cell neoplasms
B
C
may on rare occasion be stained with EMA but may be only focally positive for cytokeratins.236,237 Several EMA antibodies are available, each of which reacts to different epitopes of the large glycoprotein antigen; they include MAM-6, episialin, polymorphic epithelial mucin, CA 15-3, DF3 antigen, and breast epithelial mucin.238-244 The EMA antibodies stain skin and adnexa, breast, lung, bile ducts, pancreas, salivary gland, urothelium, endometrium and endocervix, prostate ducts, thyroid, mesothelium, and neoplasms of these tissues (Table 8-7). Many sarcomatoid carcinomas and epithelial and sarcomatoid mesotheliomas are positive. Reactive mesothelium may stain weakly compared with thick, membranous staining of mesothelioma.245,246 Many types of adenocarcinomas immunostain with EMA and must be distinguished from mesothelial cells in effusions by using a panel of immunostains that includes CK5/CK6, CEA, LeuM1, and BerEP4.59,154,247 Subsets of normal and neoplastic hematopoietic cells express EMA, including plasma cells, erythroblasts, and neoplastic cells; this includes the lymphocytic and histiocytic (L&H) cells (60% of cases) of lymphocytepredominant Hodgkin lymphoma; 5% of B-cell lymphomas; 18% of T-cell lymphomas; and approximately 60% of ALCLs.208,248-257 The EMA antibodies do not have absolute sensitivity and specificity for carcinomas and therefore should always be used with a panel of cytokeratins and other corroborating antibodies, such as LCA.258 However, in distinguishing ovarian stromal neoplasm from carcinoma, EMA is better than keratin. Most
Determining Site of Origin: Stepwise Approach
TABLE 8-7 Epithelial Membrane Antigen in Carcinomas and Nonepithelial Tissues Typically positive
Carcinomas: Skin and adnexa, breast, bile ducts, lung, pancreas, salivary gland, urothelium, endometrium, endocervix prostate, thyroid Noncarcinomatous lesions: Meningioma, mesotheliomas, solitary fibrous tumor
Focal/patchy positive
Carcinomas: Sarcomatoid carcinomas Noncarcinomatous lesions: Plasma cell tumors, lymphocyte and histiocyte cells of Hodgkin lymphoma, a few cells of non-Hodgkin lymphoma, anaplastic large cell lymphoma, malignant peripheral nerve sheath tumors, synovial sarcoma, leiomyosarcoma
Mostly negative
Germ cell tumors except choriocarcinoma, ovarian sex cord stromal tumors
ovarian stromal neoplasms show focal to patchy keratin reactivity but are almost always negative for EMA.259 In addition to epithelial neoplasms, a number of spindle cell tumors, sarcomas, CNS tumors, small round cell tumors, and a few GCTs may be positive with EMA. These tumors include solitary fibrous tumors, meningiomas, ependymomas, malignant nerve sheath tumors (MNSTs), synovial sarcoma, leiomyosarcoma, malignant fibrous histiocytoma, epithelioid sarcoma, and chordoma. With the exception of the last two tumors mentioned, EMA immunostaining is focal. Choroid plexus neoplasms and meningiomas show strong membranous EMA immunostaining. GCTs are largely negative260 except for variable EMA immunostaining in choriocarcinoma and teratoma, whereas the epithelial small round cell tumors of nephroblastoma and hepatoblastoma immunostain with EMA in the majority of cases. KEY DIAGNOSTIC POINTS
223
BerEP4, Bg8, AND MOC-31
BerEP4, Bg8, and MOC-31 are markers of epithelial differentiation. These stains are most often used to distinguish adenocarcinomas from mesothelial proliferations (Fig. 8-12).261-263 BerEP4 and MOC-31 antibodies are directed against the epitope on glycoproteins present on the surface of glandular epithelial cells of endodermal derivation. Squamous epithelium of ectodermal derivation virtually never expresses BerEP4.264 The characteristic staining with BerEP4 and MOC-31 is membranous. Bg8 antibody is directed against the Lewis Y antigen and shows cytoplasmic staining in carcinomas. When BerEP4, Bg8, and MOC-31 are combined with calretinin (nuclear and cytoplasmic staining in mesotheliomas), the combination provides the best sensitivity and specificity for distinguishing adenocarcinoma from mesothelioma.265 MOC-31 is also very useful in distinguishing metastatic tumors from primary hepatocellular carcinoma when used in a panel format. Along with HepPar1 and pCEA, MOC-31 permits distinction of metastatic carcinomas in liver from hepatocellular carcinoma 99% of the time.266-268
A
Epithelial Membrane Antigen • Used as a supplement to detect epithelial neoplasms, because CKs may rarely be focally positive or negative in undifferentiated carcinomas • Membranous, cytoplasmic, or mixed staining (both) • Positive in meningioma and ependymoma • Positive in renal cell carcinoma, negative in adrenocortical tumor • Negative in malignant melanoma • Negative in germ cell neoplasms except choriocarcinoma and teratoma • Stains plasma cells, 60% of anaplastic large cell lymphomas, lymphocyte and histiocyte cells of Hodgkin lymphoma, and a few T- and B-cell lymphomas • Strongly positive in malignant mesothelioma and in some reactive mesothelial proliferations; weak or negative for normal mesothelial cells • Almost always negative in ovarian stromal neoplasms
B Figure 8-12 This ovarian carcinoma in a pelvic wash is positive for BER-EP4 (A) and negative for calretinin (B).
224
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
KEY DIAGNOSTIC POINTS BerEP4, Bg8, and MOC-31 • Best epithelial markers to distinguish adenocarcinomas from mesothelioma • Membranous reactivity for BerEP4 and MOC-31 and cytoplasmic reactivity for Bg8 • Squamous cell carcinomas: generally negative for BerEP4 • MOC-31: especially useful in distinguishing between primary liver carcinoma and metastatic tumor
Step Five: Focusing on Tumor Differentiation—Cell-Specific Products The tissue of origin of metastases can be narrowed to a few sites with the panel approach by using CKs, CEA, EMA, and vimentin. The use of additional antibodies to cell-specific products in most instances has a very high specificity for certain tissues, which enables the pathologist to narrow the focus in the search for the origin of a metastasis. The antibodies discussed here include neuroendocrine markers, thyroglobulin, TTF-1, calretinin, WT1, GCDFP-15, mammaglobin, hormone receptors, villin, CDX-2, HepPar1, Arginase-1, SMAD4 (formerly DPC4), prostate carcinoma antigens, UROIII, thrombomodulin, renal cell carcinoma (RCC) antibody, CD10, Pax-8 and Pax-2, melan-A, inhibin, adrenal binding protein, GCT markers, and CD5. NEUROENDOCRINE ANTIBODIES
Antibodies to neuroendocrine cell components are usually used in the context of trying to distinguish tumor cell types in specific organs such as lung, thyroid, colon, and adrenal gland. The antibodies are not typically used in the initial screening panel of the workup of an undifferentiated tumor, and a paucity of literature is available that deals with this topic.269 It is critically important to use the following antibody immunostains together as a panel, because no single antibody has perfect specificity and sensitivity. It is well known that a few neuroendocrine cells can be seen with IHC in a wide variety of carcinomas; this is not to be equated with a diagnosis of NEC. Only after a complete account of the clinical findings, imaging studies, histologic studies, and IHC findings should a diagnosis be rendered. Chromogranins
The chromogranins (types A, B, and C) are a group of monomeric proteins that compose the major portion of the soluble protein extract of the neurosecretory granules of neuroendocrine cells; chromogranin A, with a molecular weight of 75 kD, is the most abundantly distributed. A strong correlation exists between the chromogranin cellular immunostaining quantity and the number of neuroendocrine-type secretory granules seen at the level of electron microscopy.270
The LK2H10 clone is a monoclonal antibody with abundant representation in the literature.270-273 Immunostaining intensity decreases with poor differentiation. The specificity of LK2H10 is close to 100%, but sensitivity is closer to 75%. Chromogranin reactivity is generally patchy compared with that of synaptophysin in neuroendocrine tumors. Synaptophysin
Synaptophysin is a glycoprotein that is an integral part of the neuroendocrine secretory granule membrane,269,274 and it is recognized by monoclonal antibody (SY38) in a variety of neuroendocrine tumors. Synaptophysin is a broad-spectrum neuroendocrine marker275 with higher sensitivity but lower specificity than antibody to chromogranin. Immunostaining for SY38 has also been documented to be most effective in identifying metastases of the neuroendocrine type.269 Synaptophysin immunostaining alone is insufficient grounds for labeling a neoplasm “neuroendocrine.” When used in the context of appropriate morphology, synaptophysin is useful to identify neuroendocrine features. Large cell undifferentiated NEC (LCNEC) can present as CUPS, and it is easy to miss the diagnosis without applying the appropriate neuroendocrine markers. The correct diagnosis of LCNEC is an important distinction, because it carries the same dismal prognosis as small cell carcinoma, whether in the lung or GI tract.276,277 Synaptophysin may be the most frequent positive marker in LCNEC.276 In one study, small cell lung carcinomas were stained by synaptophysin in as many as 79% of cases, whereas chromogranin was positive in 47% to 60% of cases, bombesin was positive in 45% of cases, and neuronspecific enolase (NSE) was seen in 33% to 60% of cases.278 Synaptophysin may be seen in 8% of non–small cell carcinomas.279 Neural Cell Adhesion Molecule (CD56) and Leu-7 (CD57)
Neural cell-adhesion molecule (NCAM [CD56]) and Leu-7 (CD57) are two very similar antigens. CD56 is a glycoprotein expressed on neurons, glial tissue, skeletal muscle, and natural killer (NK) cells.280 The CD57 antigen of a human T-cell line generates monoclonal antibody HNK-1; the differentiation antigen of the T-cell line is indicative of a NK cell activity.281 The CD57 antibody also recognizes antigen of myelinassociated glycoprotein in the myelin of the central and peripheral nervous systems. CD57 reactivity has also been found in enterochromaffin cells, pancreatic islet cells, islet cell tumors, carcinoid tumors, pheochromocytomas, and small cell carcinoma of the lung.282-285 CD56 and CD57 lack the specificity of chromogranin and synaptophysin for detecting neuroendocrine neoplasms and therefore should be used as part of a panel that includes these antibodies.286 Neuron-Specific Enolase
The enolase enzymes comprise five different forms, each of which is composed of three homodimers and two hybrids. Neuron-specific enolase (NSE) is found in a
Determining Site of Origin: Stepwise Approach
variety of normal and neoplastic neuroendocrine cells and predominates in the brain.287-290 Originally believed to be a specific marker for neuroendocrine differentiation, NSE has subsequently been observed in virtually every type of neoplasm; because of this, it is a poor antibody to use to screen for neuroendocrine differentiation. Overall a poor marker for detection of neuroendocrine differentiation because of its lack of specificity, NSE may be useful in combination with other, more specific antibodies, such as chromogranin and synaptophysin, for the appropriate neuroendocrine morphologic identification and documentation of immunostaining. Peptide Hormones
Peptide hormones are present in unique, sequestered tissues in the normal state and generally recapitulate the same hormone production in neoplasms. Endocrine neoplasms, with few exceptions, show a characteristic histologic pattern, and therefore the study of hormone production is often of academic interest only. Poorly differentiated neuroendocrine neoplasms, depending on the site of origin, may produce characteristic peptide hormones. Islet cell tumors produce insulin, glucagon, somatostatin, and gastrin; pulmonary small cell carcinoma produces bombesin in 45% of cases;278 and medullary thyroid carcinoma produces calcitonin. Cytokeratin Profile of Neuroendocrine Carcinoma
The CK profile of NECs is somewhat distinctive, in that virtually all are positive to some degree for CK8 and CK18 (e.g., CAM5.2), sometimes positive for CK7, and are negative for CK20 and HMW keratin (e.g., K903). Merkel cell carcinomas are characteristically positive for CK20 (67% of cases)291 and negative for CK7, which is the reverse for immunostaining of small cell carcinomas of lung (CK7+, CK20−).
KEY DIAGNOSTIC POINTS Neuroendocrine Differentiation • A typical neuroendocrine keratin profile is CAM5.2 and CK7 positive. • Chromogranin and synaptophysin complement each other as part of a diagnostic panel. • CD56, CD57, and other specific peptides (bombesin, glucagon, etc.) may supplement the preceding studies. • Subsets of peripheral NECs may be TTF-1 positive. • Subsets of NECs of the gastrointestinal tract may be CDX-2 positive.
THYROGLOBULIN
Thyroglobulin, a 670-kD heavily glycosylated protein, provides iodination sites for the production of thyroid hormones and is unique to the thyroid follicular epithelium. The great majority of thyroid carcinomas show immunostaining with thyroglobulin, although most of the positive cases are readily interpreted as follicular or
225
papillary carcinomas. The undifferentiated anaplastic carcinomas are generally negative for thyroglobulin. Thyroid carcinomas (except the medullary type) are almost always negative with mCEA antibody, which is a helpful feature in differential diagnosis. Thyroglobulin may be seen as scattered positive cells in medullary carcinoma and, conversely, calcitonin-positive cells may be seen in poorly differentiated follicular carcinomas.292-294 Thyroglobulin may be seen in 10% to 25% of cases of leukemic blast cells in bone marrow.295
KEY DIAGNOSTIC POINTS Thyroglobulin • Positive in almost all thyroid carcinomas, with reduced immunostaining of poorly differentiated carcinoma to complete absence in anaplastic types • Highly specific for thyroid carcinomas, with rare positivity in some leukemic blast cells
THYROID TRANSCRIPTION FACTOR 1 AND OTHER LUNG MARKERS
Thyroid transcription factor 1 (TTF-1), a nuclear tissuespecific protein transcription factor, is found only in thyroid and thyroid tumors regardless of histologic type (except for the anaplastic type). It is also found in lung carcinomas, including adenocarcinomas (75%); non–small cell carcinomas (63%); neuroendocrine and small cell carcinomas (>90%); and rare squamous cell carcinomas (<10%).296-301 Selectively expressed during embryogenesis in the thyroid, the diencephalon of the brain, and in respiratory epithelium, TTF-1 binds to and activates factors for surfactant protein derived from Clara cells.302 TTF-1 is rarely seen in carcinomas outside of the lung or thyroid (Fig. 8-13).303-305 Neuroendocrine tumors of the lung, including typical and atypical carcinoids and large cell NECs, are almost always positive for TTF-1, demonstrating a kinship with small cell carcinomas.303 Small cell and large cell NECs from origins other than the lung are also frequently TTF-1 positive.306 These sites include prostate, bladder, cervix, GI tract, thyroid, and breast.306,307 However, Merkel cell carcinomas are TTF-1 negative.306,308 The utility of TTF-1 becomes readily apparent in the differential diagnosis of primary versus metastatic carcinomas, especially in the lung or in effusions.309,310 CK7 and CK20, along with TTF-1 and CEA, are the antibodies that best discriminate primary lung carcinoma from carcinomas metastatic to the lung. In a study by Roh,311 the sensitivity of TTF-1 for metastatic lung carcinoma in lymph nodes was 69%. The specificity of TTF-1 for pulmonary lesions was confirmed by Chang and colleagues.312 TTF-1 demonstrates cytoplasmic immunostaining of hepatocellular carcinomas in 71% of cases but shows no nuclear immunostaining.313 In our opinion, nonnuclear staining is not diagnostically useful in the workup of carcinoma of unknown origin.
226
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
B
C
D
Figure 8-13 Thyroid transcription factor 1 (TTF-1) antibody identifies 70% of pulmonary adenocarcinomas (A and B) and 95% of pulmonary small cell carcinomas (C and D).
In the last few years, the specificity of TTF-1 has been challenged. Kubba and colleagues314 and Siami and colleagues,315 both from M.D. Anderson Cancer Center, have shown TTF-1 (clone 8G7G3/1) reactivity in tumors of the endocervix, endometrium, and ovary; however, the majority of the cases in their study showed only rare and focal staining. Robens and colleagues316 also showed TTF-1 reactivity in 2.4% of breast carcinomas. Other markers used for identifying lung carcinoma include surfactant apoprotein (PE10) and napsin A. PE10 is now less commonly used because of its low sensitivity; it is positive in not more than 50% of lung
carcinomas.317,318 The sensitivity and specificity of napsin A are similar to that of TTF-1 but shows cytoplasmic reactivity in lung carcinomas. Similar to TTF-1, napsin A has been reported to stain some nonpulmonary carcinomas (renal clear cell, renal papillary, endometrial, and hepatocellular carcinomas).319-321 CALRETININ AND WILMS TUMOR 1 PROTEIN
Calretinin and Wilms tumor 1 (WT1) protein are two positive mesothelial markers. Calretinin is a 29-kD intracellular calcium-binding protein that has been described in a variety of cells, including neurons,
KEY DIAGNOSTIC POINTS TTF-1 and Other Similar Markers • Nuclear immunostaining with TTF-1 is seen in all carcinomas of thyroid origin (except the anaplastic type). • Nuclear immunostaining with TTF-1 is seen in the vast majority of carcinomas of the lung: adenocarcinomas (~70% to 80%), large cell neuroendocrine carcinomas, and small cell carcinomas (95%). • Small cell carcinomas of other sites—gastrointestinal tract, bladder, cervix, and prostate—are frequently TTF-1 positive, although such entities are rare. • Merkel cell carcinomas are negative for TTF-1. • Focal to patchy TTF-1 expression is seen in gynecologic tract tumors. • PE10 lacks sensitivity for identifying lung carcinomas. • Napsin A shows cytoplasmic reactivity in lung carcinomas and has comparable sensitivity and specificity to TTF-1.
Determining Site of Origin: Stepwise Approach
A
227
B
Figure 8-14 A, Solid carcinoma of unknown origin invading the bowel wall in an elderly woman. B, Wilms tumor 1 (WT1) positivity confirms ovarian origin; the tumor was also positive for CK7 and BER-EP4 (not shown).
steroid-producing cells, renal convoluted tubules, eccrine glands, thymic keratinized cells, and mesothelial cells.322,323 Differences in immunostaining results have been reported, depending on the antibody used, with greater specificity seen with the Zymed clone.324 Calretinin may be expressed in 8% of lung adenocarcinomas, but the expression is generally focal and weak.325 Focal weak expression may also be seen in carcinoma from other sites. WT1 protein is expressed at high levels in kidney glomeruli, the gonadal ridge of developing gonads, Sertoli cells of the testis, and both epithelial and granulosa cells of the ovary, which suggests a developmental role in both the genital system and kidneys.326 WT1 nuclear expression is seen in normal mesothelium and mesothelioma and in Wilms tumor (hence the name); in desmoplastic small round cell tumor, with antibody to the carboxy-terminal end; and most notably in müllerian epithelial neoplasms, especially tubal/ovarian serous carcinoma (Fig. 8-14).327,328 The literature regarding endometrial serous carcinoma is contradictory, but it appears that nuclear expression may be seen in approximately 20% of endometrial serous carcinomas.329-334 However, the reactivity in endometrial serous carcinoma is often patchy and moderate KEY DIAGNOSTIC POINTS Calretinin and WT1 • Calretinin and WT1 are the two most sensitive mesothelial markers with high specificity. • Calretinin may be expressed in up to 8% of adenocarcinoma from various sites. • Ovarian/tubal serous carcinomas demonstrate diffuse, strong WT1 nuclear expression. • The majority of endometrial serous carcinomas are negative for WT1; positive cases generally show patchy staining. • Pure and mixed mucinous breast carcinomas may be WT1 positive; however, staining is rarely intense compared with that of serous ovarian carcinomas.
compared with that of tubal/ovarian serous carcinoma, in which it is diffuse and strong. WT1 expression is not seen in endometrioid type tumors. Domfeh and others335 showed weak to moderate WT1 expression in 64% of pure mucinous carcinomas of the breast and in 29% of breast mucinous carcinomas mixed with other subtypes. GROSS CYSTIC DISEASE FLUID PROTEIN AND MAMMAGLOBIN
Originally described by Pearlman and colleagues336 and Haagensen and associates,337 the prolactin-inducing protein identified by Murphy and coworkers338 has the same amino acid sequence as GCDFP-15 and is found in abundance in breast cystic fluid and in any cell type that has apocrine features.339,340 The latter, in addition to breast, includes acinar structures in salivary glands, apocrine glands, and sweat glands and also in Paget disease of skin, vulva, and prostate.341-345 Aside from these immunoreactivities, most other carcinomas show no appreciable immunostaining. The positive predictive value and specificity of GCDFP-15 are both reported to be 99%.345 The sensitivity for the monoclonal antibody clone D6 has been reported to be as high as 74%,345 but the experience of others has been closer to 40% to 50%342 and even lower.346 Similar results are obtained with the use of antibody BRST-2. Because the specificity of GCDFP-15 antibodies for breast carcinoma is so high, it is often used in a screening panel in the appropriate clinical situation, which often turns out to be the presentation of a woman with CUPS or a new lung mass in a patient with a history of breast cancer. Others have demonstrated the utility and specificity of GCDFP-15 antibodies in the distinction of breast carcinoma metastatic in the lung.347,348 Recently, the absolute specificity of GCDFP-15 has also been challenged. Striebel and colleagues349 reported GCDFP-15 immunoreactivity in 5.2% (11/211) of pulmonary adenocarcinomas. These tumors were characteristically of mixed acinar and papillary types with
228
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
abundant extracellular mucin production. However, 81% percent of these tumors coexpressed TTF-1, which would be helpful in their distinction from breast carcinomas. The mammaglobin gene encodes a 93–amino acid protein that is largely confined to breast tissue. Han and coworkers350 developed antibodies to mammaglobin and found high sensitivity (84.3%) and specificity (85%) for the discrimination of breast carcinoma in lymph nodes. In contrast, the sensitivity and specificity for GCDFP-15 (BRST-2) expression in their study was 44.3% and 97.9%. Among nonbreast carcinomas, convincing mammaglobin expression is seen in endometrioid carcinomas (~40% cases) and sweat and salivary gland tumors.346,350-352 This nonspecificity of mammaglobin expression in endometrioid adenocarcinomas could be used diagnostically.353 Caution is advised in interpreting weak/equivocal immunoreactivity with mammaglobin, because this pattern of staining can be seen in several nonbreast, nonendometrial carcinomas. With respect to breast carcinoma, mammaglobin is a more sensitive marker than GCDFP-15 (Fig. 8-15).346
KEY DIAGNOSTIC POINTS GCDFP-15 and Mammaglobin • GCDFP-15 has greater than 90% specificity but much lower sensitivity for breast carcinoma. • Mammaglobin has high sensitivity (~60%) for breast carcinoma, but the specificity is lower. • Both GCDFP-15 and mammaglobin should be included in the panel for diagnosing breast carcinoma (GCDFP-15+, mammaglobin+, CK7+/CK20−, ER/PR+, CEA+). • Mammaglobin expression in endometrioid carcinomas could be useful diagnostically.
HORMONE RECEPTORS: ESTROGEN AND PROGESTERONE
Intuitively, it would seem as though the estrogen receptor/progesterone receptor (ER/PR) would be confined to hormone-responsive tissues such as breast, but even the recent literature on this topic is controversial. Although some authors conclude that ER/PR is found
A
B
C
D
Figure 8-15 Adenocarcinoma involving abdominal wall (A). The tumor cells are strongly and diffusely positive for CK7 (B), patchy positive for gross cystic disease fluid protein 15 (C), and show diffuse strong staining for mammaglobin (D). In spite of negative receptor status, the morphology and immunohistochemistry profile was consistent with the patient’s known history of breast carcinoma from several years earlier.
Determining Site of Origin: Stepwise Approach
only in subsets of breast carcinomas and carcinomas of the ovary and endometrium,348 others have observed mostly ER, and rarely PR, in carcinomas of the lung,354-357 stomach, and thyroid. Vargas and colleagues358 demonstrated the estrogenrelated protein p29 in 98% of non–small cell lung cancers by IHC, suggesting that the estrogen axis may be important in this group of malignancies. In the study by Vargas and associates, these same tumors were all negative with the commercially available antibody ER1D5. Survival of this group of patients differed for men versus women, suggesting some gender-specific p29-associated factor influence. Dabbs and colleagues359 observed ER in pulmonary adenocarcinomas using antibody clone 6F11 with HIER. Nuclear ER was observed in 67% of lung adenocarcinomas, including the bronchioloalveolar variants, but it was not seen with antibody clone ER1D5. Therefore caution is advised in using ER clone 6F11 in isolation in distinguishing between a primary lung and breast carcinoma. In a systematic review of a number of studies that evaluated ER/PR expression in tumors, Wei and others360 concluded that hormone receptor expression can occasionally be seen in nonmammary, non– gynecologic tract tumors. Nevertheless, diffuse strong staining for ER (clone 1D5 or rabbit monoclonal SP1) in the right clinical context, along with the appropriate cytokeratin expression profile (CK7+/CK20−), is highly indicative of a breast or gynecologic primary tumor.361 KEY DIAGNOSTIC POINTS Hormone Receptors (Estrogen and Progesterone) • Diffuse, strong expression is suggestive of breast or gynecologic primary tumor. • Weak/moderate expression should be judged more carefully, taking into account the clinical presentation and results of other immunohistochemical stains. • ER antibody clone 6F11 gives more false-positive results compared with other clones.
VILLIN
Villin is a calcium-dependent actin-binding cytoskeletal protein found in the brush border of the intestine and in the proximal renal tubular epithelium. A brush border is characteristic of colorectal carcinomas and is recognized at the ultrastructural level by the presence of microvilli with a dense core of microfilaments, core rootlets, and surface glycocalyces. Up to 33% of pulmonary adenocarcinomas may demonstrate microvillus rootlets by ultrastructure, and their presence correlates closely with villin immunostaining.362-365 Antibodies to villin are useful for identifying its molecular presence, which is in almost all colorectal carcinomas and in more than 90% of lung carcinomas that have microvillus rootlets. CKs are a necessary part of a diagnostic panel to distinguish lung and colon carcinomas, and 90% of lung adenocarcinomas are CK20 negative, whereas colorectal carcinomas are CK20 positive. Villin may
229
stain hepatocellular neoplasms in a canalicular pattern similar to pCEA.366 Villin has also been reported to be expressed in carcinomas of the stomach, pancreas, and gall bladder and also in renal clear cell carcinoma and endometrial carcinomas.367 CDX-2 PROTEIN
CDX2 is a homeobox gene that encodes a transcription protein factor that guides development of intestinal epithelial cells from the region of the duodenum to the rectum.368 Discovered in 1983, the homeobox gene encodes proteins called homeodomains, which are very important in the developmental processes of many multicellular organisms. The homeobox is a conserved DNA motif that encodes proteins that act as transcription factors, which control the actions of other genes by binding to segments of DNA. The absence of CDX-2 protein is a lethal event in utero, and heterozygotes have GI developmental abnormalities.369-372 Using clone CDX2-88, Barbareschi and colleagues373 found a very high sensitivity and specificity for detection of colorectal carcinomas, with some CDX-2 expression in other adenocarcinomas of the GI tract and in ovarian mucinous tumors. Useful in both paraffin tissue and cytology specimens, they concluded that CDX-2 was highly sensitive and specific for intestinal differentiation (Table 8-8). Other studies have confirmed the high specificity of CDX-2 for intestinally derived adenocarcinomas, including those of the stomach, duodenum, pancreas, and biliary tree and also gastroesophageal adenocarcinomas.374,375 Colorectal and duodenal adenocarcinomas tend to have a diffuse distribution of nuclear staining in a majority of cells, whereas adenocarcinomas from other intestinal sites tend to have staining in a minority of cells. CDX-2 expression decreases dramatically in the subset of colon carcinomas that are minimally differentiated, TABLE 8-8 Percentage of Adenocarcinomas by Site with CDX-2 and Villin Immunostaining (Two to Three Positive) Carcinoma
CDX-2
Villin
Colorectal
99
82
Duodenal
100
100
Gastric
70
42
Esophagus
67
78
Pancreas
32
40
Biliary
25
60
Mucinous ovary
64
64
Urinary bladder
100
100
Thyroid
4
0
Prostate
4
0
Data from Werling RW, Yaziji H, Bacchi CE, Gown AM: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 2003;27(3):303-310.
230
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
and these are usually associated with mutations of the DNA mismatch repair genes.376 NECs of intestinal derivation showed a focal pattern of nuclear immunostaining in only 42% of cases in one study.375 In the study by Barbareschi and colleagues,377 well-differentiated NECs of the ileum/appendix showed greatest expression, whereas rectal and upper GI tumors showed lower expression. In addition, 39% of neuroendocrine tumors from sites outside the GI tract— including the bladder, breast, uterus, salivary gland, prostate, and lung—showed low expression. Not surprisingly, urinary bladder adenocarcinomas derived from the intestinal urachus are often CDX-2 positive, as are urachal cysts in the bladder (Fig. 8-16). Wang and colleagues378 studied the IHC distinction between primary adenocarcinomas of the bladder and secondary involvement of the bladder by colorectal adenocarcinoma. The key antibodies that permitted discrimination of these tumors were β-catenin (clone 14), CK7, and thrombomodulin. All colorectal tumors showed nuclear β-catenin (bladder negative), were CK7 negative, and were negative for thrombomodulin. Bladder adenocarcinomas were all thrombomodulin positive and were variably positive for CK7. Importantly, other mucinous neoplasms with morphologic intestinal features, the “colloid” carcinoma of the lung145 with goblet cells (100%) and a subset of ovarian mucinous carcinomas (64%), are CDX-2 positive.375 The majority of the colloid lung tumors are TTF-1 positive, a feature that allows distinction from metastatic colorectal mucinous carcinomas. Ovarian mucinous carcinoma may be separated from GI mucinous carcinoma by virtue of typical immunostaining for CK7 in the ovarian tumors. A prior study reported uterine cervical adenocarcinoma with intestinal features to be negative for both CDX-2 and CK20,379 however, a few recent studies have shown nuclear CDX-2 immunoreactivity in as many as 30% of cervical adenocarcinomas.380-382 This immunoreactivity is seen not only with müllerian mucinous or intestinal mucinous differentiation but also in endometrioid tumors of the uterine cervix.382 However,
A
dominant CK7 reactivity is useful in determining gynecologic origin.381 CDX-2 immunostaining may rarely be seen focally in prostate or thyroid carcinomas.375 Concomitant use of villin antibody adds specificity for intestinal differentiation. Although some CDX-2 may be seen in nonintestinal carcinomas, villin is negative in these tumors.375 CDX-2 does not immunostain liver, hepatocellular carcinoma, or carcinomas of kidney, breast, lung, or salivary gland.379 The specificity of CDX-2 for metastatic colorectal carcinoma in the liver is enhanced by the concomitant use of a CK20-positive/CK7-negative profile, because CDX-2 may be positive in upper GI carcinomas.383 Endometrioid carcinomas of the uterus or ovary may mimic colorectal carcinomas, and they may demonstrate nuclear CDX-2, in which case a panel of antibodies that includes CK7, CK20, Pax-8, villin, vimentin, and estrogen receptor would be needed to discriminate from colorectal carcinomas (Fig. 8-17). KEY DIAGNOSTIC POINTS Villin and CDX-2 • Villin is positive in colorectal and pulmonary adenocarcinomas and other gastrointestinal tumors. • Canalicular villin expression is seen in hepatocellular carcinoma. • CDX-2 is highly sensitive and specific for intestinal differentiation. • Adenocarcinomas of urinary bladder (urachal origin) are CDX-2 positive but are also positive for thrombomodulin and CK7, whereas colorectal carcinomas are negative with thrombomodulin and CK7 and have nuclear β-catenin expression. • Adenocarcinomas of the uterine cervix have been reported to be CDX-2 positive in 30% of cases. • Endometrioid adenocarcinomas of the ovary and the uterus are CDX-2 positive in as many as 25% of cases. • Other CDX-2–positive tumors include colloid lung carcinomas (also positive for TTF-1) and ovarian mucinous carcinomas (CK7 positive with CK20 expression weaker than CK7).
B
Figure 8-16 Urachal cyst. A, Hematoxylin and eosin. B, CDX-2–positive nuclei in this urachal remnant from the dome of the bladder confirm the gastrointestinal origin of this cyst.
Determining Site of Origin: Stepwise Approach
A
B
C
D
E HepPar1
Hepatocyte paraffin 1 (HepPar1) is a very sensitive and highly specific marker of hepatocytic differentiation in paraffin-embedded tissue. The antibody is directed against a mitochondrial antigen present within hepatocytes and shows a characteristic granular cytoplasmic staining (Fig. 8-18). The antibody HepPar1 is 79% specific for hepatic differentiation, and sensitivity is high.266,366,384 However, just like any other specific marker, staining is also observed in a limited number of
231
Figure 8-17 Tumor with endometrioid morphology invading the vagina. A, Hematoxylin and eosin section. Positive staining for villin (B) and CDX-2 (C) with negative staining for vimentin (D) and estrogen receptor (E) confirms the diagnosis of metastatic colon carcinoma.
other tumors.385-387 True hepatocellular carcinoma differentiation (pCEA+, HepPar1+, sinusoidal cell CD34+) has been described on rare occasions as a component of some adenocarcinomas from urinary bladder and stomach.388,389 As components of ovarian tumors, hepatoid carcinomas regularly express significant HepPar1.390 Signet-ring cell carcinomas (SRCCs) of the stomach usually show diffuse cytoplasmic staining, unlike breast or colorectal SRCCs, which are negative for HepPar1.391
232
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
B Figure 8-18 Hepatocellular carcinoma with clear cell features (A) show granular cytoplasmic positivity for HepPar1 (B).
Antibody MOC-31 detects a cell-surface glycoprotein that is found largely on epithelial cells and carcinomas. Morrison and colleagues demonstrated that, along with HepPar1 and pCEA, MOC-31 permitted distinction of metastatic carcinomas in liver from hepatocellular carcinoma 99% of the time.266-268 ARGINASE-1
Arginase-1, an enzyme used in the urea cycle, is another marker of hepatocyte differentiation. Although only a limited amount of data exists, arginase-1 appears to be quite specific for hepatocellular carcinoma. In 2010, Yan and colleagues392 demonstrated sensitivity of arginase-1 for detection of hepatocellular carcinoma to be more than 95%; both nuclear and cytoplasmic reactivity was considered a positive result. The specificity for arginase-1 was also very good, because only 2 of 557 nonhepatocellular tumors showed reactivity. In a cytologic study of 35 metastatic carcinomas, McKnight and colleagues393 did not find arginase-1 expression in any of the metastatic tumors, whereas 37 of 44 hepatocellular carcinomas were positive for the enzyme. However, in a similar cytology-based study, Fujiwara and colleagues394 showed the sensitivity of arginase-1 to be 81%, but they reported arginase-1 reactivity in 10% (6 of 61 cases) of metastatic adenocarcinomas involving the liver. The tumors that showed arginase-1 reactivity included two colorectal, three pancreatic, and one breast tumor. Only two of the three positive pancreatic cases showed strong reactivity.394 If these findings are further reproduced by other investigators, arginase-1 will become a very useful marker. DELETED IN PANCREATIC CARCINOMA, LOCUS 4: SMAD4
SMAD4, also known as DPC4, a tumor suppressor gene,395 the expression of which is lost in approximately 45% to 50% of pancreatic adenocarcinomas. SMAD4 protein expression is retained in many of the malignancies that enter into the differential diagnosis of
metastatic mucinous carcinoma with diffuse peritoneal involvement.396 A positive staining by IHC is therefore noninformative. A negative staining, which should always be interpreted with caution, is suggestive of a pancreatic primary tumor. However, some nonpancreatic tumors—such as from the ampulla, small intestine, colorectal region, and gallbladder—have also been shown to lack SMAD4 expression.397-399 KEY DIAGNOSTIC POINTS HepPar1, Arginase-1, and SMAD4 • HepPar1: A specific marker of hepatocellular differentiation, HepPar1 shows characteristic cytoplasmic granular staining. • Other HepPar1-positive tumors: HepPar1 marks signet-ring cell carcinoma of stomach and hepatoid differentiation in carcinomas from other sites (e.g., ovary). • Arginase-1: Another sensitive and specific marker of hepatocellular carcinoma, it shows nuclear and cytoplasmic reactivity. • SMAD4 (formerly DPC4): Lack of staining is specific for pancreatic primary and should be interpreted with caution with appropriate controls. • Other rare tumors sometimes negative for SMAD4: ampullary, colorectal, and gallbladder carcinoma
PROSTATE CARCINOMA ANTIGENS Prostate-Specific Antigen and Prostatic Acid Phosphatase
Together, antibodies to prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) will stain more than 95% of prostate carcinomas, but with certain caveats. The immunostaining of tumor cells falls off with increasing Gleason grade with both antibodies, and PAP is found in a wide variety of other tumors, including hindgut carcinoid tumors.400 PSA has been found as patchy staining in some salivary gland ductal carcinomas, in as many as one third of breast carcinomas and sweat gland tumors, in periurethral glands in both sexes,
Determining Site of Origin: Stepwise Approach
in cystitis glandularis of the bladder, in urachal remnants, and in some anal glands.401-408 Nevertheless, PSA is highly specific for prostate tissue, because it functions as a serine protease of the seminal fluid,409,410 and it stains the histologic subtypes of tumors that include mucinous, signet-ring, and endometrioid carcinomas.411-413 Metastatic prostate carcinoma may be immunostained to a variable degree in metastatic sites, including lymph nodes.414,415 Immunoreactivity is not diminished by brief decalcification procedures. PSA has been found to stain scattered tumor cells from cutaneous malignant melanoma and its metastases.416 This should not create a diagnostic dilemma, because prostate cancers are strongly and diffusely positive with LMW keratin antibodies such as CAM5.2, which is another illustration of the necessity of examining tumors with a panel of antibodies. Salivary gland ductal carcinomas are usually positive in a patchy distribution for both PSA and PAP, and they stain strongly for androgen receptors (ARs).417 Close clinical correlation with a PSA-, PAP-, and AR-positive profile becomes mandatory. Pro-PSA (pPSA), an antibody to the PSA precursor, is present in benign, preneoplastic, and malignant prostate epithelium with no diminution of staining in highgrade carcinomas. The antibody PS2P446 shows promise for the detection of high-grade prostate carcinoma in metastatic sites.418,419 Prostate-Specific Membrane Antigen, Prostein (p501S), NKX3-1
Prostate-specific membrane antigen (PSMA), which has a partial homologous structure with the transferrin receptor, is highly specific for prostate cells.420-422 Unlike PAP, PSMA is upregulated in prostate carcinoma, so that stronger staining is seen in higher-grade carcinomas.422,423 Extraprostatic expression of PSMA has been documented,422 and the antibodies that have been cited in the literature are 7E11-C5.3423,424 and 3F5.4G6.425 PSMA in a tumor of unknown primary is highly specific for prostate carcinoma. In addition to PSMA, two additional sensitive and specific antibodies for identifying prostate cancer are prostein and NKX3-1. Prostein is protein localized to the golgi complex and therefore shows a characteristic perinuclear golgi pattern of staining. Sheriden and colleagues426 reported this pattern of staining even in poorly differentiated tumors and metastases. NKX3-1 is a protein expressed primarily in adult prostate and has growth-suppression and differentiation effects in prostate cells. NKX3-1 typically shows nuclear reactivity. In a large tissue microarray study of 4061 samples, Gelmann and colleagues427 showed NKX3-1 expression in benign and malignant prostatic tissue; the only other tumor types that showed NKX3-1 expression included 9% of ductal breast cancers and 27% of lobular breast cancers. Subsequently, Gurel and colleagues428 showed the sensitivity of NKX3-1 to be 98.6% (68/69 cases) in identifying metastatic prostatic adenocarcinoma; specificity was 99.7% (only 1 of 349 nonprostatic tumors). The only positive nonprostatic tumor was an invasive lobular carcinoma of the breast.
233
Alpha-Methylacyl-Co-A Racemase
This mitochondrial peroxisome enzyme, alphamethylacyl-Co-A racemase (AMACR, also known as P504S), encoded by the AMACR gene, catalyzes the racemization of α-methyl–branched carboxylic coenzyme-A thioesters and is present in prostate tissue and in a wide variety of carcinomas (colorectal, ovarian, breast, bladder, lung, and renal cell), melanomas, and lymphomas.429 AMACR is very useful in prostate needle biopsies, when the differential diagnosis is carcinoma versus benign prostatic tissue, but it is not specific for prostate carcinoma in metastatic sites. Using a polyclonal antibody, Zhou and colleagues429 found AMACR to be positive in the majority of prostate carcinomas (83%). Using a monoclonal antibody (clone 13H4) to AMACR, Jiang and colleagues430 found AMACR immunoreactivity in 100% of prostatic carcinomas. Although a positive staining with AMACR in a case of CUPS is of no diagnostic use, a negative staining would make the diagnosis of prostate carcinoma unlikely.
KEY DIAGNOSTIC POINTS Prostate Carcinoma Antigens • PSMA, prostein, NKX3-1, PSA, and PAP used together provide a very sensitive and specific panel for diagnosis of prostatic carcinoma at a metastatic site (see Chapter 16). • Extraprostatic expression for all prostate carcinoma antigens has been described including breast (rare in males), salivary gland, pancreas, and anal gland tumors; rare PSA expression sometimes occurs in melanomas. • AMACR is useful in distinguishing prostatic carcinoma from benign prostatic tissue but has limited utility in a case of CUPS.
UROPLAKIN III AND THROMBOMODULIN
The asymmetric unit membrane unique to the umbrella cells of urinary tract transitional epithelium contains a transmembrane protein that is unique to urothelium. In studies performed thus far, the uroplakins (UROs) are highly specific for transitional epithelium, with moderate sensitivity,431,432 and are not seen in squamous epithelial tissue. The study by Parker and colleagues433 demonstrated a sensitivity of 57% for uroplakin III (UROIII) and a nearly perfect specificity. Unlike UROIII, thrombomodulin (TM) is highly sensitive but is not a specific marker of urothelial differentiation (Fig. 8-19). TM is an endothelial cell-surface glycoprotein that forms a 1 : 1 complex with thrombin, therefore it is expressed in vascular tumors composed of endothelial cells.434,435 TM expression is also seen in squamous carcinomas,436 it is a sensitive marker for mesotheliomas, and it is useful in the distinction of mesothelioma versus adenocarcinoma.437 Given the moderate sensitivity of UROIII and the lack of specificity of TM, it is usually necessary to use a panel of antibodies to increase the probability of identifying urothelial cell carcinoma. The best antibody
234
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
A
B
Figure 8-19 Metastatic carcinoma in a lung vascular space (A). The tumor cells were diffusely and strongly positive for thrombomodulin (B) and showed only focal weak staining with uroplakin III (not shown), confirming urothelial differentiation of the tumor cells.
panel to use to differentiate urothelial carcinoma in pelvic organs when the differential diagnosis is colorectal carcinoma, prostate carcinoma, renal cell carcinoma, or ovarian transitional cell carcinoma includes UROIII, TM, p63, CK5/6, 34βE12, and CK7/20.433,438-440 The highest sensitivities for urothelial carcinoma detection are p63 (96%), 34βE12 (88%), TM (70% to 90%), and CK20 (48%).433,440 Transitional cell components of ovarian carcinomas differ immunohistologically from urothelial carcinomas.441 TM (18%) and UROIII (6%) are rarely and focally expressed in ovarian transitional cell carcinomas, which also express WT1 (82%) and CK7 but not CK20, whereas urothelial carcinomas are WT1 negative but are positive for CK7 and CK20 (50%), TM (76%), and UROIII (50%).441-443 Brenner tumors of the ovary have an immunoprofile similar to urothelial tumors. Müllerian tumors of any morphologic type are often positive for Pax-8; however, data on urothelial tumors with respect to Pax-8 are limited. Tong and colleagues reported Pax-8 reactivity in 24% of renal pelvic urothelial carcinomas but observed no reactivity in 40 bladder urothelial carcinomas.444,445 Pax-8 and hormone receptors should also be included in the panel to distinguish between high-grade müllerian tumor and poorly differentiated urothelial tumor. KEY DIAGNOSTIC POINTS Local Pelvic Tumors • Urothelial carcinoma: Positive for UROIII, TM, p63, and cytokeratins 5/6 and 7/20; negative for WT1, and Pax-8 • Ovarian transitional cell carcinoma: Positive for WT1 and CK7; negative for CK20, UROIII, TM, and p63 • Prostate carcinoma: Positive for PSA and prostatic acid phosphatase (PAP); negative for CK7/20, UROIII, TM, p63, and Pax-8 • Colorectal carcinoma: Positive for CDX-2 and CK20; negative for CK7, UROIII, WT1, TM, p63, and Pax-8
RENAL CELL CARCINOMA ANTIGEN
Renal cell carcinoma (RCC) antigen is a 200-kD glycoprotein known as gp200, and it is present on the surface and cytoplasm of a variety of normal tissues that include the brush border of renal proximal tubules, surface of breast acini, epididymis, parathyroid, and thyroid tissue.446 McGregor and colleagues447 surveyed a number of nonrenal tumors and found that 29% of breast tumors, 28% of embryonal carcinomas, and all parathyroid adenomas were positive with RCC. In addition, 80% percent of primary RCCs were positive (clear cell 84%, papillary 96%, chromophobe 45%, sarcomatoid 25%, and collecting duct 0%) with more than 10% of cells positive in 93% of cases. No other primary renal tumors were positive, including oncocytomas. Only 67% of metastatic renal carcinomas were positive with RCC antibody, and only 2% of nonrenal metastases were positive with RCC (mostly metastatic breast carcinomas). Bakshi and colleagues448 showed that RCC may not be a very specific marker, because 76 out of 362 nonrenal tumor samples demonstrated either focal or diffuse expression for RCC. These tumors included adrenocortical neoplasms (37/170, 22%); tumors of the colon (11/29, 37.9%), breast (9/27, 33%), prostate (5/18, 27.8%), ovary (2/17, 11.8%), lung (3/21, 14.3%), and parathyroid (3/3, 100%); and melanoma (3/18, 16.7%).448 RCC antibody has a moderate degree of specificity and relatively low sensitivity for renal cell carcinoma, especially for small biopsies in which only a few tumor cells may show immunostaining. CD10
CD10 is marker of early lymphoid progenitor and normal germinal center cells. However, in the nonhematopoietic arena, it reacts mainly against proteins of the epithelium of the renal proximal tubule. It is considered a sensitive marker for renal cell carcinoma and endometrial stromal sarcomas. Although CD10 stains some other tumors as well, including transitional cell
Determining Site of Origin: Stepwise Approach
carcinoma, prostatic carcinoma, melanoma, rhabdomyosarcoma, leiomyosarcoma, hemangiopericytoma, solitary fibrous tumor, schwannomas, and hepatomas (in a canalicular pattern); given the right clinical context, it could be a specific marker of renal cell carcinoma.449 PAIRED BOX GENE 2 (PAX2)
PAX2 gene expression is required for the normal development of the kidney. Using in situ hybridization (ISH) technique on formalin-fixed human embryonic tissue, Tellier and colleagues450 demonstrated Pax-2 expression in mesonephros, metanephros, adrenals, spinal cord, optic and otic vesicles, retina, semicircular canals of the inner ear, and hindbrain. Although IHC studies of the effects of Pax-2 on human tumors are rather limited, so far Pax-2 protein expression is concordant with the findings of Tellier and colleagues.450 Pax-2 expression is mainly seen in renal tumors (positive in as many as 88% of renal clear cell carcinomas) and ovarian tumors (~65% of serous carcinoma).451-453 A study at our institution to determine sensitivity of Pax-2 in müllerian tumors has demonstrated Pax-2 reactivity in ovarian clear cell (36% positive), uterine endometrioid (28%), and uterine serous carcinomas (63%). All bladder urothelial carcinomas (11 cases) and breast carcinomas (89 cases) in that study were negative for Pax-2.454
235
negative for the purpose of identifying tumor site of origin. Apart from müllerian, renal, thyroid, thymic, and pancreatic endocrine tumors, most other carcinomas tested have been negative. Albadine and coworkers459 reported Pax-8 positivity in 21 collecting-duct carcinomas, but a majority of upper tract urothelial carcinomas (31 of 34) were negative for Pax-8. Tong and colleagues reported Pax-8 reactivity in 24% of renal pelvic urothelial carcinomas but no reactivity in 40 bladder urothelial carcinomas.444,445 For müllerian tumors, Pax-8 is more sensitive than Pax-2.460 In contrast to WT1, Pax-8 expression in müllerian tumors is not morphology specific, because Pax-8 expression is seen in all tumor types, and Pax-8 is positive in approximately 80% of all müllerian tumors.461 The lowest reactivity is seen in endocervical adenocarcinoma, and gynecologic squamous cell carcinomas are generally negative. Tumors that are consistently reported to be negative for Pax-8 protein include breast and lung. However, we have seen a case of breast carcinoma with associated in situ carcinoma to be patchy positive for Pax-8 in our clinical practice. Nevertheless, Pax-8 has now become an indispensable antibody in the workup of carcinoma of unknown primary.462 KEY DIAGNOSTIC POINTS Pax-8
KEY DIAGNOSTIC POINTS Renal Cell Carcinoma Markers (Excluding Pax-8) • Common renal clear cell carcinoma profile: Negative for CKs 7 and 20; positive for EMA, CAM5.2, CD10, renal cell carcinoma marker, Pax-2, and vimentin • Of the positive markers, none are specific, therefore a panel approach is preferred. • Other CD10-positive carcinomas are transitional cell and prostate carcinoma. • Other RCC-positive carcinomas are breast, colon, adrenocortical, and prostate. • Other Pax-2–positive carcinomas include certain gynecologic tumors (serous, clear cell, and endometrioid types); breast and transitional cell carcinomas are negative.
PAIRED BOX GENE 8 (PAX8)
Similar to PAX2, paired box gene 8 (PAX8) gene expression is also required for the normal development of the kidney. It is also required in development of the thyroid follicle and müllerian system. Therefore, Pax-8 expression is mostly seen in renal, thyroid, and müllerian tumors.445,455,456 Pax-8 is also positive in tumors of thymic origin (thymomas and thymic carcinomas) and in pancreatic endocrine tumors.457,458 The typical expression pattern is nuclear. Cytoplasmic and/or membranous reactivity is nonspecific and should be considered
• Pax-8 is positive in müllerian, thyroid, renal, thymic, and pancreatic endocrine tumors. • It is negative in lung, breast, gastrointestinal, and pancreatobiliary tract carcinomas. • Pax-8 is also negative in squamous and urothelial (bladder) carcinomas. • Typical reactivity is nuclear. • Cytoplasmic/membranous reactivity should be ignored for diagnostic purposes.
Clear cell carcinomas in metastatic sites are always problematic. The differential diagnosis of site of origin in most cases includes clear cell carcinomas of kidney, adrenal, lung, liver, and endometrium/ovary. Metastatic clear cell carcinomas to pleura are also problematic. The antibody panels most likely to yield a separation among these tumors with a high degree of certainty may include Pax-8, Pax-2, RCC, A103, CD10, CKs, inhibin, vimentin, and HepPar1.463-467 MELAN-A AND INHIBIN IN ADRENOCORTICAL TUMORS
α-Inhibin is a dimeric glycoprotein functionally similar to tumor growth factor β (TGF-β), and it has been found in ovaries (granulosa cells), testes (Sertoli cells), adrenal cortex, placenta, and pituitary.468 Useful in the diagnosis of sex cord–stromal ovarian tumors, α-inhibin is also useful to differentiate adrenal cortical adenomas and carcinomas from renal cell carcinomas
236
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
and metastases to the adrenal with high specificity and sensitivity. Melan-A, a product of the MLANA gene (also known as MART-1), is an antigen recognized by antibody A103, and although its primary utility is in the identification of melanoma cells, it also identifies more than half of adrenocortical neoplasms, especially carcinomas.469-473 Tumors other than adrenal cortical neoplasms and malignant melanoma are rarely positive for A103, rendering the antibody useful for the discrimination of adrenal cortical neoplasm from renal neoplasm and metastatic carcinoma.474,475 As with inhibin, A103 also reacts with some sex cord–stromal tumors. The A103 antibody is more specific for adrenocortical tumors, because it does not react with other carcinomas, whereas inhibin is more sensitive.473 However, if melanoma is excluded as a diagnostic possibility, a positive A103 immunostain is strong evidence in favor of an adrenocortical carcinoma.469 Adrenal 4 binding protein (Ad4BP), also known as steroid factor 1 (SF-1), is a transcription factor that is positive in virtually all adrenal cortical carcinomas but is negative in renal carcinoma, hepatocellular carcinoma, and pheochromocytoma. Ad4BP is a marker of adrenocortical malignancy.476,477
KEY DIAGNOSTIC POINTS Renal Clear Cell Carcinoma vs. Other Clear Cell Carcinomas • Renal clear cell: Positive markers include Pax-8, Pax-2, RCC marker, CAM5.2, vimentin, and CD10; CK7 is rare to negative; and inhibin, A103, and HepPar1 are negative. • Adrenocortical: Ad4BP, A103, vimentin, and inhibin are positive; CK7 and CAM5.2 are rare to negative; RCC may be positive or negative; and CD10, HepPar1, and Pax-8 are negative. • Lung: Thyroid transcription factor 1 and CK7 are positive; RCC may be positive or negative; and Pax-8, CD10, inhibin, A103, and HepPar1 are negative. • Ovarian/endometrial clear cell: Positive markers include CK7, Pax-8, estrogen receptor (may be patchy to negative), and Pax-2 (~33%); WT1 may be positive or negative, and expression of RCC is rare; CD10, inhibin, A103, and HepPar1 are negative. • Hepatic clear cell carcinoma: HepPar1, Arginase-1, and CD10 (canalicular) are positive; RCC, A103, vimentin, inhibin, CK7, and Pax-8 are negative. • Mesothelioma: Positive markers include calretinin, mesothelin, WT1, CK5/6, CK7, and CD10; negative markers include RCC, MOC-31, BerEP4, and Pax-8.
GERM CELL TUMOR MARKERS
It is important to be able to diagnose germ cell neoplasms correctly, because they are highly amenable to treatment, even in advanced stages.5,7 Germ cell tumors (GCTs) may present as tumors of unknown origin, including seminoma and its variants, embryonal carcinoma, yolk sac tumor, and choriocarcinoma. A
combination of antibodies to simple CKs, EMA, PLAP, CD117 (C-KIT), OCT3/4, SALL4, CD30, α-fetoprotein (AFP), and human chorionic gonadotropin (hCG) can be used to arrive at the correct diagnosis in most cases. GCTs are diffusely positive for CAM5.2, except seminoma, which is largely negative in most cases but may demonstrate rare focal staining.478 More than focal keratin staining should raise the suspicion of an embryonal carcinoma component arising in a seminoma. The PLAP antibodies database from the literature includes M2A, 43-9F, and TRA-1-60.479-481 PLAP is strongly positive with crisp membranous and cytoplasmic staining in classic seminoma, negative in the spermatocytic variant,482,483 and variably positive in embryonal, yolk sac, and choriocarcinomas.483-488 PLAP is not 100% specific for GCTs, because 10% to 15% of non–germ cell carcinomas will be positive, and this group includes müllerian carcinomas and also GI, lung, and rare breast and renal carcinomas.489 However, these carcinomas are EMA positive, whereas GCTs are negative. EMA is negative in all these tumors except choriocarcinoma, in which it stains approximately 50% of cases.478,483,490 PLAP is increasingly replaced by more specific markers such as OCT3/4 and SALL4. AFP is present in most yolk sac tumors in patchy distribution but is only present focally in some cases of embryonal carcinoma.478,483,490-492 Hepatoid differentiation in GCTs, especially with yolk sac tumors, typically immunostains with AFP and does not show the immunoprofile of true hepatocytic differentiation, that is, the biliary pattern of immunostaining with pCEA. OCT3/4 is a transcription factor encoded by the POU5F1 gene, and it is involved in the initiation, maintenance, and differentiation of pluripotent and germline cells. OCT3/4 is a highly sensitive and specific antibody that detects seminoma/dysgerminoma and embryonal carcinoma.493-495 Immunostaining for OCT3/4 is nuclear and typically stains 90% of nuclei in seminoma/ dysgerminoma and embryonal carcinoma. Mixed GCTs with components of seminoma are positive, whereas OCT3/4 is negative in yolk sac tumors and immature teratomas. SALL4 is another specific germ cell marker. It is a zinc finger transcription factor that plays a role in the maintenance and pluripotency of embryonic stem cells. In contrast to OCT3/4, it stains all types of GCTs.496-498 The specificity of SALL4 has been reported to be greater than 95%.497 SALL4 also appears to be a useful marker of GCTs that arise in extragonadal tissue, such as mediastinum, retroperitoneum, and CNS.499 Although some of the above markers are helpful in distinguishing between different GCTs, they are not entirely specific. CD117, or C-KIT, is a marker of interstitial cell of Cajal and therefore stains GISTs, mast cells, and melanocytes in addition to germ cells.500,501 CD30, which is positive in embryonal carcinoma, is an activation marker often seen in Hodgkin lymphoma and ALCL.502 AFP can be positive in hepatocytic or hepatoid tumors, and hCG-positive cells have been described in several carcinomas.503
Special Clinical Presentations
237
KEY DIAGNOSTIC POINTS Germ Cell Tumor Markers • Seminoma: CAM5.2 is rare to negative; positive markers include placental alkaline phosphatase (PLAP), OCT3/4, SALL4, and CD117; EMA is negative. • Embryonal carcinoma: CAM5.2, PLAP, OCT3/4, SALL4, and CD30 are positive; EMA is negative, and α-fetoprotein is rare to negative. • Yolk sac tumor: Positive markers include PLAP, α-fetoprotein, and SALL4; EMA and OCT3/4 are negative • Choriocarcinoma: CAM5.2, EMA, and human chorionic gonadotropin (hCG) are positive; PLAP and SALL4 may be positive or negative; and OCT3/4 is negative. • PLAP immunostains 10% to 15% of non–germ cell carcinomas, which are also EMA positive.
CD5
CD5 is a 67-kD glycoprotein receptor that may be present on a variety of T lymphocytes and mantle zone lymphocytes. Hishima and colleagues504 found CD5 expression in thymic carcinoma, and subsequent reports verified and expanded those findings. In the studies published, the clones of antibodies used include NCL-CD5 (CD5/54/B4)348,505 with sensitivities of 29% to 67% for immunostaining of thymic carcinomas506,507; for clone NCL-CD5-4C7, sensitivities of 62% to 100% are reported.507,508 For mediastinal carcinomas, the usual differential diagnosis includes metastatic squamous carcinoma and other poorly differentiated metastatic lung carcinomas and GCTs, none of which stain with clone NCL-CD5-4C7.508 Although some normal epithelia and carcinoma of GI, breast, and urologic sites react with this clone, this fact is largely irrelevant to the focused study on the mediastinum.508 A CD5-positive mediastinal tumor is strong evidence for thymic carcinoma, although some atypical thymomas and thymic carcinomas that arise in a thymoma are also positive.508,509 Spindle cell carcinomas have been nonreactive with CD5 antibody.510 KEY DIAGNOSTIC POINTS CD5 • CD5 is positive in the majority of thymic carcinomas of most histologic types. • CD5 is negative in spindle cell thymic carcinoma. • Lung carcinomas are largely negative for CD5.
Combined Antibody (Panel) Approach to Solving Diagnostic Problems When antibodies to CK7, CK20, CK5, or other keratins are combined in panels with various antibodies to other intermediate filaments (vimentin), antibodies to supplemental epithelial antigens (CEA, EMA), or antibodies to specific cell products or transcription factors (neuroendocrine granules, Pax-8, TTF-1, WT1, etc.), a more specific identification of cell type may be rendered. An algorithmic approach as shown in Figure 8-20 may be used in arriving at the correct diagnosis. Over the last
few years, several specific markers of tumor origin have been described, but it is important to remember that the tumors that often present as CUPS, but that lack specific markers, include upper GI tumors (gastric and gastroesophageal junction carcinoma), tumors of gallbladder, cholangiocarcinoma, and pancreatic ductal carcinoma. For the latter, SMAD4 is helpful if negative (seen in 50% of cases of pancreatic cancers). MUC stains, once thought to be helpful, are not very distinctive between these tumor types.511 If these sites are suspected based on tumor morphology, clinical presentation, and differential cytokeratin expression, site-specific imaging can accurately determine the site of origin.
Special Clinical Presentations Determination of tumor site of origin in certain special circumstances is discussed below.
Metastatic Carcinoma in the Pleura Versus Epithelial Mesothelioma As discussed in the opening of this chapter, the anatomic site of metastasis of a neoplasm is the prime starting point in determining the origin of the neoplasm. Neoplasms metastatic to the pleura are most often due to primary lung carcinomas but may be due to carcinomas that originate in other anatomic sites. Metastasis to the pleura from distant sites is more common, because patients are living longer with their disease. Therefore the differential diagnosis of a malignant epithelial neoplasm in the pleura includes metastatic lung carcinoma, metastatic nonpulmonary carcinoma, and malignant mesothelioma. The single best antibody to use to make the distinction between lung carcinoma and mesothelioma is TTF-1, which has a high sensitivity and very high specificity for lung carcinomas, especially adenocarcinoma, and small cell carcinoma.299,300 In addition, CK5/6, which is largely restricted to mesothelial cells, also stain squamous cell carcinomas, lung carcinomas, and nonpulmonary carcinomas to a focal degree. A panel approach is generally used to arrive at the correct diagnosis. Most tumors can be reliably identified as mesothelioma or adenocarcinoma based on clinical history and available IHC markers (Table 8-9). Electron microscopy is still a very useful “accessory” technique that may be helpful for equivocal cases that include mesothelioma variants.512
238
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
Poorly differentiated malignant neoplasm
Diffuse strong pankeratin
Weak/equivocal or patchy pankeratin
See part B
vimentin /
Epithelioid morphology
Only vimentin
Use sarcoma differentiation markers (see Table 8.1)
LCA B or T cell markers
S100 HMB45 melanA
Lymphoma
Melanoma
Small round blue cell tumor
Spindle cell morphology
Other epithelial markers* and/or specific keratin
Germ cell markers
Other epithelial markers / and/or specific keratin
Neuroendocrine (NE) markers often
NE markers often negative
Carcinoma (see parts B through D
Malignant germ cell tumor
Sarcoma with epithelioid morphology (see Table 8.1)
• NE carcinoma • Neuroblastoma (but keratin )
Small round blue cell sarcoma (see Table 8.1)
Sarcomatoid carcinoma (other epithelial markers ) Sarcoma (see Table 8.1)
*Other epithelial markers: EMA, CEA, MOC31, BER-EP4, B72.3 Specific cytokeratins: CK7, CK20, CK5, CK14, CK17
A
Pankeratin (AE1/3 and/or CAM5.2) positive epithelioid tumor
CK7 /CK20
CK7 /CK20
CK7 /CK20
CK7 /CK20
see part C
see part D CDX2 Villin
CDX2
Uroplakin III Thrombomodulin
• Colorectal • Some upper GI
• Some upper GI • Merkel cell ( NE markers)
Urothelial CA (CK5 and p63 )
Uroplakin III Thrombomodulin CDX2 /
SMAD4
MUC5AC • Cholangiocarcinoma • Gallbladder carcinoma • Pancreas (50%) • Some upper GI
SMAD4
MUC5AC • Ovarian mucinous • Some upper GI
MUC5AC± Pancreas (50%)
B Figure 8-20 Algorithmic approaches. A, To determine line of differentiation in a poorly differentiated malignant neoplasm. B, To determine site of origin in an adenocarcinoma or a poorly differentiated carcinoma.
CK7 /CK20 , pankeratin (AE1/3 and/or CAM5.2)-positive CA
TTF1
TTF1 Use panels with specific markers Thyroglobulin PAX8
Thyroid
Thyroglobulin PAX8 Napsin A Lung
Breast: GCDFP15 , MGB , ER , CEA , GATA3 ,† PAX8 , WT1 , CDX2 Salivary gland tumors: May stain exactly like breast; use clinicopathologic features to distinguish. Salivary duct CA often ER , but AR Mullerian serous CA: PAX8 , WT1 , GCDFP15 , MGB , ER / , calretinin Mullerian endometrioid CA: PAX8 , ER , vimentin , MGB / , WT1 , CEA Endocervix: CEA , vimentin , ER , p16 (if p16+, confirm with HPV ISH/PCR) Cholangiocarcinoma: CDX2 / , MUC5AC , SMAD4 , PAX8 , GATA3 † Upper GI: CDX2 / , MUC5AC / , SMAD4 , PAX8 , GATA3 † Pancreatic: MUC5AC , PAX8 , CDX2 / , SMAD4 , GATA3 † Papillary renal cell carcinoma: CD10 , RCC , PAX8 Thymic carcinoma: CD5 , PAX8 Mesothelioma: WT1 , calretinin , CK5/6 , MOC31 , BER-EP4 , B72.3
C CK7 /CK20 , pankeratin (AE1/3 and/or CAM5.2)-positive CA
EMA
EMA
CD10 RCC PAX2 PAX8
Hep Par1 Arginase-1 AFP+ CEA (p) Albumin ISH
PSA PAP PSMA Prostein NKX3.1
Melan A Inhibin Ad4BP (SF-1) PAX8
Renal cell CA
Hepatocellular CA*
Prostatic CA
Adrenocortical CA*
*Hepatocellular carcinomas may be EMA–and adrenocortical carcinomas may show only focal reactivity with pankeratins.
D Figure 8-20, cont’d C, Further workup of a CK7-positive, CK20-negative tumor. D, Further workup of a CK7-negative, CK20-negative tumor. *Hepatocellular carcinomas may be EMA–, and adrenocortical carcinomas may show only focal reactivity with pankeratins. †Based on some recent studies559 and the authors’ preliminary data, GATA3 is expressed predominantly in breast and urothelial carcinomas. AFP, α-fetoprotein; CEA, carcinoembryonic antigen; ER, estrogen receptor; EMA, epithelial membrane antigen; GCDFP, gross cystic disease fluid protein; GI, gastrointestinal; PSA, prostate-specific antigen; PAP, prostatic acid phosphatase; PSMA, prostate-specific membrane antigen; TTF1, thyroid transcription factor 1; WT1, Wilms tumor 1.
KEY DIAGNOSTIC POINTS Metastatic Carcinoma in Pleura and Abdomen vs. Epithelial Mesothelioma • Positive mesothelioma markers: calretinin, WT1, CK 5/6, thrombomodulin, mesothelin • Negative mesothelioma markers: MOC-31, Bg8, BerEP4, B72.3, CEA, CD15 • Pitfall: CK5/6 is positive in squamous and transitional cell carcinomas; WT1 is positive in ovarian/tubal papillary serous carcinomas. • Lung carcinoma: TTF-1, napsin A, and CEA are positive; thyroglobulin is negative; and CK7 is positive, whereas CK20 is negative. • Breast carcinoma: GCDFP-15, mammaglobin, CEA, and estrogen and progesterone receptors are positive; CK7 is also positive, but CK20 is negative. • Thyroid carcinoma: Pax-8, TTF-1, and thyroglobulin are positive; CEA is negative (except in medullary thyroid carcinoma); and CK7 is positive, whereas CK20 is negative. • Müllerian carcinomas: Pax-8 is positive, but WT1 is only positive in tubal/ovarian serous carcinoma; estrogen and progesterone receptor staining is variable, depending on tumor type; and CK7 is positive, whereas CK20 is negative.
240
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
TABLE 8-9 Antibody Panels: Mesothelioma Versus Lung Carcinoma Mesothelioma
Carcinoma
Calretinin
+
R
Wilms tumor 1
+
N
Mesothelin
+
N
Cytokeratin 5
+
S
Biliary glycoprotein 8
N
+
BerEP4
N
+
MOC-31
N
+
Reactivity: +, positive; N, negative; R, rare cells show staining; S, sometimes positive.
Mediastinal Tumors: Type and Site of Origin Tumors that may be confused with CUPS within the mediastinum include thymic neoplasm (thymoma, thymic carcinoid, or thymic carcinoma), thyroid tumors, lymphomas, paragangliomas, and GCTs. Thymomas are generally easy to recognize because of their characteristic admixture of neoplastic thymic epithelial cells with nonneoplastic lymphocytes. The neoplastic thymic epithelial cells are positive for keratins, but the expression profile may vary according to thymoma subtype.513 These cells are also positive for CEA and EMA.514,515 The thymic lymphocytes in a thymoma are of T-cell derivation but do not stain with markers of mature (peripheral) T cells. Instead, they are positive for terminal deoxynucleotidyl transferase (TdT), CD1a, and CD99.516,517 In contrast, the thymic carcinomas rarely resemble a thymoma and are morphologically more similar to carcinoma types in other organs. The microscopic types of thymic carcinoma recognized by the World Health Organization (WHO) are keratinizing and nonkeratinizing epidermoid carcinoma (most predominant group), lymphoepithelioma-like, sarcomatoid, clear cell, basaloid, mucoepidermoid, papillary, mucinous, small cell, and undifferentiated carcinomas. Therefore their identification as thymic neoplasms is very difficult or sometimes impossible. One stain that is very specific to thymic carcinoma is CD5, which is present in most thymic carcinomas but is absent in thymomas and carcinomas of nonthymic origin.504,506,507 Tumors of thyroid origin may occur in the mediastinum as primary tumors as a result of a retrosternal location of the thyroid gland. The major subtypes of thyroid carcinomas are referred to as the papillary, follicular, Hürthle cell, poorly differentiated, anaplastic, and medullary types. All carcinomas are generally positive for pankeratin stains. The usual profile is CK7 positive and CK20 negative, and the two stains that have been traditionally used for determining site of origin are thyroglobulin and TTF-1.518 All carcinoma subtypes are positive for TTF-1 except anaplastic carcinoma, and all carcinomas are positive for thyroglobulin
except anaplastic and medullary types. Because medullary carcinomas are endocrine tumors, they also express other endocrine markers such as chromogranin, synaptophysin, and the specific product of C-cells, calcitonin. Another feature of medullary thyroid carcinoma is the consistent positivity for CEA. As far as anaplastic carcinoma is concerned, it is not only difficult to prove the site of origin, it is also difficult to prove that it is a carcinoma and not a sarcoma. Keratin stains are generally helpful in this regard. Vimentin positivity is the rule in the spindle cell component, and EMA and CEA reactivity may be identified, particularly in the squamoid component.519 Now with the availability of Pax-8, it can be used to confirm thyroid origin in such a scenario, because Pax-8 stains all types of thyroid cancers, including a significant number of anaplastic carcinomas. Nonaka and colleagues456 reported the diagnostic utility of Pax-8 antibody, which stained 79% (22 of 28 cases) of anaplastic thyroid carcinoma but was negative in all lung carcinomas tested (147 cases). Pax-8 was also expressed in renal tubules, fallopian tubes, ovarian inclusion cysts, and lymphoid follicles and was found in renal carcinoma, nephroblastoma, seminoma, and ovarian carcinoma, but it was not present in normal tissue or in carcinomas of the lung. Pax-8 could be a useful marker of anaplastic thyroid carcinoma if the differential includes a lung carcinoma.456 Mediastinal germ cells tumors are often primary in this location and likely arise from extragonadal germ cells. Although the possibility of a metastasis from the gonads should always be considered, the presence of a single lesion in the mediastinum without retroperitoneal involvement argues against a gonadal primary. All varieties of GCTs can be seen within the mediastinum. Dysgerminomas are practically never seen in females, and other tumors—seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratocarcinoma—show a high male predilection.520 Mature cystic teratomas are seen with equal frequency in both males and females, and the IHC profile is generally similar to that of their gonadal counterparts, with some occasional differences.521 Seminoma is often positive for PLAP, SALL4, OCT3/4, CD117, and CD57 and is generally negative for keratins. Although focal reactivity for CAM5.2 may be seen in seminomas, they are often negative for AE1/AE3.522-524 In contrast, embryonal carcinomas often show AE1/AE3 reactivity and are also positive for CD30, SALL4, and OCT3/4, and they may show reactivity for PLAP.525,526 Yolk sac tumors are positive for AFP and SALL4 but are negative for OCT3/4, and choriocarcinomas show reactivity for hCG.492 However, it is important to note that immunostains for AFP and hCG often show high background and should be interpreted with caution and in the right context. Serum elevation of AFP and hCG is commonly associated with nonseminomatous germ cell neoplasms, therefore measuring serum levels is another alternative to IHC in confirming the diagnosis. Another important feature of all GCTs, regardless of origin or microscopic type, is the presence of a cytogenetic aberration, isochromosome 12p (i[12p]).527 This aberration results in a gain of 12p, which can be identified by routine cytogenetic techniques even in FFPE material.528 Finally, an
Special Clinical Presentations
attempt should be made to classify a tumor as germ cell before rendering a diagnosis of undifferentiated malignant neoplasm. PERITONEAL CARCINOMATOSIS
It is not uncommon to receive a biopsy from a patient with tumor that extensively involves the abdomen and peritoneum without a known primary. In a female patient, serous papillary morphology is most suggestive of an ovarian or tubal primary. The IHC profile of so-called primary peritoneal serous carcinoma (PPSC) is identical to ovarian/tubal primary tumor. The differential diagnosis is more challenging when the tumor shows a mucinous morphology. The source of metastases in a mucinous carcinoma in both sexes is often gastrointestinal in origin, and it is worth looking at appendix, bowel, pancreas, and stomach clinically or radiographically. In a female patient, mucinous carcinoma that involves both ovaries may also be metastatic from the uterine cervix.529 A combination of CK7, CK20, CDX2,** SMAD4, MUC stains, p16, and vimentin may be helpful in arriving at the correct diagnosis.530,531 Pax-8 stain may also be used, because it stains approximately 60% of cervical adenocarcinomas, but it is negative in tumors of GI tract origin.461 Most ovarian mucinous carcinomas have been reported to be negative for Pax-8,532 but we have seen patchy reactivity on
241
whole tissue sections used in diagnostic pathology. Therefore Pax-8 should also be included in the panel, because a positive result is quite valuable in determining site of origin. When the morphology is unrevealing, the differential diagnosis should not only include carcinomas but also mesothelioma and, in females, ovarian stromal tumors. The ovarian stromal tumors may show keratin positivity but are almost always EMA negative. They are positive for inhibin, calretinin, and CD99.259,533,534 PAGET DISEASE
Paget disease occurs in mammary and extramammary (EM) forms. Paget disease of the breast is almost always indicative of an underlying breast carcinoma,535-537 whereas EM Paget disease may be an indicator of metastatic carcinoma. Paget disease of the breast manifests as CK7-positive malignant cells that infiltrate the epidermis of the nipple. Tumor cells are conspicuous by their infiltrative “shotgun” pattern, large size, abundant cytoplasm, signet-ring forms, and, sometimes, mucin positivity. Epidermal keratinocytes are negative with CK7, whereas most Paget cells are positive for CK7, GCDFP-15, and CEA (Fig. 8-21). Toker cells are CK7 positive and may be present in the skin of the normal nipple, but generally they are inconspicuous compared with Paget cells538
A
B
C
D
Figure 8-21 A typical case of primary perineal Paget disease (A), which stains for CK7 (B), carcinoembryonic antigen (C), and gross cystic disease fluid protein 15 (D).
242
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
and should not cause diagnostic problems; they are bland cytologically and difficult to find in the normal epithelium. The presence of CK7-positive cells in the nipple epidermis does not equate with Paget disease, even in circumstances in which a benign nipple lesion is evident, such as nipple adenoma. The presence of benignappearing CK7-positive cells in the epidermis may be a manifestation of the extension of cells from the lactiferous ducts or benign nipple epithelial proliferations into the epidermis.539 The Paget cells may be assessed for ER540 or the HER2 oncoprotein.541 However, HER2 is a better marker of Paget disease, because the underlying carcinoma is often a high-grade ductal carcinoma in situ with comedonecrosis, which is generally ER negative and HER2 positive. The differential diagnosis for Paget disease includes melanoma and Bowen disease (squamous cell carcinoma), for which appropriate IHC stains could be used. For diagnosing melanoma, at least two melanoma markers should be used, because S-100 staining can be seen in as many as 18% of breast carcinoma cases. The EM forms of Paget disease occur predominantly in females as vulvar or perianal disease but may also occur in males and at other sites. Primary vulvar Paget disease is a localized carcinoma of sweat duct origin that may be in situ or invasive; histologically, it is composed of large cells with voluminous cytoplasm and mucin positivity, and they are universally positive for both CK7 and GCDFP-15.338 The extravulvar form of the disease occurs in the perianal areas as metastatic disease from sites that may include the rectum, cervix, or urinary bladder; therefore the diagnostic problem inherent with a histologic diagnosis of extravulvar Paget disease is the differential diagnosis of adnexal skin neoplasm versus metastatic carcinoma from those three sites. Colorectal carcinomas presenting as Paget disease are often mucin positive with signet-ring forms and intraluminal “dirty” necrosis.342 They are GCDFP-15 negative, CEA positive, and strongly positive for CK20; and although they are largely CK7 negative, they may be focally or weakly positive for CK7.338,340-342,344 Transitional cell carcinomas are typically strongly positive for both CK7 and CK20 and are also positive for CK5 and p63; they are GCDFP-15 negative, they lack signet cells and mucin, and they may stain with URO antibody.
Molecular Approach for Determining Site of Origin Several different types of molecular techniques can be applied for identifying tumors of unknown origin. These could range from identifying a single molecular event to transcriptional profiling that examines the expression of hundreds and thousands of genes. The examples of the former are summarized in Table 8-10. Molecular assays to determine a single fusion transcript or to identify a virus by PCR have been in diagnostic use for a number of years; however, with recent technologic advancements, gene-expression profiling technology has also come to the forefront of diagnostic testing. Both complementary DNA (cDNA) and oligonucleotide microarrays are now used extensively for a variety of different applications. TABLE 8-10 Molecular (Single Gene/Event) Assays for Determining Site of Origin Technique
Determinant
Tumor Type/Site
RT-PCR
Fusion transcripts
Various sarcomas (see Table 8-1)
PCR
HPV
Uterine, cervix, and some head and neck tumors
ISH
HPV
Uterine, cervix, and some head and neck tumors
ISH
Albumin
Hepatocellular carcinoma
ISH
EBV
Nasopharyngeal carcinomas, lymphomas (Burkitt, Hodgkin, some T-cell lymphomas), subset of gastric cancers, immunodeficiencyassociated tumors
FISH
Isochromosome 12p [i(12p)]
Germ cell tumors
Mutation analysis
KIT gene
Gastrointestinal stromal tumors
EBV, Epstein-Barr virus; FISH, fluorescence in-situ hybridization; HPV, human papilloma virus; ISH, in-situ hybridization; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR.
KEY DIAGNOSTIC POINTS Paget Disease • Mammary Paget disease is positive for CK7, GCDFP-15, CEA, and HER2. • Pagetoid squamous carcinoma is positive for p63 and CK5 and negative for CK7. • Vulvar Paget disease (primary adnexal carcinoma) is positive for CK7, GCDFP-15, and CEA. • Metastatic colorectal carcinoma presenting as perianal-vulvar Paget disease is positive for CK20, CDX-2, and CEA; CK7 and GCDFP-15 are negative. • Metastatic transitional carcinoma presenting as perianal-vulvar Paget disease: CK5, CK7, CK20, CEA, URO, and p63 are positive; GCDFP-15 is negative.
Molecular Approach for Determining Site of Origin
For the first time in 2001, Ramaswamy and colleagues542 provided a proof of the principle that geneexpression analysis could be used in identifying tumor site of origin; they subjected 218 tumors of 14 different morphologic types and 90 normal tissue samples to gene-expression analysis. Expression levels of more than 16,000 genes were used to test the accuracy of a multiclass classifier based on a support vector machine algorithm. An overall classification accuracy of 78% was achieved. They were also able to correctly classify metastatic samples, which indicated that cancers retain their tissue of origin identity throughout metastatic evolution, and prediction is driven by cancer-intrinsic geneexpression patterns and not by the gene-expression signature of contaminating nonmalignant tissue. However, Ramaswamy and associates were unable to classify the majority of poorly differentiated carcinomas correctly. Several smaller studies that followed showed somewhat similar results but were unable to provide a test that could be practically useful, and they lacked a broad coverage of all tumor types.543-547 Subsequently, Ma and colleagues548 translated their microarray findings in designing a 92-gene RT-PCR assay for determining tumor site of origin. They achieved an overall success rate of 87% in classifying 32 different tumor classes in the validation set of 119 FFPE tumor samples. This RT-PCR–based assay (CancerTYPE ID) for tumor of unknown origin is now offered by bioTheranostics in the United States. At the same time as the CancerTYPE ID test was introduced, a similar gene-expression–based assay to determine site of origin in a centralized format (CUP Print) was introduced by Agendia BV in the Netherlands. This test used cDNA microarrays for 495 genes to determine 49 tumor subtypes by using “k-nearest neighbor” bioinformatics strategy. The test was reported to have 88% overall accuracy for determining site of origin.549 However, this commercial assay was later taken off the market. Recently, another gene-expression–based test in a decentralized format has become available, offered by Pathwork Diagnostics (Sunnyvale, CA). The test measures the expression of more than 1500 genes to identify 15 different tumor types that represent 60 different morphologies. The 15 tumors types identified in this assay are carcinomas (bladder, breast, colorectal, gastric, hepatocellular, kidney, non–small cell lung, ovarian, pancreatic, prostate, and thyroid), germ cell neoplasms, melanoma, non-Hodgkin lymphoma, and soft tissue sarcomas. The test requires specimen processing to be performed in a Clinical Laboratory Improvement Amendments (CLIA) certified lab by using a standardized protocol and a proprietary microarray chip. The chip is then scanned, and the data file is submitted via the Internet to the company, where fully automated software generates a report. The report contains a similarity score that ranges from 0 to 100 for each of the 15 tumor types, and the final interpretation is performed by the molecular pathologist who initiated the testing. In a clinical validation study of 487 metastatic, poorly differentiated, and undifferentiated tumors that had been identified as one of the 15 tumor types on the
243
panel using existing methods, the test demonstrated 89% positive percent agreement (sensitivity) and 99% negative percent agreement (specificity) with available diagnoses.550 Another study that examined the Pathwork Tissue of Origin test has shown high reproducibility among different laboratories despite numerous sources of variability.551 It should be noted that the initial panel of carcinomas in the Pathwork assay did not contain endometrial carcinomas. Recently, the company introduced a second test to distinguish between endometrial and ovarian carcinomas but did not clarify whether the test distinguishes among similar subtypes. This test is performed if the most likely site is of ovarian origin on initial testing. According to the company Web site, the test analyzes 14 different morphologic subtypes by using a 316-gene classification model, but it does not provide the specifics; therefore it is not clear whether the test can adequately separate endometrial endometrioid tumors from ovarian endometrioid tumors or an ovarian clear cell carcinoma from endometrial clear cell carcinoma. If this test was developed by using random or consecutive ovarian and endometrial carcinomas, it is likely that the ovarian profile is representative of ovarian serous carcinoma, the most common ovarian tumor, and the endometrial profile is probably representative of endometrial endometrioid carcinoma, the most common endometrial tumor. If this is true, then the second assay is of no clinical value, because these two tumors—ovarian serous carcinoma and endometrial endometrioid carcinoma—can be easily distinguished on morphology and/or with IHC. Another approach to identifying tissue of origin is by using microRNA (miRNA)-based methodology, because miRNAs are noncoding regulatory RNAs considered to be highly tissue-specific biomarkers based on the current knowledge.552 They have a role in cellular differentiation during tissue development and have also been thought to play a role in development of specific malignancies.553 Rosenfeld and colleagues554 recently demonstrated the enormous potential of miRNA in identifying tissue of origin. Using a decision tree–based classification, they used 48 miRNA markers to achieve an overall accuracy of 89% among 22 different tumor tissues. This study was performed by using FFPE tissues, and the technique has the potential for incorporation into diagnostic pathology in dealing with challenging cases. Another study validated these initial findings.555 The same authors have now recently published a second-generation miRNA-based assay for determining tumor site of origin. In a study of 509 independent samples, the investigators showed the overall sensitivity of the assay to be 85%.556 These studies led to the development of a commercial assay that claims to separate 42 different tumor types based on 64 miRNA profiles. The multigene molecular assays available to predict primary tumor site are summarized in Table 8-11. All the above molecular assays have been designed by using robust techniques, but it is useful to remember that these have been compared with the existing validation technologies: clinical suspicion, radiographic evaluation, morphology, IHC, and possibly autopsy findings. Therefore these molecular tests may complement our current diagnostic tools
244
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
TABLE 8-11 Multigene Expression Assays for Determining Site of Origin CancerTYPE ID
miRview mets2
Tissue of Origin†
Company
bioTheranostics,* San Diego, CA
Rosetta Genomics, Philadelphia, PA
Pathwork Diagnostics, Sunnyvale CA
Technique
RT-PCR
miRNA profile
Oligonucleotide array
Platform used
TaqMan, Applied Biosystems
Proprietary
Affymetrix
Number of genes analyzed
92
64 miRNAs
>1500
Specimen requirement
FFPE
FFPE
FFPE
Number of tumor types/subtypes identified
32 tumor types
42 tumor types
15 tumor types
Classification accuracy
87%
85%
84%
In-house testing requirement
None
None
Specimen processing required
FDA Approval
No
No
Yes
*(bioTheranostics was formerly AviaraDx (originally Arcturus Bioscience). † Pathworks Diagnostics ceased operations in April 2013 (after this chapter was written); the future of the Tissue of Origin test is currently uncertain. FFPE, Fresh-frozen paraffin-embedded; miRNA, microRNA; RT-PCR, reverse transcription polymerase chain reaction.
but are unlikely to replace the existing method of evaluation for CUPS. Another prohibitive factor for using these molecular tests in routine practice is their high cost, which is generally $3000 or more. Recently, Hainsworth and colleagues557 reported the result of a prospective trial in which tumor biopsy specimens from untreated patients with CUPS were tested with bioTheranostics’ 92-gene RT-PCR assay. In total, 194 patients received assay-directed, site-specific firstline therapy, and the median survival for these patients was 12.5 months (95% confidence interval [CI], 9.1 to 15.4 months). The median survival was significantly better if the predicted tumor type was a therapy “responsive,” compared with prediction of a more “resistant” tumor type (13.4 vs. 7.6 months respectively; P = .04). The authors concluded that molecular tumor profiling contributes to patient management and should be included in standard evaluation. However, it is unfortunate that the authors did not directly compare a carefully performed morphoimmunohistologic evaluation with molecular profiling in this trial. The question remains whether the newly available molecular assays can replace the current gold standard of morphology and immunohistologic evaluation. It is true that pathologic interpretation is somewhat subjective, because it not only depends on the expertise of the pathologist but also on the available clinical information and the willingness of the pathologist to acquire more information. In contrast, molecular assays are offered by companies that provide site of origin based purely on the genomic content of the tumor, so the results are not altered by other (i.e., nontissue) variables. Although molecular assays can be considered to be more reproducible, they are not more accurate compared with carefully performed morphoimmunohistologic evaluation by a knowledgeable and diligent pathologist.
Moreover, molecular profiling is not independent of morphology and IHC profile. Many of the IHC markers used in evaluation of CUPS have been derived from investigational molecular profiling and represent the best markers that have been picked from a plethora of nonspecific genomic markers. Both IHC and molecular profiling work best when the tumors are well differentiated and are less helpful when tumors are poorly differentiated. Given these facts, it is unlikely that commercially available molecular assays will replace standard morphologic and IHC evaluation. The molecular assays will likely be requested by hospitals and practices where IHC and oncologic pathology expertise is lacking, and much will also depend on the submission of the medical oncologist to marketing executives. No matter what principal method of evaluation is finally chosen, concordance among different methodologies is the goal; otherwise, an inaccurate assessment has the potential to harm the patient.558
Summary By working closely with the clinician who performs a careful clinical history and assessment, the pathologist should develop a working differential diagnosis based on tumor location, radiologic assessment of the tumor, or both. This information is key to utilizing IHC and other ancillary techniques as a cost-effective tool in patient care. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Rosai J: Cytokeratin expression in cauda equina paragangliomas. Author’s response to letter. Am J Surg Pathol. 23:491, 1999. 2. Blaszyk H, Hartmann A, Bjornsson J: Cancer of unknown primary: clinicopathologic correlations. Apmis. 111:1089–1094, 2003. 3. Greco FA, Burris HA 3rd, Erland JB, et al: Carcinoma of unknown primary site. Cancer. 89:2655–2660, 2000. 4. Yakushiji S, Ando M, Yonemori K, et al: Cancer of unknown primary site: review of consecutive cases at the National Cancer Center Hospital of Japan. Int J Clin Oncol. 11:421–425, 2006. 5. Haskell CM, Cochran AJ, Barsky SH, et al: Metastasis of unknown origin. Curr Probl Cancer. 12:5–58, 1988. 6. Krementz ET, Cerise EJ, Foster DS, et al: Metastases of undetermined source. Curr Probl Cancer. 4:4–37, 1979. 7. Schapira DV, Jarrett AR: The need to consider survival, outcome, and expense when evaluating and treating patients with unknown primary carcinoma. Arch Intern Med. 155:2050–2054, 1995. 8. Pavlidis N, Briasoulis E, Hainsworth J, et al: Diagnostic and therapeutic management of cancer of an unknown primary. Eur J Cancer. 39:1990–2005, 2003. 9. van de Wouw AJ, Jansen RL, Griffioen AW, et al: Clinical and immunohistochemical analysis of patients with unknown primary tumour. A search for prognostic factors in UPT. Anticancer Res. 24:297–301, 2004. 10. Pisharodi LR, Lavoie R, Bedrossian CW: Differential diagnostic dilemmas in malignant fine-needle aspirates of liver: a practical approach to final diagnosis. Diagn Cytopathol. 12:364–370; discussion 370-361, 1995. 11. Debevec M: Management of patients with brain metastases of unknown origin. Neoplasma. 37:601–606, 1990. 12. Merchut MP: Brain metastases from undiagnosed systemic neoplasms. Arch Intern Med. 149:1076–1080, 1989. 13. Nussbaum ES, Djalilian HR, Cho KH, et al: Brain metastases. Histology, multiplicity, surgery, and survival. Cancer. 78:1781– 1788, 1996. 14. Bartelt S, Lutterbach J: Brain metastases in patients with cancer of unknown primary. J Neurooncol. 64:249–253, 2003. 15. Kambhu SA, Kelsen DP, Fiore J, et al: Metastatic adenocarcinomas of unknown primary site. Prognostic variables and treatment results. Am J Clin Oncol. 13:55–60, 1990. 16. Lagerwaard FJ, Levendag PC, Nowak PJ, et al: Identification of prognostic factors in patients with brain metastases: a review of 1292 patients. Int J Radiat Oncol Biol Phys. 43:795–803, 1999. 17. Nguyen LN, Maor MH, Oswald MJ: Brain metastases as the only manifestation of an undetected primary tumor. Cancer. 83:2181– 2184, 1998. 18. Rougraff BT, Kneisl JS, Simon MA: Skeletal metastases of unknown origin. A prospective study of a diagnostic strategy. J Bone Joint Surg Am. 75:1276–1281, 1993. 19. Sears D, Hajdu SI: The cytologic diagnosis of malignant neoplasms in pleural and peritoneal effusions. Acta Cytol. 31:85–97, 1987. 20. Ringenberg QS, Doll DC, Loy TS, et al: Malignant ascites of unknown origin. Cancer. 64:753–755, 1989. 21. Sadeghi B, Arvieux C, Glehen O, et al: Peritoneal carcinomatosis from non-gynecologic malignancies: results of the EVOCAPE 1 multicentric prospective study. Cancer. 88:358–363, 2000. 22. Cho KR, Epstein JI: Metastatic prostatic carcinoma to supradiaphragmatic lymph nodes. A clinicopathologic and immunohistochemical study. Am J Surg Pathol. 11:457–463, 1987. 23. Nguyen C, Shenouda G, Black MJ, et al: Metastatic squamous cell carcinoma to cervical lymph nodes from unknown primary mucosal sites. Head Neck. 16:58–63, 1994. 24. Conessa C, Clement P, Foehrenbach H, et al: Positron emission tomography in head and neck squamous cell carcinomas. Ann Otolaryngol Chir Cervicofac. 123:227–239, 2006. 25. Schmalbach CE, Miller FR: Occult primary head and neck carcinoma. Curr Oncol Rep. 9:139–146, 2007. 26. Silva P, Hulse P, Sykes AJ, et al: Should FDG-PET scanning be routinely used for patients with an unknown head and neck squamous primary? J Laryngol Otol. 121:149–153, 2007.
244.e1
27. Gluckman JL, Robbins KT, Fried MP: Cervical metastatic squamous carcinoma of unknown or occult primary source. Head Neck. 12:440–443, 1990. 28. Jereczek-Fossa BA, Jassem J, Orecchia R: Cervical lymph node metastases of squamous cell carcinoma from an unknown primary. Cancer Treat Rev. 30:153–164, 2004. 29. Calabrese L, Jereczek-Fossa BA, Jassem J, et al: Diagnosis and management of neck metastases from an unknown primary. Acta Otorhinolaryngol Ital. 25:2–12, 2005. 30. Zaren HA, Copeland EM 3rd: Inguinal node metastases. Cancer. 41:919–923, 1978. 31. Ayoub JP, Hess KR, Abbruzzese MC, et al: Unknown primary tumors metastatic to liver. J Clin Oncol. 16:2105–2112, 1998. 32. Greco FA, Hainsworth JD: The management of patients with adenocarcinoma and poorly differentiated carcinoma of unknown primary site. Semin Oncol. 16:116–122, 1989. 33. Guthrie TH Jr: Treatable carcinoma of unknown origin. Am J Med Sci. 298:74–78, 1989. 34. Hainsworth JD, Greco FA: Managing carcinomas of unknown primary site. Oncology (Williston Park). 2:43–49, 52–44, 1988. 35. Lenzi R, Hess KR, Abbruzzese MC, et al: Poorly differentiated carcinoma and poorly differentiated adenocarcinoma of unknown origin: favorable subsets of patients with unknownprimary carcinoma? J Clin Oncol. 15:2056–2066, 1997. 36. Kirsten F, Chi CH, Leary JA, et al: Metastatic adeno or undifferentiated carcinoma from an unknown primary site–natural history and guidelines for identification of treatable subsets. Q J Med. 62:143–161, 1987. 37. Perchalski JE, Hall KL, Dewar MA: Metastasis of unknown origin. Prim Care. 19:747–757, 1992. 38. Jakobsen JH, Johansen J, Jorgensen KE: Neck lymph node metastases from an unknown primary tumor. Ugeskr Laeger. 153:428–430, 1991. 39. van der Gaast A, Verwij J, Planting AS, et al: The value of immunohistochemistry in patients with poorly differentiated adenocarcinomas and undifferentiated carcinomas of unknown primary. J Cancer Res Clin Oncol. 122:181–185, 1996. 40. Matthews P, Ellis IO: Use of immunocytochemistry in the diagnosis of metastatic carcinoma. Ann Med. 28:297–300, 1996. 41. Abbruzzese JL, Abbruzzese MC, Lenzi R, et al: Analysis of a diagnostic strategy for patients with suspected tumors of unknown origin. J Clin Oncol. 13:2094–2103, 1995. 42. Bartsch R, Wenzel C, Zielinski CC, et al: HER-2-Positive Breast Cancer: Hope Beyond Trastuzumab. BioDrugs. 21:69–77, 2007. 43. Molina JR, Adjei AA, Jett JR: Advances in chemotherapy of non-small cell lung cancer. Chest. 130:1211–1219, 2006. 44. Ramalingam S, Belani CP: Recent advances in targeted therapy for non-small cell lung cancer. Expert Opin Ther Targets. 11:245– 257, 2007. 45. Hess KR, Abbruzzese MC, Lenzi R, et al: Classification and regression tree analysis of 1000 consecutive patients with unknown primary carcinoma. Clin Cancer Res. 5:3403–3410, 1999. 46. Osteen RT, Kopf G, Wilson RE: In pursuit of the unknown primary. Am J Surg. 135:494–497, 1978. 47. Song SY, Kim WS, Lee HR, et al: Adenocarcinoma of unknown primary site. Korean J Intern Med. 17:234–239, 2002. 48. Mackay B, Ordonez NG: Pathological evaluation of neoplasms with unknown primary tumor site. Semin Oncol. 20:206–228, 1993. 49. Kagan AR, Steckel RJ: The limited role of radiologic imaging in patients with unknown tumor primary. Semin Oncol. 18:170– 173, 1991. 50. Steckel RJ, Kagan AR: Metastatic tumors of unknown origin. Cancer. 67:1242–1244, 1991. 51. Jonk A, Kroon BB, Rumke P, et al: Lymph node metastasis from melanoma with an unknown primary site. Br J Surg. 77:665– 668, 1990. 52. Le Chevalier T, Cvitkovic E, Caille P, et al: Early metastatic cancer of unknown primary origin at presentation. A clinical study of 302 consecutive autopsied patients. Arch Intern Med. 148:2035–2039, 1988. 53. Hainsworth JD, Wright EP, Johnson DH, et al: Poorly differentiated carcinoma of unknown primary site: clinical usefulness of immunoperoxidase staining. J Clin Oncol. 9:1931–1938, 1991.
244.e2
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
54. Hainsworth JD, Greco FA: Poorly differentiated carcinoma and poorly differentiated adenocarcinoma of unknown primary tumor site. Semin Oncol. 20:279–286, 1993. 55. Greco FA, Gray J, Burris HA 3rd, et al: Taxane-based chemotherapy for patients with carcinoma of unknown primary site. Cancer J. 7:203–212, 2001. 56. Mukai H, Watanabe T, Ando M, et al: Unknown primary carcinoma: a feasibility assessment of combination chemotherapy with cisplatin and docetaxel. Int J Clin Oncol. 8:23–25, 2003. 57. Martelli G, Pilotti S, Lepera P, et al: Fine needle aspiration cytology in superficial lymph nodes: an analysis of 266 cases. Eur J Surg Oncol. 15:13–16, 1989. 58. Ascoli V, Taccogna S, Scalzo CC, et al: Utility of cytokeratin 20 in identifying the origin of metastatic carcinomas in effusions. Diagn Cytopathol. 12:303–308, 1995. 59. Bedrossian CW: Special stains, the old and the new: the impact of immunocytochemistry in effusion cytology. Diagn Cytopathol. 18:141–149, 1998. 60. Bonnefoi H, Smith IE: How should cancer presenting as a malignant pleural effusion be managed? Br J Cancer. 74:832–835, 1996. 61. DiBonito L, Falconieri G, Colautti I, et al: The positive peritoneal effusion. A retrospective study of cytopathologic diagnoses with autopsy confirmation. Acta Cytol. 37:483–488, 1993. 62. Lai CR, Pan CC, Tsay SH: Contribution of immunocytochemistry in routine diagnostic cytology. Diagn Cytopathol. 14:221– 225, 1996. 63. Lidang Jensen M, Johansen P: Immunocytochemical staining of serous effusions: an additional method in the routine cytology practice? Cytopathology. 5:93–103, 1994. 64. Longatto Filho A, Bisi H, Alves VA, et al: Adenocarcinoma in females detected in serous effusions. Cytomorphologic aspects and immunocytochemical reactivity to cytokeratins 7 and 20. Acta Cytol. 41:961–971, 1997. 65. Frayling IM: Methods of molecular analysis: mutation detection in solid tumours. Mol Pathol. 55:73–79, 2002. 66. Leader M, Collins M, Patel J, et al: Vimentin: an evaluation of its role as a tumour marker. Histopathology. 11:63–72, 1987. 67. Azumi N, Battifora H: The distribution of vimentin and keratin in epithelial and nonepithelial neoplasms. A comprehensive immunohistochemical study on formalin- and alcohol-fixed tumors. Am J Clin Pathol. 88:286–296, 1987. 68. Kurtin PJ, Pinkus GS: Leukocyte common antigen–a diagnostic discriminant between hematopoietic and nonhematopoietic neoplasms in paraffin sections using monoclonal antibodies: correlation with immunologic studies and ultrastructural localization. Hum Pathol. 16:353–365, 1985. 69. Ries S, Barr R, LeBoit P, et al: Cutaneous sarcomatoid B-cell lymphoma. Am J Dermatopathol. 29:96–98, 2007. 70. Colella G, Tirelli A, Capone R, et al: Myeloid sarcoma occurring in the maxillary gingiva: a case without leukemic manifestations. Int J Hematol. 81:138–141, 2005. 71. Menasce LP, Banerjee SS, Beckett E, et al: Extra-medullary myeloid tumour (granulocytic sarcoma) is often misdiagnosed: a study of 26 cases. Histopathology. 34:391–398, 1999. 72. Sadahira Y, Sugihara T, Yawata Y, et al: Cutaneous granulocytic sarcoma mimicking immunoblastic large cell lymphoma. Pathol Int. 49:347–353, 1999. 73. Chen J, Yanuck RR 3rd, Abbondanzo SL, et al: c-Kit (CD117) reactivity in extramedullary myeloid tumor/granulocytic sarcoma. Arch Pathol Lab Med. 125:1448–1452, 2001. 74. Palomino-Portilla EA, Valbuena JR, Quinones-Avila Mdel P, et al: Myeloid sarcoma of appendix mimicking acute appendicitis. Arch Pathol Lab Med. 129:1027–1031, 2005. 75. Roth MJ, Medeiros LJ, Elenitoba-Johnson K, et al: Extramedullary myeloid cell tumors. An immunohistochemical study of 29 cases using routinely fixed and processed paraffin-embedded tissue sections. Arch Pathol Lab Med. 119:790–798, 1995. 76. Hudock J, Chatten J, Miettinen M: Immunohistochemical evaluation of myeloid leukemia infiltrates (granulocytic sarcomas) in formaldehyde-fixed, paraffin-embedded tissue. Am J Clin Pathol. 102:55–60, 1994. 77. Traweek ST, Arber DA, Rappaport H, et al: Extramedullary myeloid cell tumors. An immunohistochemical and morphologic study of 28 cases. Am J Surg Pathol. 17:1011–1019, 1993.
78. Blessing K, Sanders DS, Grant JJ: Comparison of immunohistochemical staining of the novel antibody melan-A with S100 protein and HMB-45 in malignant melanoma and melanoma variants. Histopathology. 32:139–146, 1998. 79. DeYoung BR, Wick MR: Immunohistologic evaluation of metastatic carcinomas of unknown origin: an algorithmic approach. Semin Diagn Pathol. 17:184–193, 2000. 80. Drlicek M, Bodenteich A, Urbanits S, et al: Immunohistochemical panel of antibodies in the diagnosis of brain metastases of the unknown primary. Pathol Res Pract. 200:727–734, 2004. 81. Drier JK, Swanson PE, Cherwitz DL, et al: S100 protein immunoreactivity in poorly differentiated carcinomas. Immunohistochemical comparison with malignant melanoma. Arch Pathol Lab Med. 111:447–452, 1987. 82. Stroup RM, Pinkus GS: S-100 immunoreactivity in primary and metastatic carcinoma of the breast: a potential source of error in immunodiagnosis. Hum Pathol. 19:949–953, 1988. 83. Hashimoto H, Daimaru Y, Enjoji M: S-100 protein distribution in liposarcoma. An immunoperoxidase study with special reference to the distinction of liposarcoma from myxoid malignant fibrous histiocytoma. Virchows Arch A Pathol Anat Histopathol. 405:1–10, 1984. 84. Nakajima T, Watanabe S, Sato Y, et al: An immunoperoxidase study of S-100 protein distribution in normal and neoplastic tissues. Am J Surg Pathol. 6:715–727, 1982. 85. Miettinen M, Franssila K: Immunohistochemical spectrum of malignant melanoma. The common presence of keratins. Lab Invest. 61:623–628, 1989. 86. Selby WL, Nance KV, Park HK: CEA immunoreactivity in metastatic malignant melanoma. Mod Pathol. 5:415–419, 1992. 87. Zarbo RJ, Gown AM, Nagle RB, et al: Anomalous cytokeratin expression in malignant melanoma: one- and two-dimensional western blot analysis and immunohistochemical survey of 100 melanomas. Mod Pathol. 3:494–501, 1990. 88. Laskin WB, Miettinen M: Epithelioid sarcoma: new insights based on an extended immunohistochemical analysis. Arch Pathol Lab Med. 127:1161–1168, 2003. 89. Machen SK, Fisher C, Gautam RS, et al: Utility of cytokeratin subsets for distinguishing poorly differentiated synovial sarcoma from peripheral primitive neuroectodermal tumour. Histopathology. 33:501–507, 1998. 90. Olsen SH, Thomas DG, Lucas DR: Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol. 19:659– 668, 2006. 91. Ordonez NG, Mahfouz SM, Mackay B: Synovial sarcoma: an immunohistochemical and ultrastructural study. Hum Pathol. 21:733–749, 1990. 92. Sebire NJ, Gibson S, Rampling D, et al: Immunohistochemical findings in embryonal small round cell tumors with molecular diagnostic confirmation. Appl Immunohistochem Mol Morphol. 13:1–5, 2005. 93. Folpe AL, Goldblum JR, Rubin BP, et al: Morphologic and immunophenotypic diversity in Ewing family tumors: a study of 66 genetically confirmed cases. Am J Surg Pathol. 29:1025–1033, 2005. 94. Lazar A, Abruzzo LV, Pollock RE, et al: Molecular diagnosis of sarcomas: chromosomal translocations in sarcomas. Arch Pathol Lab Med. 130:1199–1207, 2006. 95. Fritsch MK, Bridge JA, Schuster AE, et al: Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue. Pediatr Dev Pathol. 6:43–53, 2003. 96. Scicchitano MS, Dalmas DA, Bertiaux MA, et al: Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples. J Histochem Cytochem. 54:1229– 1237, 2006. 97. Hammar SP: Metastatic adenocarcinoma of unknown primary origin. Hum Pathol. 29:1393–1402, 1998. 98. Spagnolo DV, Michie SA, Crabtree GS, et al: Monoclonal antikeratin (AE1) reactivity in routinely processed tissue from 166 human neoplasms. Am J Clin Pathol. 84:697–704, 1985. 99. Al-Abbadi MA, Almasri NM, Al-Quran S, et al: Cytokeratin and epithelial membrane antigen expression in angiosarcomas: an
References immunohistochemical study of 33 cases. Arch Pathol Lab Med. 131:288–292, 2007. 100. Bhargava R, Shia J, Hummer AJ, et al: Distinction of endometrial stromal sarcomas from “hemangiopericytomatous” tumors using a panel of immunohistochemical stains. Mod Pathol. 18:40–47, 2005. 101. Fuchs U, Kivela T, Summanen P, et al: An immunohistochemical and prognostic analysis of cytokeratin expression in malignant uveal melanoma. Am J Pathol. 141:169–181, 1992. 102. Gustmann C, Altmannsberger M, Osborn M, et al: Cytokeratin expression and vimentin content in large cell anaplastic lymphomas and other non-Hodgkin’s lymphomas. Am J Pathol. 138:1413–1422, 1991. 103. Korabiowska M, Fischer G, Steinacker A, et al: Cytokeratin positivity in paraffin-embedded malignant melanomas: comparative study of KL1, A4 and Lu5 antibodies. Anticancer Res. 24:3203– 3207, 2004. 104. Srivastava A, Rosenberg AE, Selig M, et al: Keratin-positive Ewing’s sarcoma: an ultrastructural study of 12 cases. Int J Surg Pathol. 13:43–50, 2005. 105. Traweek ST, Liu J, Battifora H: Keratin gene expression in nonepithelial tissues. Detection with polymerase chain reaction. Am J Pathol. 142:1111–1118, 1993. 106. Vakar-Lopez F, Ayala AG, Raymond AK, et al: Epithelial Phenotype in Ewing’s Sarcoma/Primitive Neuroectodermal Tumor. Int J Surg Pathol. 8:59–65, 2000. 107. Miettinen M: Keratin immunohistochemistry: update of applications and pitfalls. Pathol Annu. 28 (Pt 2):113–143, 1993. 108. Moll R: Cytokeratins in the histological diagnosis of malignant tumors. Int J Biol Markers. 9:63–69, 1994. 109. Quinlan RA, Schiller DL, Hatzfeld M, et al: Patterns of expression and organization of cytokeratin intermediate filaments. Ann N Y Acad Sci. 455:282–306, 1985. 110. Moll R, Franke WW, Schiller DL, et al: The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 31:11–24, 1982. 111. Schaafsma HE, Ramaekers FC: Cytokeratin subtyping in normal and neoplastic epithelium: basic principles and diagnostic applications. Pathol Annu. 29(Pt 1):21–62, 1994. 112. Romano V, Bosco P, Rocchi M, et al: Chromosomal assignments of human type I and type II cytokeratin genes to different chromosomes. Cytogenet Cell Genet. 48:148–151, 1988. 113. Rosenberg M, Fuchs E, Le Beau MM, et al: Three epidermal and one simple epithelial type II keratin genes map to human chromosome 12. Cytogenet Cell Genet. 57:33–38, 1991. 114. Rosenberg M, RayChaudhury A, Shows TB, et al: A group of type I keratin genes on human chromosome 17: characterization and expression. Mol Cell Biol. 8:722–736, 1988. 115. Franke WW, Schmid E, Schiller DL, et al: Differentiation-related patterns of expression of proteins of intermediate-size filaments in tissues and cultured cells. Cold Spring Harb Symp Quant Biol. 46(Pt 1):431–453, 1982. 116. Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 13:962–972, 2000. 117. Judkins AR, Montone KT, LiVolsi VA, et al: Sensitivity and specificity of antibodies on necrotic tumor tissue. Am J Clin Pathol. 110:641–646, 1998. 118. Stasiak PC, Purkis PE, Leigh IM, et al: Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins. J Invest Dermatol. 92:707– 716, 1989. 119. Bartek J, Bartkova J, Taylor-Papadimitriou J, et al: Differential expression of keratin 19 in normal human epithelial tissues revealed by monospecific monoclonal antibodies. Histochem J. 18:565–575, 1986. 120. Ramaekers F, Huysmans A, Schaart G, et al: Tissue distribution of keratin 7 as monitored by a monoclonal antibody. Exp Cell Res. 170:235–249, 1987. 121. Ramaekers F, van Niekerk C, Poels L, et al: Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocarcinomas. Am J Pathol. 136:641–655, 1990. 122. van Niekerk CC, Jap PH, Ramaekers FC, et al: Immunohistochemical demonstration of keratin 7 in routinely fixed paraffinembedded human tissues. J Pathol. 165:145–152, 1991.
244.e3
123. Loy TS, Calaluce RD: Utility of cytokeratin immunostaining in separating pulmonary adenocarcinomas from colonic adenocarcinomas. Am J Clin Pathol. 102:764–767, 1994. 124. van de Molengraft FJ, van Niekerk CC, Jap PH, et al: OV-TL 12/30 (keratin 7 antibody) is a marker of glandular differentiation in lung cancer. Histopathology. 22:35–38, 1993. 125. Berezowski K, Stastny JF, Kornstein MJ: Cytokeratins 7 and 20 and carcinoembryonic antigen in ovarian and colonic carcinoma. Mod Pathol. 9:426–429, 1996. 126. Guerrieri C, Franlund B, Boeryd B: Expression of cytokeratin 7 in simultaneous mucinous tumors of the ovary and appendix. Mod Pathol. 8:573–576, 1995. 127. Loy TS, Calaluce RD, Keeney GL: Cytokeratin immunostaining in differentiating primary ovarian carcinoma from metastatic colonic adenocarcinoma. Mod Pathol. 9:1040–1044, 1996. 128. Osborn M, van Lessen G, Weber K, et al: Differential diagnosis of gastrointestinal carcinomas by using monoclonal antibodies specific for individual keratin polypeptides. Lab Invest. 55:497– 504, 1986. 129. Prayson RA, Hart WR, Petras RE: Pseudomyxoma peritonei. A clinicopathologic study of 19 cases with emphasis on site of origin and nature of associated ovarian tumors. Am J Surg Pathol. 18:591–603, 1994. 130. Ronnett BM, Kurman RJ, Shmookler BM, et al: The morphologic spectrum of ovarian metastases of appendiceal adenocarcinomas: a clinicopathologic and immunohistochemical analysis of tumors often misinterpreted as primary ovarian tumors or metastatic tumors from other gastrointestinal sites. Am J Surg Pathol. 21:1144–1155, 1997. 131. Ronnett BM, Shmookler BM, Diener-West M, et al: Immunohistochemical evidence supporting the appendiceal origin of pseudomyxoma peritonei in women. Int J Gynecol Pathol. 16:1– 9, 1997. 132. Ueda G, Sawada M, Ogawa H, et al: Immunohistochemical study of cytokeratin 7 for the differential diagnosis of adenocarcinomas in the ovary. Gynecol Oncol. 51:219–223, 1993. 133. Van Eyken P, Sciot R, Brock P, et al: Abundant expression of cytokeratin 7 in fibrolamellar carcinoma of the liver. Histopathology. 17:101–107, 1990. 134. Ramalingam P, Hart WR, Goldblum JR: Cytokeratin subset immunostaining in rectal adenocarcinoma and normal anal glands. Arch Pathol Lab Med. 125:1074–1077, 2001. 135. Tot T: Cytokeratins 20 and 7 as biomarkers: usefulness in discriminating primary from metastatic adenocarcinoma. Eur J Cancer. 38:758–763, 2002. 136. Moll R, Schiller DL, Franke WW: Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns. J Cell Biol. 111:567– 580, 1990. 137. Miettinen M: Keratin 20: immunohistochemical marker for gastrointestinal, urothelial, and Merkel cell carcinomas. Mod Pathol. 8:384–388, 1995. 138. Moll R, Lowe A, Laufer J, et al: Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol. 140:427–447, 1992. 139. Moll R, Zimbelmann R, Goldschmidt MD, et al: The human gene encoding cytokeratin 20 and its expression during fetal development and in gastrointestinal carcinomas. Differentiation. 53:75–93, 1993. 140. Tot T: Adenocarcinomas metastatic to the liver: the value of cytokeratins 20 and 7 in the search for unknown primary tumors. Cancer. 85:171–177, 1999. 141. Chan JK, Suster S, Wenig BM, et al: Cytokeratin 20 immunoreactivity distinguishes Merkel cell (primary cutaneous neuroendocrine) carcinomas and salivary gland small cell carcinomas from small cell carcinomas of various sites. Am J Surg Pathol. 21:226–234, 1997. 142. Moll I, Kuhn C, Moll R: Cytokeratin 20 is a general marker of cutaneous Merkel cells while certain neuronal proteins are absent. J Invest Dermatol. 104:910–915, 1995. 143. Goldstein NS, Thomas M: Mucinous and nonmucinous bronchioloalveolar adenocarcinomas have distinct staining patterns with thyroid transcription factor and cytokeratin 20 antibodies. Am J Clin Pathol. 116:319–325, 2001.
244.e4
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
144. Lau SK, Desrochers MJ, Luthringer DJ: Expression of thyroid transcription factor-1, cytokeratin 7, and cytokeratin 20 in bronchioloalveolar carcinomas: an immunohistochemical evaluation of 67 cases. Mod Pathol. 15:538–542, 2002. 145. Rossi G, Murer B, Cavazza A, et al: Primary mucinous (so-called colloid) carcinomas of the lung: a clinicopathologic and immunohistochemical study with special reference to CDX-2 homeobox gene and MUC2 expression. Am J Surg Pathol. 28:442–452, 2004. 146. Tot T: Patterns of distribution of cytokeratins 20 and 7 in special types of invasive breast carcinoma: a study of 123 cases. Ann Diagn Pathol. 3:350–356, 1999. 147. Rullier A, Le Bail B, Fawaz R, et al: Cytokeratin 7 and 20 expression in cholangiocarcinomas varies along the biliary tract but still differs from that in colorectal carcinoma metastasis. Am J Surg Pathol. 24:870–876, 2000. 148. Tot T, Samii S: The clinical relevance of cytokeratin phenotyping in needle biopsy of liver metastasis. Apmis. 111:1075–1082, 2003. 149. Nikitakis NG, Tosios KI, Papanikolaou VS, et al: Immunohistochemical expression of cytokeratins 7 and 20 in malignant salivary gland tumors. Mod Pathol. 17:407–415, 2004. 150. Nandedkar MA, Palazzo J, Abbondanzo SL, et al: CD45 (leukocyte common antigen) immunoreactivity in metastatic undifferentiated and neuroendocrine carcinoma: a potential diagnostic pitfall. Mod Pathol. 11:1204–1210, 1998. 151. Gown AM, Vogel AM: Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues. Am J Pathol. 114:309–321, 1984. 152. Gown AM, Vogel AM: Anti-intermediate filament monoclonal antibodies: tissue-specific tools in tumor diagnosis. Surv Synth Pathol Res. 3:369–385, 1984. 153. Gown AM, Vogel AM: Monoclonal antibodies to human intermediate filament proteins. III. Analysis of tumors. Am J Clin Pathol. 84:413–424, 1985. 154. Clover J, Oates J, Edwards C: Anti-cytokeratin 5/6: a positive marker for epithelioid mesothelioma. Histopathology. 31:140– 143, 1997. 155. Kaufmann O, Fietze E, Mengs J, et al: Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol. 116:823–830, 2001. 156. Ordonez NG: Value of cytokeratin 5/6 immunostaining in distinguishing epithelial mesothelioma of the pleura from lung adenocarcinoma. Am J Surg Pathol. 22:1215–1221, 1998. 157. Reis-Filho JS, Simpson PT, Martins A, et al: Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray. Virchows Arch. 443:122–132, 2003. 158. Chu PG, Weiss LM: Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod Pathol. 15:6–10, 2002. 159. Argani P, Rosai J: Hyperplastic mesothelial cells in lymph nodes: report of six cases of a benign process that can stimulate metastatic involvement by mesothelioma or carcinoma. Hum Pathol. 29:339–346, 1998. 160. Bhargava R, Beriwal S, McManus K, et al: CK5 is more sensitive than CK5/6 in identifying “basal-like” phenotype of breast carcinoma. Am J Clin Pathol. 2008, In Press. 161. Litzky LA, Brooks JJ: Cytokeratin immunoreactivity in malignant fibrous histiocytoma and spindle cell tumors: comparison between frozen and paraffin-embedded tissues. Mod Pathol. 5:30–34, 1992. 162. Miettinen M: Immunoreactivity for cytokeratin and epithelial membrane antigen in leiomyosarcoma. Arch Pathol Lab Med. 112:637–640, 1988. 163. Miettinen M: Keratin subsets in spindle cell sarcomas. Keratins are widespread but synovial sarcoma contains a distinctive keratin polypeptide pattern and desmoplakins. Am J Pathol. 138:505–513, 1991. 164. Rosenberg AE, O’Connell JX, Dickersin GR, et al: Expression of epithelial markers in malignant fibrous histiocytoma of the musculoskeletal system: an immunohistochemical and electron microscopic study. Hum Pathol. 24:284–293, 1993.
165. Swanson PE: Heffalumps, jagulars, and cheshire cats. A commentary on cytokeratins and soft tissue sarcomas. Am J Clin Pathol. 95:S2–S7, 1991. 166. Aubry MC, Myers JL, Colby TV, et al: Endometrial stromal sarcoma metastatic to the lung: a detailed analysis of 16 patients. Am J Surg Pathol. 26:440–449, 2002. 167. Brown DC, Theaker JM, Banks PM, et al: Cytokeratin expression in smooth muscle and smooth muscle tumours. Histopathology. 11:477–486, 1987. 168. Eusebi V, Carcangiu ML, Dina R, et al: Keratin-positive epithelioid angiosarcoma of thyroid. A report of four cases. Am J Surg Pathol. 14:737–747, 1990. 169. Fletcher CD, Beham A, Bekir S, et al: Epithelioid angiosarcoma of deep soft tissue: a distinctive tumor readily mistaken for an epithelial neoplasm. Am J Surg Pathol. 15:915–924, 1991. 170. Goldblum JR, Rice TW: Epithelioid angiosarcoma of the pulmonary artery. Hum Pathol. 26:1275–1277, 1995. 171. Gown AM, Boyd HC, Chang Y, et al: Smooth muscle cells can express cytokeratins of “simple” epithelium. Immunocytochemical and biochemical studies in vitro and in vivo. Am J Pathol. 132:223–232, 1988. 172. Hasegawa T, Fujii Y, Seki K, et al: Epithelioid angiosarcoma of bone. Hum Pathol. 28:985–989, 1997. 173. Hirose T, Kudo E, Hasegawa T, et al: Expression of intermediate filaments in malignant fibrous histiocytomas. Hum Pathol. 20:871–877, 1989. 174. Jochum W, Schroder S, Risti B, et al: Cytokeratin-positive angiosarcoma of the adrenal gland. Pathologe. 15:181–186, 1994. 175. Knapp AC, Franke WW: Spontaneous losses of control of cytokeratin gene expression in transformed, non-epithelial human cells occurring at different levels of regulation. Cell. 59:67–79, 1989. 176. Kwaspen FH, Smedts FM, Broos A, et al: Reproducible and highly sensitive detection of the broad spectrum epithelial marker keratin 19 in routine cancer diagnosis. Histopathology. 31:503–516, 1997. 177. McCluggage WG, Clarke R, Toner PG: Cutaneous epithelioid angiosarcoma exhibiting cytokeratin positivity. Histopathology. 27:291–294, 1995. 178. Meis-Kindblom JM, Kindblom LG: Angiosarcoma of soft tissue: a study of 80 cases. Am J Surg Pathol. 22:683–697, 1998. 179. Miettinen M, Rapola J: Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcoma-like tumors. Expression of cytokeratin and the 68-kD neurofilament protein. Am J Surg Pathol. 13:120–132, 1989. 180. Rizeq MN, van de Rijn M, Hendrickson MR, et al: A comparative immunohistochemical study of uterine smooth muscle neoplasms with emphasis on the epithelioid variant. Hum Pathol. 25:671–677, 1994. 181. Frisman DM, McCarthy WF, Schleiff P, et al: Immunocytochemistry in the differential diagnosis of effusions: use of logistic regression to select a panel of antibodies to distinguish adenocarcinomas from mesothelial proliferations. Mod Pathol. 6:179– 184, 1993. 182. Banks ER, Jansen JF, Oberle E, et al: Cytokeratin positivity in fine-needle aspirates of melanomas and sarcomas. Diagn Cytopathol. 12:230–233, 1995. 183. Gerharz CD, Moll R, Meister P, et al: Cytoskeletal heterogeneity of an epithelioid sarcoma with expression of vimentin, cytokeratins, and neurofilaments. Am J Surg Pathol. 14:274–283, 1990. 184. Heikinheimo K, Persson S, Kindblom LG, et al: Expression of different cytokeratin subclasses in human chordoma. J Pathol. 164:145–150, 1991. 185. Ordonez NG, Tornos C: Malignant peripheral nerve sheath tumor of the pleura with epithelial and rhabdomyoblastic differentiation: report of a case clinically simulating mesothelioma. Am J Surg Pathol. 21:1515–1521, 1997. 186. Rosai J, Pinkus GS: Immunohistochemical demonstration of epithelial differentiation in adamantinoma of the tibia. Am J Surg Pathol. 6:427–434, 1982. 187. Smith KJ, Skelton HG 3rd, Morgan AM, et al: Spindle cell neoplasms coexpressing cytokeratin and vimentin (metaplastic squamous cell carcinoma). J Cutan Pathol. 19:286–293, 1992.
References 188. Gatter KC, Ralfkiaer E, Skinner J, et al: An immunocytochemical study of malignant melanoma and its differential diagnosis from other malignant tumours. J Clin Pathol. 38:1353–1357, 1985. 189. Ben-Izhak O, Stark P, Levy R, et al: Epithelial markers in malignant melanoma. A study of primary lesions and their metastases. Am J Dermatopathol. 16:241–246, 1994. 190. Mooi WJ, Deenik W, Peterse JL, et al: Keratin immunoreactivity in melanoma of soft parts (clear cell sarcoma). Histopathology. 27:61–65, 1995. 191. Kriho VK, Yang HY, Moskal JR, et al: Keratin expression in astrocytomas: an immunofluorescent and biochemical reassessment. Virchows Arch. 431:139–147, 1997. 192. Cosgrove M, Fitzgibbons PL, Sherrod A, et al: Intermediate filament expression in astrocytic neoplasms. Am J Surg Pathol. 13:141–145, 1989. 193. Artlich A, Schmidt D: Immunohistochemical profile of meningiomas and their histological subtypes. Hum Pathol. 21:843–849, 1990. 194. Meis JM, Ordonez NG, Bruner JM: Meningiomas. An immunohistochemical study of 50 cases. Arch Pathol Lab Med. 110:934– 937, 1986. 195. Probst-Cousin S, Villagran-Lillo R, Lahl R, et al: Secretory meningioma: clinical, histologic, and immunohistochemical findings in 31 cases. Cancer. 79:2003–2015, 1997. 196. Radley MG, di Sant’Agnese PA, Eskin TA, et al: Epithelial differentiation in meningiomas. An immunohistochemical, histochemical, and ultrastructural study–with review of the literature. Am J Clin Pathol. 92:266–272, 1989. 197. Carbone A, Manconi R, Poletti A, et al: Heterogeneous immunostaining patterns of follicular dendritic reticulum cells in human lymphoid tissue with selected antibodies reactive with different cell lineages. Hum Pathol. 19:51–56, 1988. 198. Cho J, Gong G, Choe G, et al: Extrafollicular reticulum cells in pathologic lymph nodes. J Korean Med Sci. 9:9–15, 1994. 199. Doglioni C, Dell’Orto P, Zanetti G, et al: Cytokeratinimmunoreactive cells of human lymph nodes and spleen in normal and pathological conditions. An immunocytochemical study. Virchows Arch A Pathol Anat Histopathol. 416:479–490, 1990. 200. Franke WW, Moll R: Cytoskeletal components of lymphoid organs. I. Synthesis of cytokeratins 8 and 18 and desmin in subpopulations of extrafollicular reticulum cells of human lymph nodes, tonsils, and spleen. Differentiation. 36:145–163, 1987. 201. Gould VE, Bloom KJ, Franke WW, et al: Increased numbers of cytokeratin-positive interstitial reticulum cells (CIRC) in reactive, inflammatory and neoplastic lymphadenopathies: hyperplasia or induced expression? Virchows Arch. 425:617–629, 1995. 202. Iuzzolino P, Bontempini L, Doglioni C, et al: Keratin immunoreactivity in extrafollicular reticular cells of the lymph node. Am J Clin Pathol. 91:239–240, 1989. 203. Lasota J, Hyjek E, Koo CH, et al: Cytokeratin-positive large-cell lymphomas of B-cell lineage. A study of five phenotypically unusual cases verified by polymerase chain reaction. Am J Surg Pathol. 20:346–354, 1996. 204. Ramaekers F, Haag D, Jap P, et al: Immunochemical demonstration of keratin and vimentin in cytologic aspirates. Acta Cytol. 28:385–392, 1984. 205. Zoltowska A: Immunohistochemical comparative investigations of lymphatic tissue in reactive processes, myasthenic thymuses and Hodgkin’s disease. Arch Immunol Ther Exp (Warsz). 43:15– 22, 1995. 206. Xu X, Roberts SA, Pasha TL, et al: Undesirable cytokeratin immunoreactivity of native nonepithelial cells in sentinel lymph nodes from patients with breast carcinoma. Arch Pathol Lab Med. 124:1310–1313, 2000. 207. Battifora H, Kopinski M: The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J Histochem Cytochem. 34:1095–1100, 1986. 208. Delsol G, Al Saati T, Gatter KC, et al: Coexpression of epithelial membrane antigen (EMA), Ki-1, and interleukin-2 receptor by anaplastic large cell lymphomas. Diagnostic value in so-called malignant histiocytosis. Am J Pathol. 130:59–70, 1988.
244.e5
209. Frierson HF Jr, Bellafiore FJ, Gaffey MJ, et al: Cytokeratin in anaplastic large cell lymphoma. Mod Pathol. 7:317–321, 1994. 210. Petruch UR, Horny HP, Kaiserling E: Frequent expression of haemopoietic and non-haemopoietic antigens by neoplastic plasma cells: an immunohistochemical study using formalinfixed, paraffin-embedded tissue. Histopathology. 20:35–40, 1992. 211. Wotherspoon AC, Norton AJ, Isaacson PG: Immunoreactive cytokeratins in plasmacytomas. Histopathology. 14:141–150, 1989. 212. Miettinen M: Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity. Pathol Res Pract. 184:431–436, 1989. 213. Battifora H: Assessment of antigen damage in immunohistochemistry. The vimentin internal control. Am J Clin Pathol. 96:669–671, 1991. 214. Ramaekers FC, Haag D, Kant A, et al: Coexpression of keratinand vimentin-type intermediate filaments in human metastatic carcinoma cells. Proc Natl Acad Sci U S A. 80:2618–2622, 1983. 215. Geisinger KR, Dabbs DJ, Marshall RB: Malignant mixed mullerian tumors. An ultrastructural and immunohistochemical analysis with histogenetic considerations. Cancer. 59:1781– 1790, 1987. 216. Livasy CA, Karaca G, Nanda R, et al: Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol. 19:264–271, 2006. 217. McNutt MA, Bolen JW, Gown AM, et al: Coexpression of intermediate filaments in human epithelial neoplasms. Ultrastruct Pathol. 9:31–43, 1985. 218. Dabbs DJ, Geisinger KR, Norris HT: Intermediate filaments in endometrial and endocervical carcinomas. The diagnostic utility of vimentin patterns. Am J Surg Pathol. 10:568–576, 1986. 219. Dabbs DJ, Sturtz K, Zaino RJ: The immunohistochemical discrimination of endometrioid adenocarcinomas. Hum Pathol. 27:172–177, 1996. 220. Jones MW, Onisko A, Dabbs DJ, et al: The value of immunohistochemistry in distinction between endocervical adenocarcinoma and adenocarcinoma of endometrium, endometrioid type. A comparative tissue microarray study of 76 cases. International Academy of Pathology. 2008, XXVII International Congress:Abstract. 221. Buchegger F, Schreyer M, Carrel S, et al: Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD. Int J Cancer. 33:643–649, 1984. 222. Nagura H, Tsutsumi Y, Watanabe K, et al: Immunohistochemistry of carcinoembryonic antigen, secretory component and lysozyme in benign and malignant common bile duct tissues. Virchows Arch A Pathol Anat Histopathol. 403:271–280, 1984. 223. Svenberg T: Carcinoembryonic antigen-like substances of human bile. Isolation and partial characterization. Int J Cancer. 17:588– 596, 1976. 224. Chedid A, Chejfec G, Eichorst M, et al: Antigenic markers of hepatocellular carcinoma. Cancer. 65:84–87, 1990. 225. Ferrandez-Izquierdo A, Llombart-Bosch A: Immunohistochemical characterization of 130 cases of primary hepatic carcinomas. Pathol Res Pract. 182:783–791, 1987. 226. Maeda T, Kajiyama K, Adachi E, et al: The expression of cytokeratins 7, 19, and 20 in primary and metastatic carcinomas of the liver. Mod Pathol. 9:901–909, 1996. 227. Ronnett BM, Kurman RJ, Zahn CM, et al: Pseudomyxoma peritonei in women: a clinicopathologic analysis of 30 cases with emphasis on site of origin, prognosis, and relationship to ovarian mucinous tumors of low malignant potential. Hum Pathol. 26:509–524, 1995. 228. Sheahan K, O’Brien MJ, Burke B, et al: Differential reactivities of carcinoembryonic antigen (CEA) and CEA-related monoclonal and polyclonal antibodies in common epithelial malignancies. Am J Clin Pathol. 94:157–164, 1990. 229. Balaton AJ, Nehama-Sibony M, Gotheil C, et al: Distinction between hepatocellular carcinoma, cholangiocarcinoma, and metastatic carcinoma based on immunohistochemical staining for carcinoembryonic antigen and for cytokeratin 19 on paraffin sections. J Pathol. 156:305–310, 1988.
244.e6
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
230. Ma CK, Zarbo RJ, Frierson HF Jr, et al: Comparative immunohistochemical study of primary and metastatic carcinomas of the liver. Am J Clin Pathol. 99:551–557, 1993. 231. Van Eyken P, Sciot R, Paterson A, et al: Cytokeratin expression in hepatocellular carcinoma: an immunohistochemical study. Hum Pathol. 19:562–568, 1988. 232. Cui S, Hano H, Sakata A, et al: Enhanced CD34 expression of sinusoid-like vascular endothelial cells in hepatocellular carcinoma. Pathol Int. 46:751–756, 1996. 233. Hilkens J, Buijs F, Hilgers J, et al: Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors. Int J Cancer. 34:197–206, 1984. 234. McGuckin MA, Walsh MD, Hohn BG, et al: Prognostic significance of MUC1 epithelial mucin expression in breast cancer. Hum Pathol. 26:432–439, 1995. 235. Hilkens J, Ligtenberg MJ, Vos HL, et al: Cell membraneassociated mucins and their adhesion-modulating property. Trends Biochem Sci. 17:359–363, 1992. 236. Gatter KC, Alcock C, Heryet A, et al: Clinical importance of analysing malignant tumours of uncertain origin with immunohistological techniques. Lancet. 1:1302–1305, 1985. 237. Pinkus GS, Etheridge CL, O’Connor EM: Are keratin proteins a better tumor marker than epithelial membrane antigen? A comparative immunohistochemical study of various paraffinembedded neoplasms using monoclonal and polyclonal antibodies. Am J Clin Pathol. 85:269–277, 1986. 238. Gendler S, Taylor-Papadimitriou J, Duhig T, et al: A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem. 263:12820–12823, 1988. 239. Hayes DF, Zurawski VR Jr, Kufe DW: Comparison of circulating CA15-3 and carcinoembryonic antigen levels in patients with breast cancer. J Clin Oncol. 4:1542–1550, 1986. 240. Hilkens J, Buijs F: Biosynthesis of MAM-6, an epithelial sialomucin. Evidence for involvement of a rare proteolytic cleavage step in the endoplasmic reticulum. J Biol Chem. 263:4215–4222, 1988. 241. Hilkens J, Buijs F, Ligtenberg M: Complexity of MAM-6, an epithelial sialomucin associated with carcinomas. Cancer Res. 49:786–793, 1989. 242. Kufe D, Inghirami G, Abe M, et al: Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma. 3:223–232, 1984. 243. Peterson JA, Couto JR, Taylor MR, et al: Selection of tumorspecific epitopes on target antigens for radioimmunotherapy of breast cancer. Cancer Res. 55:s5847–s5851, 1995. 244. Peterson JA, Zava DT, Duwe AK, et al: Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule. Hybridoma. 9:221–235, 1990. 245. al-Nafussi A, Carder PJ: Monoclonal antibodies in the cytodiagnosis of serous effusions. Cytopathology. 1:119–128, 1990. 246. Leong AS, Parkinson R, Milios J: “Thick” cell membranes revealed by immunocytochemical staining: a clue to the diagnosis of mesothelioma. Diagn Cytopathol. 6:9–13, 1990. 247. Singh HK, Silverman JF, Berns L, et al: Significance of epithelial membrane antigen in the work-up of problematic serous effusions. Diagn Cytopathol. 13:3–7, 1995. 248. Al Saati T, Caveriviere P, Gorguet B, et al: Epithelial membrane antigen in hematopoietic neoplasms. Hum Pathol. 17:533–534, 1986. 249. Chittal SM, Caveriviere P, Schwarting R, et al: Monoclonal antibodies in the diagnosis of Hodgkin’s disease. The search for a rational panel. Am J Surg Pathol. 12:9–21, 1988. 250. Chittal SM, Delsol G: The interface of Hodgkin’s disease and anaplastic large cell lymphoma. Cancer Surv. 30:87–105, 1997. 251. Delsol G, Gatter KC, Stein H, et al: Human lymphoid cells express epithelial membrane antigen. Implications for diagnosis of human neoplasms. Lancet. 2:1124–1129, 1984. 252. Fujimoto J, Hata J, Ishii E, et al: Ki-1 lymphomas in childhood: immunohistochemical analysis and the significance of epithelial membrane antigen (EMA) as a new marker. Virchows Arch A Pathol Anat Histopathol. 412:307–314, 1988.
253. Gatter KC, Abdulaziz Z, Beverley P, et al: Use of monoclonal antibodies for the histopathological diagnosis of human malignancy. J Clin Pathol. 35:1253–1267, 1982. 254. Hall PA, d’Ardenne AJ, Stansfeld AG: Paraffin section immunohistochemistry. I. Non-Hodgkin’s lymphoma. Histopathology. 13:149–160, 1988. 255. Sarker AB, Akagi T, Yoshino T, et al: Expression of vimentin and epithelial membrane antigen in human malignant lymphomas. Acta Pathol Jpn. 40:581–587, 1990. 256. Stein H, Hansmann ML, Lennert K, et al: Reed-Sternberg and Hodgkin cells in lymphocyte-predominant Hodgkin’s disease of nodular subtype contain J chain. Am J Clin Pathol. 86:292–297, 1986. 257. Strickler JG, Weiss LM, Copenhaver CM, et al: Monoclonal antibodies reactive in routinely processed tissue sections of malignant lymphoma, with emphasis on T-cell lymphomas. Hum Pathol. 18:808–814, 1987. 258. Thomas P, Battifora H: Keratins versus epithelial membrane antigen in tumor diagnosis: an immunohistochemical comparison of five monoclonal antibodies. Hum Pathol. 18:728–734, 1987. 259. McCluggage WG, Young RH: Immunohistochemistry as a diagnostic aid in the evaluation of ovarian tumors. Semin Diagn Pathol. 22:3–32, 2005. 260. Shek TW, Yuen ST, Luk IS, et al: Germ cell tumour as a diagnostic pitfall of metastatic carcinoma. J Clin Pathol. 49:223–225, 1996. 261. Ordonez NG: Value of the MOC-31 monoclonal antibody in differentiating epithelial pleural mesothelioma from lung adenocarcinoma. Hum Pathol. 29:166–169, 1998. 262. Ordonez NG: Value of the Ber-EP4 antibody in differentiating epithelial pleural mesothelioma from adenocarcinoma. The M.D. Anderson experience and a critical review of the literature. Am J Clin Pathol. 109:85–89, 1998. 263. Riera JR, Astengo-Osuna C, Longmate JA, et al: The immunohistochemical diagnostic panel for epithelial mesothelioma: a reevaluation after heat-induced epitope retrieval. Am J Surg Pathol. 21:1409–1419, 1997. 264. Rossen K, Thomsen HK: Ber-EP4 immunoreactivity depends on the germ layer origin and maturity of the squamous epithelium. Histopathology. 39:386–389, 2001. 265. Yaziji H, Battifora H, Barry TS, et al: Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: identification of a three-antibody immunohistochemical panel with maximal sensitivity and specificity. Mod Pathol. 19:514–523, 2006. 266. Morrison C, Marsh W Jr, Frankel WL: A comparison of CD10 to pCEA, MOC-31, and hepatocyte for the distinction of malignant tumors in the liver. Mod Pathol. 15:1279–1287, 2002. 267. Porcell AI, De Young BR, Proca DM, et al: Immunohistochemical analysis of hepatocellular and adenocarcinoma in the liver: MOC31 compares favorably with other putative markers. Mod Pathol. 13:773–778, 2000. 268. Siddiqui MT, Saboorian MH, Gokaslan ST, et al: Diagnostic utility of the HepPar1 antibody to differentiate hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration samples. Cancer. 96:49–52, 2002. 269. Wiedenmann B, Kuhn C, Schwechheimer K, et al: Synaptophysin identified in metastases of neuroendocrine tumors by immunocytochemistry and immunoblotting. Am J Clin Pathol. 88:560–569, 1987. 270. Wilson BS, Lloyd RV: Detection of chromogranin in neuroendocrine cells with a monoclonal antibody. Am J Pathol. 115:458– 468, 1984. 271. DeStephano DB, Lloyd RV, Pike AM, et al: Pituitary adenomas. An immunohistochemical study of hormone production and chromogranin localization. Am J Pathol. 116:464–472, 1984. 272. Lloyd RV, Mervak T, Schmidt K, et al: Immunohistochemical detection of chromogranin and neuron-specific enolase in pancreatic endocrine neoplasms. Am J Surg Pathol. 8:607–614, 1984. 273. Lloyd RV, Wilson BS: Specific endocrine tissue marker defined by a monoclonal antibody. Science. 222:628–630, 1983. 274. Wiedenmann B, Franke WW: Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell. 41:1017–1028, 1985.
References 275. Thomas L, Hartung K, Langosch D, et al: Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane. Science. 242:1050–1053, 1988. 276. Piehl MR, Gould VE, Warren WH, et al: Immunohistochemical identification of exocrine and neuroendocrine subsets of large cell lung carcinomas. Pathol Res Pract. 183:675–682, 1988. 277. Staren ED, Gould VE, Warren WH, et al: Neuroendocrine carcinomas of the colon and rectum: a clinicopathologic evaluation. Surgery. 104:1080–1089, 1988. 278. Guinee DG Jr, Fishback NF, Koss MN, et al: The spectrum of immunohistochemical staining of small-cell lung carcinoma in specimens from transbronchial and open-lung biopsies. Am J Clin Pathol. 102:406–414, 1994. 279. Kayser K, Schmid W, Ebert W, et al: Expression of neuroendocrine markers (neuronspecific enolase, synaptophysin and bombesin) in carcinoma of the lung. Pathol Res Pract. 183:412– 417, 1988. 280. Tsang WY, Chan JK, Ng CS, et al: Utility of a paraffin sectionreactive CD56 antibody (123C3) for characterization and diagnosis of lymphomas. Am J Surg Pathol. 20:202–210, 1996. 281. Abo T, Balch CM: A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J Immunol. 127:1024–1029, 1981. 282. Baylin SB, Jackson RD, Goodwin G, et al: Neuroendocrinerelated biochemistry in the spectrum of human lung cancers. Exp Lung Res. 3:209–223, 1982. 283. Caillaud JM, Benjelloun S, Bosq J, et al: HNK-1-defined antigen detected in paraffin-embedded neuroectoderm tumors and those derived from cells of the amine precursor uptake and decarboxylation system. Cancer Res. 44:4432–4439, 1984. 284. Cole SP, Mirski S, McGarry RC, et al: Differential expression of the Leu-7 antigen on human lung tumor cells. Cancer Res. 45:4285–4290, 1985. 285. Shioda Y, Nagura H, Tsutsumi Y, et al: Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody. Histochem J. 16:843– 854, 1984. 286. Wick MR: Immunohistology of neuroendocrine and neuroectodermal tumors. Semin Diagn Pathol. 17:194–203, 2000. 287. Battifora H, Silva EG: The use of antikeratin antibodies in the immunohistochemical distinction between neuroendocrine (Merkel cell) carcinoma of the skin, lymphoma, and oat cell carcinoma. Cancer. 58:1040–1046, 1986. 288. Osborn M, Dirk T, Kaser H, et al: Immunohistochemical localization of neurofilaments and neuron-specific enolase in 29 cases of neuroblastoma. Am J Pathol. 122:433–442, 1986. 289. Tsokos M, Linnoila RI, Chandra RS, et al: Neuron-specific enolase in the diagnosis of neuroblastoma and other small, round-cell tumors in children. Hum Pathol. 15:575–584, 1984. 290. Vinores SA, Bonnin JM, Rubinstein LJ, et al: Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other tissues. Arch Pathol Lab Med. 108:536– 540, 1984. 291. Nicholson SA, McDermott MB, Swanson PE, et al: CD99 and cytokeratin-20 in small-cell and basaloid tumors of the skin. Appl Immunohistochem Mol Morphol. 8:37–41, 2000. 292. Jiang C, Tan Y, Li E: Histopathological and immunohistochemical studies on medullary thyroid carcinoma. Zhonghua Bing Li Xue Za Zhi. 25:332–335, 1996. 293. Kargi A, Yorukoglu ••, Aktas S, et al: Neuroendocrine differentiation in non-neuroendocrine thyroid carcinoma. Thyroid. 6:207–210, 1996. 294. Kovacs CS, Mase RM, Kovacs K, et al: Thyroid medullary carcinoma with thyroglobulin immunoreactivity in sporadic multiple endocrine neoplasia type 2-B. Cancer. 74:928–932, 1994. 295. Ruck P, Horny HP, Greschniok A, et al: Nonspecific immunostaining of blast cells of acute leukemia by antibodies against nonhemopoietic antigens. Hematol Pathol. 9:49–56, 1995. 296. Bejarano PA, Baughman RP, Biddinger PW, et al: Surfactant proteins and thyroid transcription factor-1 in pulmonary and breast carcinomas. Mod Pathol. 9:445–452, 1996. 297. Di Loreto C, Di Lauro V, Puglisi F, et al: Immunocytochemical expression of tissue specific transcription factor-1 in lung carcinoma. J Clin Pathol. 50:30–32, 1997.
244.e7
298. Di Loreto C, Puglisi F, Di Lauro V, et al: TTF-1 protein expression in pleural malignant mesotheliomas and adenocarcinomas of the lung. Cancer Lett. 124:73–78, 1998. 299. Fabbro D, Di Loreto C, Stamerra O, et al: TTF-1 gene expression in human lung tumours. Eur J Cancer. 32A:512–517, 1996. 300. Lazzaro D, Price M, de Felice M, et al: The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development. 113:1093–1104, 1991. 301. Stahlman MT, Gray ME, Whitsett JA: Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung. J Histochem Cytochem. 44:673–678, 1996. 302. Guazzi S, Price M, De Felice M, et al: Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. Embo J. 9:3631–3639, 1990. 303. Folpe AL, Gown AM, Lamps LW, et al: Thyroid transcription factor-1: immunohistochemical evaluation in pulmonary neuroendocrine tumors. Mod Pathol. 12:5–8, 1999. 304. Holzinger A, Dingle S, Bejarano PA, et al: Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis. Hybridoma. 15:49–53, 1996. 305. Khoor A, Whitsett JA, Stahlman MT, et al: Utility of surfactant protein B precursor and thyroid transcription factor 1 in differentiating adenocarcinoma of the lung from malignant mesothelioma. Hum Pathol. 30:695–700, 1999. 306. Kaufmann O, Dietel M: Expression of thyroid transcription factor-1 in pulmonary and extrapulmonary small cell carcinomas and other neuroendocrine carcinomas of various primary sites. Histopathology. 36:415–420, 2000. 307. Ordonez NG: Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol. 24:1217–1223, 2000. 308. Agoff SN, Lamps LW, Philip AT, et al: Thyroid transcription factor-1 is expressed in extrapulmonary small cell carcinomas but not in other extrapulmonary neuroendocrine tumors. Mod Pathol. 13:238–242, 2000. 309. Afify AM, al-Khafaji BM: Diagnostic utility of thyroid transcription factor-1 expression in adenocarcinomas presenting in serous fluids. Acta Cytol. 46:675–678, 2002. 310. Jang KY, Kang MJ, Lee DG, et al: Utility of thyroid transcription factor-1 and cytokeratin 7 and 20 immunostaining in the identification of origin in malignant effusions. Anal Quant Cytol Histol. 23:400–404, 2001. 311. Roh MS, Hong SH: Utility of thyroid transcription factor-1 and cytokeratin 20 in identifying the origin of metastatic carcinomas of cervical lymph nodes. J Korean Med Sci. 17:512–517, 2002. 312. Chang YL, Lee YC, Liao WY, et al: The utility and limitation of thyroid transcription factor-1 protein in primary and metastatic pulmonary neoplasms. Lung Cancer. 44:149–157, 2004. 313. Wieczorek TJ, Pinkus JL, Glickman JN, et al: Comparison of thyroid transcription factor-1 and hepatocyte antigen immunohistochemical analysis in the differential diagnosis of hepato cellular carcinoma, metastatic adenocarcinoma, renal cell carcinoma, and adrenal cortical carcinoma. Am J Clin Pathol. 118:911–921, 2002. 314. Kubba LA, McCluggage WG, Liu J, et al: Thyroid transcription factor-1 expression in ovarian epithelial neoplasms. Mod Pathol. 21:485–490, 2008. 315. Siami K, McCluggage WG, Ordonez NG, et al: Thyroid transcription factor-1 expression in endometrial and endocervical adenocarcinomas. Am J Surg Pathol. 31:1759–1763, 2007. 316. Robens J, Goldstein L, Gown AM, et al: Thyroid transcription factor-1 expression in breast carcinomas. Am J Surg Pathol. 34:1881–1885, 2010. 317. Nicholson AG, McCormick CJ, Shimosato Y, et al: The value of PE-10, a monoclonal antibody against pulmonary surfactant, in distinguishing primary and metastatic lung tumours. Histopathology. 27:57–60, 1995. 318. Yang M, Nonaka D: A study of immunohistochemical differential expression in pulmonary and mammary carcinomas. Mod Pathol. 23:654–661, 2010. 319. Bishop JA, Sharma R, Illei PB: Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum pathol. 41:20–25, 2010.
244.e8
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
320. Mukhopadhyay S, Katzenstein AL: Comparison of monoclonal napsin A, polyclonal napsin A, and TTF-1 for determining lung origin in metastatic adenocarcinomas. Am J Clin Pathol. 138:703–711, 2012. 321. Ye J, Findeis-Hosey JJ, Yang Q, et al: Combination of napsin A and TTF-1 immunohistochemistry helps in differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung. Appl Immunohistochem Mol Morphol. 19:313–317, 2011. 322. Andressen C, Blumcke I, Celio MR: Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res. 271:181–208, 1993. 323. Doglioni C, Tos AP, Laurino L, et al: Calretinin: a novel immunocytochemical marker for mesothelioma. Am J Surg Pathol. 20:1037–1046, 1996. 324. Ordonez NG: Value of calretinin immunostaining in differentiating epithelial mesothelioma from lung adenocarcinoma. Mod Pathol. 11:929–933, 1998. 325. Ordonez NG: The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. Am J Surg Pathol. 27:1031–1051, 2003. 326. Pritchard-Jones K, Fleming S, Davidson D, et al: The candidate Wilms’ tumour gene is involved in genitourinary development. Nature. 346:194–197, 1990. 327. Shimizu M, Toki T, Takagi Y, et al: Immunohistochemical detection of the Wilms’ tumor gene (WT1) in epithelial ovarian tumors. Int J Gynecol Pathol. 19:158–163, 2000. 328. Waldstrom M, Grove A: Immunohistochemical expression of wilms tumor gene protein in different histologic subtypes of ovarian carcinomas. Arch Pathol Lab Med. 129:85–88, 2005. 329. Acs G, Pasha T, Zhang PJ: WT1 is differentially expressed in serous, endometrioid, clear cell, and mucinous carcinomas of the peritoneum, fallopian tube, ovary, and endometrium. Int J Gynecol Pathol. 23:110–118, 2004. 330. Al-Hussaini M, Stockman A, Foster H, et al: WT-1 assists in distinguishing ovarian from uterine serous carcinoma and in distinguishing between serous and endometrioid ovarian carcinoma. Histopathology. 44:109–115, 2004. 331. Dupont J, Wang X, Marshall DS, et al: Wilms Tumor Gene (WT1) and p53 expression in endometrial carcinomas: a study of 130 cases using a tissue microarray. Gynecol Oncol. 94:449– 455, 2004. 332. Egan JA, Ionescu MC, Eapen E, et al: Differential expression of WT1 and p53 in serous and endometrioid carcinomas of the endometrium. Int J Gynecol Pathol. 23:119–122, 2004. 333. Goldstein NS, Uzieblo A: WT1 immunoreactivity in uterine papillary serous carcinomas is different from ovarian serous carcinomas. Am J Clin Pathol. 117:541–545, 2002. 334. Hashi A, Yuminamochi T, Murata S, et al: Wilms tumor gene immunoreactivity in primary serous carcinomas of the fallopian tube, ovary, endometrium, and peritoneum. Int J Gynecol Pathol. 22:374–377, 2003. 335. Domfeh AB, Carley AL, Striebel JM, et al: WT1 immunoreactivity in breast carcinoma: selective expression in pure and mixed mucinous subtypes. Mod Pathol. 2008, In Press. 336. Pearlman WH, Gueriguian JL, Sawyer ME: A specific progesterone-binding component of human breast cyst fluid. J Biol Chem. 248:5736–5741, 1973. 337. Haagensen DE Jr, Mazoujian G, Holder WD Jr, et al: Evaluation of a breast cyst fluid protein detectable in the plasma of breast carcinoma patients. Ann Surg. 185:279–285, 1977. 338. Murphy LC, Lee-Wing M, Goldenberg GJ, et al: Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo: correlation with steroid receptor status. Cancer Res. 47:4160–4164, 1987. 339. Eusebi V, Magalhaes F, Azzopardi JG: Pleomorphic lobular carcinoma of the breast: an aggressive tumor showing apocrine differentiation. Hum Pathol. 23:655–662, 1992. 340. Mazoujian G, Parish TH, Haagensen DE Jr: Immunoperoxidase localization of GCDFP-15 with mouse monoclonal antibodies versus rabbit antiserum. J Histochem Cytochem. 36:377–382, 1988. 341. Mazoujian G, Margolis R: Immunohistochemistry of gross cystic disease fluid protein (GCDFP-15) in 65 benign sweat gland tumors of the skin. Am J Dermatopathol. 10:28–35, 1988. 342. Mazoujian G, Pinkus GS, Davis S, et al: Immunohistochemistry of a gross cystic disease fluid protein (GCDFP-15) of the breast.
A marker of apocrine epithelium and breast carcinomas with apocrine features. Am J Pathol. 110:105–112, 1983. 343. Swanson PE, Pettinato G, Lillemoe TJ, et al: Gross cystic disease fluid protein-15 in salivary gland tumors. Arch Pathol Lab Med. 115:158–163, 1991. 344. Viacava P, Naccarato AG, Bevilacqua G: Spectrum of GCDFP15 expression in human fetal and adult normal tissues. Virchows Arch. 432:255–260, 1998. 345. Wick MR, Lillemoe TJ, Copland GT, et al: Gross cystic disease fluid protein-15 as a marker for breast cancer: immunohistochemical analysis of 690 human neoplasms and comparison with alpha-lactalbumin. Hum Pathol. 20:281–287, 1989. 346. Bhargava R, Beriwal S, Dabbs DJ: Mammaglobin vs GCDFP-15: an immunohistologic validation survey for sensitivity and specificity. Am J Clin Pathol. 127:103–113, 2007. 347. Kaufmann O, Deidesheimer T, Muehlenberg M, et al: Immunohistochemical differentiation of metastatic breast carcinomas from metastatic adenocarcinomas of other common primary sites. Histopathology. 29:233–240, 1996. 348. Raab SS, Berg LC, Swanson PE, et al: Adenocarcinoma in the lung in patients with breast cancer. A prospective analysis of the discriminatory value of immunohistology. Am J Clin Pathol. 100:27–35, 1993. 349. Striebel JM, Dacic S, Yousem SA: Gross cystic disease fluid protein-(GCDFP-15): expression in primary lung adenocarcinoma. Am J Surg Pathol. 32:426–432, 2008. 350. Han JH, Kang Y, Shin HC, et al: Mammaglobin expression in lymph nodes is an important marker of metastatic breast carcinoma. Arch Pathol Lab Med. 127:1330–1334, 2003. 351. Ciampa A, Fanger G, Khan A, et al: Mammaglobin and CRxA-01 in pleural effusion cytology: potential utility of distinguishing metastatic breast carcinomas from other cytokeratin 7-positive/ cytokeratin 20-negative carcinomas. Cancer. 102:368–372, 2004. 352. Sasaki E, Tsunoda N, Hatanaka Y, et al: Breast-specific expression of MGB1/mammaglobin: an examination of 480 tumors from various organs and clinicopathological analysis of MGB1-positive breast cancers. Mod Pathol. 20:208–214, 2007. 353. Onuma K, Dabbs DJ, Bhargava R: Mammaglobin expression in the female genital tract: immunohistochemical analysis in benign and neoplastic endocervix and endometrium. Int J Gynecol Pathol. 27:418–425, 2008. 354. Beattie CW, Hansen NW, Thomas PA: Steroid receptors in human lung cancer. Cancer Res. 45:4206–4214, 1985. 355. Cagle PT, Mody DR, Schwartz MR: Estrogen and progesterone receptors in bronchogenic carcinoma. Cancer Res. 50:6632– 6635, 1990. 356. Kaiser U, Hofmann J, Schilli M, et al: Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer. Int J Cancer. 67:357–364, 1996. 357. Su JM, Hsu HK, Chang H, et al: Expression of estrogen and progesterone receptors in non-small-cell lung cancer: immunohistochemical study. Anticancer Res. 16:3803–3806, 1996. 358. Vargas SO, Leslie KO, Vacek PM, et al: Estrogen-receptor-related protein p29 in primary nonsmall cell lung carcinoma: pathologic and prognostic correlations. Cancer. 82:1495–1500, 1998. 359. Dabbs DJ, Landreneau RJ, Liu Y, et al: Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg. 73:403–405; discussion 406, 2002. 360. Wei S, Said-Al-Naief N, Hameed O: Estrogen and progesterone receptor expression is not always specific for mammary and gynecologic carcinomas: a tissue microarray and pooled literature review study. Appl Immunohistochem Mol Morphol. 17:393– 402, 2009. 361. Tot T: The role of cytokeratins 20 and 7 and estrogen receptor analysis in separation of metastatic lobular carcinoma of the breast and metastatic signet ring cell carcinoma of the gastrointestinal tract. Apmis. 108:467–472, 2000. 362. Bacchi CE, Gown AM: Distribution and pattern of expression of villin, a gastrointestinal-associated cytoskeletal protein, in human carcinomas: a study employing paraffin-embedded tissue. Lab Invest. 64:418–424, 1991. 363. Nambu Y, Iannettoni MD, Orringer MB, et al: Unique expression patterns and alterations in the intestinal protein villin in primary and metastatic pulmonary adenocarcinomas. Mol Carcinog. 23:234–242, 1998.
References 364. Sharma S, Tan J, Sidhu G, et al: Lung adenocarcinomas metastatic to the brain with and without ultrastructural evidence of rootlets: an electron microscopic and immunohistochemical study using cytokeratins 7 and 20 and villin. Ultrastruct Pathol. 22:385–391, 1998. 365. Tan J, Sidhu G, Greco MA, et al: Villin, cytokeratin 7, and cytokeratin 20 expression in pulmonary adenocarcinoma with ultrastructural evidence of microvilli with rootlets. Hum Pathol. 29:390–396, 1998. 366. Lau SK, Prakash S, Geller SA, et al: Comparative immunohistochemical profile of hepatocellular carcinoma, cholangio carcinoma, and metastatic adenocarcinoma. Hum Pathol. 33:1175–1181, 2002. 367. Moll R, Robine S, Dudouet B, et al: Villin: a cytoskeletal protein and a differentiation marker expressed in some human adenocarcinomas. Virchows Arch B Cell Pathol Incl Mol Pathol. 54:155– 169, 1987. 368. Drummond F, Putt W, Fox M, et al: Cloning and chromosome assignment of the human CDX2 gene. Ann Hum Genet. 61:393– 400, 1997. 369. Beck F, Chawengsaksophak K, Waring P, et al: Reprogramming of intestinal differentiation and intercalary regeneration in Cdx2 mutant mice. Proc Natl Acad Sci U S A. 96:7318–7323, 1999. 370. Chawengsaksophak K, James R, Hammond VE, et al: Homeosis and intestinal tumours in Cdx2 mutant mice. Nature. 386:84– 87, 1997. 371. Mallo GV, Rechreche H, Frigerio JM, et al: Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis. Int J Cancer. 74:35–44, 1997. 372. Tamai Y, Nakajima R, Ishikawa T, et al: Colonic hamartoma development by anomalous duplication in Cdx2 knockout mice. Cancer Res. 59:2965–2970, 1999. 373. Barbareschi M, Murer B, Colby TV, et al: CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs. Am J Surg Pathol. 27:141–149, 2003. 374. Bai YQ, Yamamoto H, Akiyama Y, et al: Ectopic expression of homeodomain protein CDX2 in intestinal metaplasia and carcinomas of the stomach. Cancer Lett. 176:47–55, 2002. 375. Werling RW, Yaziji H, Bacchi CE, et al: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 27:303–310, 2003. 376. Hinoi T, Tani M, Lucas PC, et al: Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon. Am J Pathol. 159:2239–2248, 2001. 377. Barbareschi M, Roldo C, Zamboni G, et al: CDX-2 homeobox gene product expression in neuroendocrine tumors: its role as a marker of intestinal neuroendocrine tumors. Am J Surg Pathol. 28:1169–1176, 2004. 378. Wang HL, Lu DW, Yerian LM, et al: Immunohistochemical distinction between primary adenocarcinoma of the bladder and secondary colorectal adenocarcinoma. Am J Surg Pathol. 25:1380–1387, 2001. 379. Raspollini MR, Baroni G, Taddei A, et al: Primary cervical adenocarcinoma with intestinal differentiation and colonic carcinoma metastatic to cervix: an investigation using Cdx-2 and a limited immunohistochemical panel. Arch Pathol Lab Med. 127:1586– 1590, 2003. 380. McCluggage WG, Shah R, Connolly LE, et al: Intestinal-type cervical adenocarcinoma in situ and adenocarcinoma exhibit a partial enteric immunophenotype with consistent expression of CDX2. Int J Gynecol Pathol. 27:92–100, 2008. 381. Park KJ, Bramlage MP, Ellenson LH, et al: Immunoprofile of Adenocarcinomas of the Endometrium, Endocervix, and Ovary With Mucinous Differentiation. Appl Immunohistochem Mol Morphol. 2008. 382. Sullivan LM, Smolkin ME, Frierson HF Jr, et al: Comprehensive Evaluation of CDX2 in Invasive Cervical Adenocarcinomas: Immunopositivity in the Absence of Overt Colorectal Morphology. Am J Surg Pathol. 2008.
244.e9
383. Tot T: Identifying colorectal metastases in liver biopsies: the novel CDX2 antibody is less specific than the cytokeratin 20+/7− phenotype. Med Sci Monit. 10:BR139–143, 2004. 384. Wennerberg AE, Nalesnik MA, Coleman WB: Hepatocyte paraffin 1: a monoclonal antibody that reacts with hepatocytes and can be used for differential diagnosis of hepatic tumors. Am J Pathol. 143:1050–1054, 1993. 385. Chu PG, Ishizawa S, Wu E, et al: Hepatocyte antigen as a marker of hepatocellular carcinoma: an immunohistochemical comparison to carcinoembryonic antigen, CD10, and alpha-fetoprotein. Am J Surg Pathol. 26:978–988, 2002. 386. Fan Z, van de Rijn M, Montgomery K, et al: Hep par 1 antibody stain for the differential diagnosis of hepatocellular carcinoma: 676 tumors tested using tissue microarrays and conventional tissue sections. Mod Pathol. 16:137–144, 2003. 387. Kakar S, Muir T, Murphy LM, et al: Immunoreactivity of Hep Par 1 in hepatic and extrahepatic tumors and its correlation with albumin in situ hybridization in hepatocellular carcinoma. Am J Clin Pathol. 119:361–366, 2003. 388. Ishikura H, Fukasawa Y, Ogasawara K, et al: An AFP-producing gastric carcinoma with features of hepatic differentiation. A case report. Cancer. 56:840–848, 1985. 389. Sinard J, Macleay L Jr, Melamed J: Hepatoid adenocarcinoma in the urinary bladder. Unusual localization of a newly recognized tumor type. Cancer. 73:1919–1925, 1994. 390. Pitman MB, Triratanachat S, Young RH, et al: Hepatocyte paraffin 1 antibody does not distinguish primary ovarian tumors with hepatoid differentiation from metastatic hepatocellular carcinoma. Int J Gynecol Pathol. 23:58–64, 2004. 391. Chu PG, Weiss LM: Immunohistochemical characterization of signet-ring cell carcinomas of the stomach, breast, and colon. Am J Clin Pathol. 121:884–892, 2004. 392. Yan BC, Gong C, Song J, et al: Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol. 34:1147–1154, 2010. 393. McKnight R, Nassar A, Cohen C, et al: Arginase-1: a novel immunohistochemical marker of hepatocellular differentiation in fine needle aspiration cytology. Cancer Cytopathol. 120:223– 229, 2012. 394. Fujiwara M, Kwok S, Yano H, et al: Arginase-1 is a more sensitive marker of hepatic differentiation than HepPar-1 and glypican-3 in fine-needle aspiration biopsies. Cancer Cytopathol. 120:230– 237, 2012. 395. Hahn SA, Schutte M, Hoque AT, et al: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 271:350–353, 1996. 396. Ji H, Isacson C, Seidman JD, et al: Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 21:391–400, 2002. 397. McCarthy DM, Hruban RH, Argani P, et al: Role of the DPC4 tumor suppressor gene in adenocarcinoma of the ampulla of Vater: analysis of 140 cases. Mod Pathol. 16:272–278, 2003. 398. Parwani AV, Geradts J, Caspers E, et al: Immunohistochemical and genetic analysis of non-small cell and small cell gallbladder carcinoma and their precursor lesions. Mod Pathol. 16:299–308, 2003. 399. Svrcek M, Jourdan F, Sebbagh N, et al: Immunohistochemical analysis of adenocarcinoma of the small intestine: a tissue microarray study. J Clin Pathol. 56:898–903, 2003. 400. Lowe FC, Trauzzi SJ: Prostatic acid phosphatase in 1993. Its limited clinical utility. Urol Clin North Am. 20:589–595, 1993. 401. Alanen KA, Kuopio T, Koskinen PJ, et al: Immunohistochemical labelling for prostate specific antigen in non-prostatic tissues. Pathol Res Pract. 192:233–237, 1996. 402. Bostwick DG: Prostate-specific antigen. Current role in diagnostic pathology of prostate cancer. Am J Clin Pathol. 102:S31–S37, 1994. 403. Elgamal AA, Van de Voorde W, Van Poppel H, et al: Immunohistochemical localization of prostate-specific markers within the accessory male sex glands of Cowper, Littre, and Morgagni. Urology. 44:84–90, 1994. 404. Frazier HA, Humphrey PA, Burchette JL, et al: Immunoreactive prostatic specific antigen in male periurethral glands. J Urol. 147:246–248, 1992.
244.e10
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
405. Golz R, Schubert GE: Prostatic specific antigen: immunoreactivity in urachal remnants. J Urol. 141:1480–1482, 1989. 406. Kamoshida S, Tsutsumi Y: Extraprostatic localization of prostatic acid phosphatase and prostate-specific antigen: distribution in cloacogenic glandular epithelium and sex-dependent expression in human anal gland. Hum Pathol. 21:1108–1111, 1990. 407. Nowels K, Kent E, Rinsho K, et al: Prostate specific antigen and acid phosphatase-reactive cells in cystitis cystica and glandularis. Arch Pathol Lab Med. 112:734–737, 1988. 408. van Krieken JH: Prostate marker immunoreactivity in salivary gland neoplasms. A rare pitfall in immunohistochemistry. Am J Surg Pathol. 17:410–414, 1993. 409. Kuriyama M, Wang MC, Lee CL, et al: Multiple marker evaluation in human prostate cancer with the use of tissue-specific antigens. J Natl Cancer Inst. 68:99–105, 1982. 410. Nadji M, Tabei SZ, Castro A, et al: Prostatic origin of tumors. An immunohistochemical study. Am J Clin Pathol. 73:735–739, 1980. 411. Lee SS: Endometrioid adenocarcinoma of the prostate: a clinicopathologic and immunohistochemical study. J Surg Oncol. 55:235–238, 1994. 412. Leong FJ, Leong AS, Swift J: Signet-ring carcinoma of the prostate. Pathol Res Pract. 192:1232–1238; discussion 1239–1241, 1996. 413. Millar EK, Sharma NK, Lessells AM: Ductal (endometrioid) adenocarcinoma of the prostate: a clinicopathological study of 16 cases. Histopathology. 29:11–19, 1996. 414. Esteban JM, Battifora H: Tumor immunophenotype: comparison between primary neoplasm and its metastases. Mod Pathol. 3:192–197, 1990. 415. Kramer SA, Farnham R, Glenn JF, et al: Comparative morphology of primary and secondary deposits of prostatic adenocarcinoma. Cancer. 48:271–273, 1981. 416. Bodey B, Bodey B Jr, Kaiser HE: Immunocytochemical detection of prostate specific antigen expression in human primary and metastatic melanomas. Anticancer Res. 17:2343–2346, 1997. 417. Fan CY, Wang J, Barnes EL: Expression of androgen receptor and prostatic specific markers in salivary duct carcinoma: an immunohistochemical analysis of 13 cases and review of the literature. Am J Surg Pathol. 24:579–586, 2000. 418. Beach R, Gown AM, De Peralta-Venturina MN, et al: P504S immunohistochemical detection in 405 prostatic specimens including 376 18-gauge needle biopsies. Am J Surg Pathol. 26:1588–1596, 2002. 419. Chan TY, Mikolajczyk SD, Lecksell K, et al: Immunohistochemical staining of prostate cancer with monoclonal antibodies to the precursor of prostate-specific antigen. Urology. 62:177–181, 2003. 420. Horoszewicz JS, Kawinski E, Murphy GP: Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients. Anticancer Res. 7:927–935, 1987. 421. Murphy GP, Elgamal AA, Su SL, et al: Current evaluation of the tissue localization and diagnostic utility of prostate specific membrane antigen. Cancer. 83:2259–2269, 1998. 422. Silver DA, Pellicer I, Fair WR, et al: Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 3:81–85, 1997. 423. Murphy GP, Barren RJ, Erickson SJ, et al: Evaluation and comparison of two new prostate carcinoma markers. Free-prostate specific antigen and prostate specific membrane antigen. Cancer. 78:809–818, 1996. 424. Troyer JK, Beckett ML, Wright GL Jr: Location of prostatespecific membrane antigen in the LNCaP prostate carcinoma cell line. Prostate. 30:232–242, 1997. 425. Murphy GP, Tino WT, Holmes EH, et al: Measurement of prostate-specific membrane antigen in the serum with a new antibody. Prostate. 28:266–271, 1996. 426. Sheridan T, Herawi M, Epstein JI, et al: The role of P501S and PSA in the diagnosis of metastatic adenocarcinoma of the prostate. Am J Surg Pathol. 31:1351–1355, 2007. 427. Gelmann EP, Bowen C, Bubendorf L: Expression of NKX3.1 in normal and malignant tissues. The Prostate. 55:111–117, 2003. 428. Gurel B, Ali TZ, Montgomery EA, et al: NKX3.1 as a marker of prostatic origin in metastatic tumors. Am J Surg Pathol. 34:1097– 1105, 2010.
429. Zhou M, Chinnaiyan AM, Kleer CG, et al: Alpha-MethylacylCoA racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions. Am J Surg Pathol. 26:926–931, 2002. 430. Jiang Z, Fanger GR, Woda BA, et al: Expression of alphamethylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: astudy of 761 cases. Hum Pathol. 34:792–796, 2003. 431. Moll R, Wu XR, Lin JH, et al: Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas. Am J Pathol. 147:1383– 1397, 1995. 432. Wu RL, Osman I, Wu XR, et al: Uroplakin II gene is expressed in transitional cell carcinoma but not in bilharzial bladder squamous cell carcinoma: alternative pathways of bladder epithelial differentiation and tumor formation. Cancer Res. 58:1291–1297, 1998. 433. Parker DC, Folpe AL, Bell J, et al: Potential utility of uroplakin III, thrombomodulin, high molecular weight cytokeratin, and cytokeratin 20 in noninvasive, invasive, and metastatic urothelial (transitional cell) carcinomas. Am J Surg Pathol. 27:1–10, 2003. 434. Appleton MA, Attanoos RL, Jasani B: Thrombomodulin as a marker of vascular and lymphatic tumours. Histopathology. 29:153–157, 1996. 435. Yonezawa S, Maruyama I, Sakae K, et al: Thrombomodulin as a marker for vascular tumors. Comparative study with factor VIII and Ulex europaeus I lectin. Am J Clin Pathol. 88:405–411, 1987. 436. Lager DJ, Callaghan EJ, Worth SF, et al: Cellular localization of thrombomodulin in human epithelium and squamous malignancies. Am J Pathol. 146:933–943, 1995. 437. Kushitani K, Takeshima Y, Amatya VJ, et al: Immunohistochemical marker panels for distinguishing between epithelioid mesothelioma and lung adenocarcinoma. Pathol Int. 57:190–199, 2007. 438. Jiang J, Ulbright TM, Younger C, et al: Cytokeratin 7 and cytokeratin 20 in primary urinary bladder carcinoma and matched lymph node metastasis. Arch Pathol Lab Med. 125:921–923, 2001. 439. Langner C, Ratschek M, Tsybrovskyy O, et al: P63 immunoreactivity distinguishes upper urinary tract transitional-cell carcinoma and renal-cell carcinoma even in poorly differentiated tumors. J Histochem Cytochem. 51:1097–1099, 2003. 440. Ordonez NG: Value of thrombomodulin immunostaining in the diagnosis of mesothelioma. Histopathology. 31:25–30, 1997. 441. Logani S, Oliva E, Amin MB, et al: Immunoprofile of ovarian tumors with putative transitional cell (urothelial) differentiation using novel urothelial markers: histogenetic and diagnostic implications. Am J Surg Pathol. 27:1434–1441, 2003. 442. Mhawech P, Uchida T, Pelte MF: Immunohistochemical profile of high-grade urothelial bladder carcinoma and prostate adenocarcinoma. Hum Pathol. 33:1136–1140, 2002. 443. Ordonez NG: Transitional cell carcinomas of the ovary and bladder are immunophenotypically different. Histopathology. 36:433–438, 2000. 444. Tong GX, Weeden EM, Hamele-Bena D, et al: Expression of PAX8 in nephrogenic adenoma and clear cell adenocarcinoma of the lower urinary tract: evidence of related histogenesis? Am J Surg Pathol. 32:1380–1387, 2008. 445. Tong GX, Yu WM, Beaubier NT, et al: Expression of PAX8 in normal and neoplastic renal tissues: an immunohistochemical study. Mod Pathol. 22:1218–1227, 2009. 446. Yoshida SO, Imam A: Monoclonal antibody to a proximal nephrogenic renal antigen: immunohistochemical analysis of formalin-fixed, paraffin-embedded human renal cell carcinomas. Cancer Res. 49:1802–1809, 1989. 447. McGregor DK, Khurana KK, Cao C, et al: Diagnosing primary and metastatic renal cell carcinoma: the use of the monoclonal antibody “Renal Cell Carcinoma Marker”. Am J Surg Pathol. 25:1485–1492, 2001. 448. Bakshi N, Kunju LP, Giordano T, et al: Expression of renal cell carcinoma antigen (RCC) in renal epithelial and nonrenal tumors: diagnostic Implications. Appl Immunohistochem Mol Morphol. 15:310–315, 2007. 449. Chu P, Arber DA: Paraffin-section detection of CD10 in 505 nonhematopoietic neoplasms. Frequent expression in renal cell
References carcinoma and endometrial stromal sarcoma. Am J Clin Pathol. 113:374–382, 2000. 450. Tellier AL, Amiel J, Delezoide AL, et al: Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome. Am J Med Genet. 93:85–88, 2000. 451. Mazal PR, Stichenwirth M, Koller A, et al: Expression of aquaporins and PAX-2 compared to CD10 and cytokeratin 7 in renal neoplasms: a tissue microarray study. Mod Pathol. 18:535–540, 2005. 452. Memeo L, Jhang J, Assaad AM, et al: Immunohistochemical analysis for cytokeratin 7, KIT, and PAX2: value in the differential diagnosis of chromophobe cell carcinoma. Am J Clin Pathol. 127:225–229, 2007. 453. Tong GX, Chiriboga L, Hamele-Bena D, et al: Expression of PAX2 in papillary serous carcinoma of the ovary: immunohistochemical evidence of fallopian tube or secondary Mullerian system origin? Mod Pathol. 20:856–863, 2007. 454. Chivukula M, Dabbs DJ, O’Connor S, et al: PAX 2: a novel Mullerian marker for serous papillary carcinomas to differentiate from micropapillary breast carcinoma. Int J Gynecol Pathol. 28:570–578, 2009. 455. Laury AR, Perets R, Piao H, et al: A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 35:816–826, 2011. 456. Nonaka D, Tang Y, Chiriboga L, et al: Diagnostic utility of thyroid transcription factors Pax8 and TTF-2 (FoxE1) in thyroid epithelial neoplasms. Mod Pathol. 21:192–200, 2008. 457. Long KB, Srivastava A, Hirsch MS, et al: PAX8 Expression in well-differentiated pancreatic endocrine tumors: correlation with clinicopathologic features and comparison with gastrointestinal and pulmonary carcinoid tumors. Am J Surg Pathol. 34:723–729, 2010. 458. Weissferdt A, Moran CA: Pax8 expression in thymic epithelial neoplasms: an immunohistochemical analysis. Am J Surg Pathol. 35:1305–1310, 2011. 459. Albadine R, Schultz L, Illei P, et al: PAX8 (+)/p63 (−) immunostaining pattern in renal collecting duct carcinoma (CDC): a useful immunoprofile in the differential diagnosis of CDC versus urothelial carcinoma of upper urinary tract. Am J Surg Pathol. 34:965–969, 2010. 460. Ozcan A, Liles N, Coffey D, et al: PAX2 and PAX8 expression in primary and metastatic mullerian epithelial tumors: a comprehensive comparison. Am J Surg Pathol. 35:1837–1847, 2011. 461. Woodard AH, Yu J, Dabbs DJ, et al: NY-BR-1 and PAX8 immunoreactivity in breast, gynecologic tract, and other CK7+ carcinomas: potential use for determining site of origin. Am J Clin Pathol. 136:428–435, 2011. 462. Ordonez NG: Value of PAX 8 immunostaining in tumor diagnosis: a review and update. Adv Anat Pathol. 19:140–151, 2012. 463. Avery AK, Beckstead J, Renshaw AA, et al: Use of antibodies to RCC and CD10 in the differential diagnosis of renal neoplasms. Am J Surg Pathol. 24:203–210, 2000. 464. Cameron RI, Ashe P, O’Rourke DM, et al: A panel of immunohistochemical stains assists in the distinction between ovarian and renal clear cell carcinoma. Int J Gynecol Pathol. 22:272–276, 2003. 465. Fetsch PA, Powers CN, Zakowski MF, et al: Anti-alpha-inhibin: marker of choice for the consistent distinction between adrenocortical carcinoma and renal cell carcinoma in fine-needle aspiration. Cancer. 87:168–172, 1999. 466. Ghorab Z, Jorda M, Ganjei P, et al: Melan A (A103) is expressed in adrenocortical neoplasms but not in renal cell and hepatocellular carcinomas. Appl Immunohistochem Mol Morphol. 11:330– 333, 2003. 467. Murakata LA, Ishak KG, Nzeako UC: Clear cell carcinoma of the liver: a comparative immunohistochemical study with renal clear cell carcinoma. Mod Pathol. 13:874–881, 2000. 468. Meunier H, Rivier C, Evans RM, et al: Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci U S A. 85:247–251, 1988. 469. Busam KJ, Iversen K, Coplan KA, et al: Immunoreactivity for A103, an antibody to melan-A (Mart-1), in adrenocortical and other steroid tumors. Am J Surg Pathol. 22:57–63, 1998.
244.e11
470. Jungbluth AA, Busam KJ, Gerald WL, et al: A103: An antimelan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues. Am J Surg Pathol. 22:595–602, 1998. 471. Busam KJ, Jungbluth AA: Melan-A, a new melanocytic differentiation marker. Adv Anat Pathol. 6:12–18, 1999. 472. Hofbauer GF, Kamarashev J, Geertsen R, et al: Melan A/MART-1 immunoreactivity in formalin-fixed paraffin-embedded primary and metastatic melanoma: frequency and distribution. Melanoma Res. 8:337–343, 1998. 473. Renshaw AA, Granter SR: A comparison of A103 and inhibin reactivity in adrenal cortical tumors: distinction from hepatocellular carcinoma and renal tumors. Mod Pathol. 11:1160–1164, 1998. 474. Loy TS, Phillips RW, Linder CL: A103 immunostaining in the diagnosis of adrenal cortical tumors: an immunohistochemical study of 316 cases. Arch Pathol Lab Med. 126:170–172, 2002. 475. Shin SJ, Hoda RS, Ying L, et al: Diagnostic utility of the monoclonal antibody A103 in fine-needle aspiration biopsies of the adrenal. Am J Clin Pathol. 113:295–302, 2000. 476. Sasano H, Shizawa S, Suzuki T, et al: Transcription factor adrenal 4 binding protein as a marker of adrenocortical malignancy. Hum Pathol. 26:1154–1156, 1995. 477. Sasano H, Suzuki T, Moriya T: Recent advances in histopathology and immunohistochemistry of adrenocortical carcinoma. Endocr Pathol. 17:345–354, 2006. 478. Fogel M, Lifschitz-Mercer B, Moll R, et al: Heterogeneity of intermediate filament expression in human testicular seminomas. Differentiation. 45:242–249, 1990. 479. Giwercman A, Andrews PW, Jorgensen N, et al: Immunohistochemical expression of embryonal marker TRA-1-60 in carcinoma in situ and germ cell tumors of the testis. Cancer. 72:1308–1314, 1993. 480. Giwercman A, Marks A, Bailey D, et al: A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis. Apmis. 96:667–670, 1988. 481. Jacobsen GK, Norgaard-Pedersen B: Placental alkaline phosphatase in testicular germ cell tumours and in carcinoma-in-situ of the testis. An immunohistochemical study. Acta Pathol Microbiol Immunol Scand [A]. 92:323–329, 1984. 482. Burke AP, Mostofi FK: Placental alkaline phosphatase immunohistochemistry of intratubular malignant germ cells and associated testicular germ cell tumors. Hum Pathol. 19:663–670, 1988. 483. Cummings OW, Ulbright TM, Eble JN, et al: Spermatocytic seminoma: an immunohistochemical study. Hum Pathol. 25:54– 59, 1994. 484. Bailey D, Marks A, Stratis M, et al: Immunohistochemical staining of germ cell tumors and intratubular malignant germ cells of the testis using antibody to placental alkaline phosphatase and a monoclonal anti-seminoma antibody. Mod Pathol. 4:167– 171, 1991. 485. Hustin J, Collette J, Franchimont P: Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumours. Int J Androl. 10:29–35, 1987. 486. Niehans GA, Manivel JC, Copland GT, et al: Immunohistochemistry of germ cell and trophoblastic neoplasms. Cancer. 62:1113–1123, 1988. 487. Uchida T, Shimoda T, Miyata H, et al: Immunoperoxidase study of alkaline phosphatase in testicular tumor. Cancer. 48:1455– 1462, 1981. 488. Wick MR, Swanson PE, Manivel JC: Placental-like alkaline phosphatase reactivity in human tumors: an immunohistochemical study of 520 cases. Hum Pathol. 18:946–954, 1987. 489. Watanabe H, Tokuyama H, Ohta H, et al: Expression of placental alkaline phosphatase in gastric and colorectal cancers. An immunohistochemical study using the prepared monoclonal antibody. Cancer. 66:2575–2582, 1990. 490. Mostofi FK, Sesterhenn IA, Davis CJ Jr: Immunopathology of germ cell tumors of the testis. Semin Diagn Pathol. 4:320–341, 1987. 491. Eglen DE, Ulbright TM: The differential diagnosis of yolk sac tumor and seminoma. Usefulness of cytokeratin, alphafetoprotein, and alpha-1-antitrypsin immunoperoxidase reactions. Am J Clin Pathol. 88:328–332, 1987.
244.e12
Immunohistology of Metastatic Carcinoma of Unknown Primary Site
492. Jacobsen GK, Jacobsen M: Alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) in testicular germ cell tumours. A prospective immunohistochemical study. Acta Pathol Microbiol Immunol Scand [A]. 91:165–176, 1983. 493. Cheng L, Thomas A, Roth LM, et al: OCT4: a novel biomarker for dysgerminoma of the ovary. Am J Surg Pathol. 28:1341–1346, 2004. 494. Jones TD, Ulbright TM, Eble JN, et al: OCT4 staining in testicular tumors: a sensitive and specific marker for seminoma and embryonal carcinoma. Am J Surg Pathol. 28:935–940, 2004. 495. Hattab EM, Tu PH, Wilson JD, et al: OCT4 immunohistochemistry is superior to placental alkaline phosphatase (PLAP) in the diagnosis of central nervous system germinoma. Am J Surg Pathol. 29:368–371, 2005. 496. Cao D, Guo S, Allan RW, et al: SALL4 is a novel sensitive and specific marker of ovarian primitive germ cell tumors and is particularly useful in distinguishing yolk sac tumor from clear cell carcinoma. Am J Surg Pathol. 33:894–904, 2009. 497. Cao D, Humphrey PA, Allan RW: SALL4 is a novel sensitive and specific marker for metastatic germ cell tumors, with particular utility in detection of metastatic yolk sac tumors. Cancer. 115:2640–2651, 2009. 498. Cao D, Li J, Guo CC, et al: SALL4 is a novel diagnostic marker for testicular germ cell tumors. Am J Surg Pathol. 33:1065–1077, 2009. 499. Camparo P, Comperat EM: SALL4 is a useful marker in the diagnostic work-up of germ cell tumors in extra-testicular locations. Virchows Arch. 2012. 500. Miettinen M, Lasota J: KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol. 13:205–220, 2005. 501. Miettinen M, Sobin LH, Sarlomo-Rikala M: Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with a reference to CD117 (KIT). Mod Pathol. 13:1134–1142, 2000. 502. Al-Shamkhani A: The role of CD30 in the pathogenesis of haematopoietic malignancies. Curr Opin Pharmacol. 4:355–359, 2004. 503. Grammatico D, Grignon DJ, Eberwein P, et al: Transitional cell carcinoma of the renal pelvis with choriocarcinomatous differentiation. Immunohistochemical and immunoelectron microscopic assessment of human chorionic gonadotropin production by transitional cell carcinoma of the urinary bladder. Cancer. 71:1835–1841, 1993. 504. Hishima T, Fukayama M, Fujisawa M, et al: CD5 expression in thymic carcinoma. Am J Pathol. 145:268–275, 1994. 505. Berezowski K, Grimes MM, Gal A, et al: CD5 immunoreactivity of epithelial cells in thymic carcinoma and CASTLE using paraffin-embedded tissue. Am J Clin Pathol. 106:483–486, 1996. 506. Dorfman DM, Shahsafaei A, Chan JK: Thymic carcinomas, but not thymomas and carcinomas of other sites, show CD5 immunoreactivity. Am J Surg Pathol. 21:936–940, 1997. 507. Kornstein MJ, Rosai J: CD5 labeling of thymic carcinomas and other nonlymphoid neoplasms. Am J Clin Pathol. 109:722–726, 1998. 508. Tateyama H, Eimoto T, Tada T, et al: Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma. Am J Clin Pathol. 111:235–240, 1999. 509. Kuo TT, Chan JK: Thymic carcinoma arising in thymoma is associated with alterations in immunohistochemical profile. Am J Surg Pathol. 22:1474–1481, 1998. 510. Suster S, Moran CA: Spindle cell thymic carcinoma: clinicopathologic and immunohistochemical study of a distinctive variant of primary thymic epithelial neoplasm. Am J Surg Pathol. 23:691–700, 1999. 511. Lau SK, Weiss LM, Chu PG: Differential expression of MUC1, MUC2, and MUC5AC in carcinomas of various sites: an immunohistochemical study. Am J Clin Pathol. 122:61–69, 2004. 512. Hammar SP: Macroscopic, histologic, histochemical, immunohistochemical, and ultrastructural features of mesothelioma. Ultrastruct Pathol. 30:3–17, 2006. 513. Kodama T, Watanabe S, Sato Y, et al: An immunohistochemical study of thymic epithelial tumors. I. Epithelial component. Am J Surg Pathol. 10:26–33, 1986.
514. Fukai I, Masaoka A, Hashimoto T, et al: The distribution of epithelial membrane antigen in thymic epithelial neoplasms. Cancer. 70:2077–2081, 1992. 515. Truong LD, Mody DR, Cagle PT, et al: Thymic carcinoma. A clinicopathologic study of 13 cases. Am J Surg Pathol. 14:151– 166, 1990. 516. Chan JK, Tsang WY, Seneviratne S, et al: The MIC2 antibody 013. Practical application for the study of thymic epithelial tumors. Am J Surg Pathol. 19:1115–1123, 1995. 517. Pomplun S, Wotherspoon AC, Shah G, et al: Immunohistochemical markers in the differentiation of thymic and pulmonary neoplasms. Histopathology. 40:152–158, 2002. 518. Bejarano PA, Nikiforov YE, Swenson ES, et al: Thyroid transcription factor-1, thyroglobulin, cytokeratin 7, and cytokeratin 20 in thyroid neoplasms. Appl Immunohistochem Mol Morphol. 8:189– 194, 2000. 519. Ordonez NG, El-Naggar AK, Hickey RC, et al: Anaplastic thyroid carcinoma. Immunocytochemical study of 32 cases. Am J Clin Pathol. 96:15–24, 1991. 520. Weidner N: Germ-cell tumors of the mediastinum. Semin Diagn Pathol. 16:42–50, 1999. 521. Suster S, Moran CA: Applications and limitations of immunohistochemistry in the diagnosis of malignant mesothelioma. Adv Anat Pathol. 13:316–329, 2006. 522. Cossu-Rocca P, Jones TD, Roth LM, et al: Cytokeratin and CD30 expression in dysgerminoma. Hum Pathol. 37:1015–1021, 2006. 523. Moran CA, Suster S, Przygodzki RM, et al: Primary germ cell tumors of the mediastinum: II. Mediastinal seminomas—a clinicopathologic and immunohistochemical study of 120 cases. Cancer. 80:691–698, 1997. 524. Ulbright TM: Germ cell tumors of the gonads: a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod Pathol. 18(Suppl 2):S61–S79, 2005. 525. Cheng L: Establishing a germ cell origin for metastatic tumors using OCT4 immunohistochemistry. Cancer. 101:2006–2010, 2004. 526. Sung MT, Jones TD, Beck SD, et al: OCT4 is superior to CD30 in the diagnosis of metastatic embryonal carcinomas after chemotherapy. Hum Pathol. 37:662–667, 2006. 527. Bosl GJ, Ilson DH, Rodriguez E, et al: Clinical relevance of the i(12p) marker chromosome in germ cell tumors. J Natl Cancer Inst. 86:349–355, 1994. 528. Kernek KM, Brunelli M, Ulbright TM, et al: Fluorescence in situ hybridization analysis of chromosome 12p in paraffin-embedded tissue is useful for establishing germ cell origin of metastatic tumors. Mod Pathol. 17:1309–1313, 2004. 529. Elishaev E, Gilks CB, Miller D, et al: Synchronous and metachronous endocervical and ovarian neoplasms: evidence supporting interpretation of the ovarian neoplasms as metastatic endocervical adenocarcinomas simulating primary ovarian surface epithelial neoplasms. Am J Surg Pathol. 29:281–294, 2005. 530. Nonaka D, Kusamura S, Baratti D, et al: CDX-2 expression in pseudomyxoma peritonei: a clinicopathological study of 42 cases. Histopathology. 49:381–387, 2006. 531. Chu PG, Chung L, Weiss LM, et al: Determining the site of origin of mucinous adenocarcinoma: an immunohistochemical study of 175 cases. Am J Surg Pathol. 35:1830–1836, 2011. 532. Tabrizi AD, Kalloger SE, Kobel M, et al: Primary ovarian mucinous carcinoma of intestinal type: significance of pattern of invasion and immunohistochemical expression profile in a series of 31 cases. Int J Gynecol Pathol. 29:99–107, 2010. 533. Baker PM, Oliva E: Immunohistochemistry as a tool in the differential diagnosis of ovarian tumors: an update. Int J Gynecol Pathol. 24:39–55, 2005. 534. Deavers MT, Malpica A, Liu J, et al: Ovarian sex cord-stromal tumors: an immunohistochemical study including a comparison of calretinin and inhibin. Mod Pathol. 16:584–590, 2003. 535. Ashikari R, Park K, Huvos AG, et al: Paget’s disease of the breast. Cancer. 26:680–685, 1970. 536. Kister SJ, Haagensen CD: Paget’s disease of the breast. Am J Surg. 119:606–609, 1970. 537. Salvadori B, Fariselli G, Saccozzi R: Analysis of 100 cases of Paget’s disease of the breast. Tumori. 62:529–535, 1976.
References 538. Lundquist K, Kohler S, Rouse RV: Intraepidermal cytokeratin 7 expression is not restricted to Paget cells but is also seen in Toker cells and Merkel cells. Am J Surg Pathol. 23:212–219, 1999. 539. Yao DX, Hoda SA, Chiu A, et al: Intraepidermal cytokeratin 7 immunoreactive cells in the non-neoplastic nipple may represent interepithelial extension of lactiferous duct cells. Histopathology. 40:230–236, 2002. 540. Tani EM, Skoog L: Immunocytochemical detection of estrogen receptors in mammary Paget cells. Acta Cytol. 32:825–828, 1988. 541. Meissner K, Riviere A, Haupt G, et al: Study of neu-protein expression in mammary Paget’s disease with and without underlying breast carcinoma and in extramammary Paget’s disease. Am J Pathol. 137:1305–1309, 1990. 542. Ramaswamy S, Tamayo P, Rifkin R, et al: Multiclass cancer diagnosis using tumor gene expression signatures. Proc Natl Acad Sci U S A. 98:15149–15154, 2001. 543. Bloom G, Yang IV, Boulware D, et al: Multi-platform, multi-site, microarray-based human tumor classification. Am J Pathol. 164:9–16, 2004. 544. Buckhaults P, Zhang Z, Chen YC, et al: Identifying tumor origin using a gene expression-based classification map. Cancer Res. 63:4144–4149, 2003. 545. Shedden KA, Taylor JM, Giordano TJ, et al: Accurate molecular classification of human cancers based on gene expression using a simple classifier with a pathological tree-based framework. Am J Pathol. 163:1985–1995, 2003. 546. Talantov D, Baden J, Jatkoe T, et al: A quantitative reverse transcriptase-polymerase chain reaction assay to identify metastatic carcinoma tissue of origin. J Mol Diagn. 8:320–329, 2006. 547. Tothill RW, Kowalczyk A, Rischin D, et al: An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin. Cancer Res. 65:4031–4040, 2005. 548. Ma XJ, Patel R, Wang X, et al: Molecular classification of human cancers using a 92-gene real-time quantitative polymerase chain reaction assay. Arch Pathol Lab Med. 130:465–473, 2006. 549. Horlings HM, van Laar RK, Kerst JM, et al: Gene expression profiling to identify the histogenetic origin of metastatic
244.e13
adenocarcinomas of unknown primary. J Clin Oncol. 26:4435– 4441, 2008. 550. Monzon FA, Dumur CI, Lyons-Weiler M, et al: Validation of a gene expression-based tissue of origin test applied to poorly differentiated and undifferentiated cancers. Association for Molecular Pathology. 2007, Annual Meeting:Abstract. 551. Dumur CI, Lyons-Weiler M, Sciulli C, et al: Interlaboratory performance of a microarray-based gene expression test to determine tissue of origin in poorly differentiated and undifferentiated cancers. J Mol Diagn. 10:67–77, 2008. 552. Landgraf P, Rusu M, Sheridan R, et al: A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 129:1401–1414, 2007. 553. Lu J, Getz G, Miska EA, et al: MicroRNA expression profiles classify human cancers. Nature. 435:834–838, 2005. 554. Rosenfeld N, Aharonov R, Meiri E, et al: MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol. 26:462–469, 2008. 555. Varadhachary GR, Spector Y, Abbruzzese JL, et al: Prospective gene signature study using microRNA to identify the tissue of origin in patients with carcinoma of unknown primary. Clin Cancer Res. 17:4063–4070, 2011. 556. Meiri E, Mueller WC, Rosenwald S, et al: A second-generation microRNA-based assay for diagnosing tumor tissue origin. Oncologist. 17:801–812, 2012. 557. Hainsworth JD, Rubin MS, Spigel DR, et al: Molecular Gene Expression Profiling to Predict the Tissue of Origin and Direct Site-Specific Therapy in Patients With Carcinoma of Unknown Primary Site: A Prospective Trial of the Sarah Cannon Research Institute. J Clin Oncol. 2012. 558. Chiang WM, Kapadia M, Laver NV, et al: Cancer of unknown primary: from immunohistochemistry to gene expression profiling. J Clin Oncol. 30:e300–e302, 2012. 559. Liu J, Shi J, Wilkerson ML, et al: Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol. 138:57–61, 2012.
C H A P T E R 9
IMMUNOHISTOLOGY OF HEAD AND NECK LESIONS LESTER D.R. THOMPSON
Overview 245 Squamoproliferative Lesions 245 Nasal Cavity and Paranasal Sinuses 254 Nasopharynx 278 Oral Cavity 284 Larynx/Hypopharynx 287 Salivary Glands 290 Ear and Temporal Bone 310 Metastatic Tumors 319 Summary 321
reactions similar to skin melanoma. One of the most commonly used antibodies in the head and neck is cytokeratin (CK) stains. Three antibodies are particularly useful in head and neck tumors and deserve special mention. The first of these, p63, represents a group of different isotypes of a protein that are homologous to p53.1 This protein product will highlight squamous epithelia and is also an excellent marker for myoepithelial/ basal cells. As a surrogate marker for human papilloma virus (HPV) for oropharyngeal carcinoma specifically, p16 has gained popularity, but it is not specific to this tumor. Androgen receptor (AR) may help with the differential diagnosis of salivary duct carcinoma, metastatic prostate carcinoma, or selected sebaceous neoplasms of salivary glands or skin.
Squamoproliferative Lesions Overview The head and neck, by convention defined as the area above the clavicles and below the cranial cavity, is an anatomically complex region composed of a heterogeneous array of tissues and organs. Among the various tissues are mucosal surface epithelia, salivary glands, odontogenic apparatus, bone, cartilage, soft tissues, peripheral and central nervous systems, paraganglia, lymphoid tissue, endocrine organs, and skin. The latter three are discussed elsewhere in this volume. This chapter focuses on unique head and neck lesions or lesions in which immunohistochemistry (IHC) and/or molecular workup may be useful for establishing the diagnosis or for providing additional prognostic or predictive information. It is not intended to be an exhaustive catalog of all head and neck neoplasms, especially not those recognized by routine hematoxylin and eosin (H&E)–stained slides, except as they may warrant in differential diagnosis. Nearly all types of processes and neoplasms may develop in the head and neck, and a consequently broad spectrum of antigens and antibodies are used in diagnosis (Table 9-1). For example, mucosal melanoma is a unique tumor, but S-100 protein, human melanoma black 45 (HMB-45), tyrosinase, and/or melan-A have
Reactive Changes Reactive epithelial changes can have a variety of histologic patterns and cytomorphonuclear features. However, no IHC stains help identify or classify reactive changes, including pseudoepitheliomatous hyperplasia, nor are they able to reliably distinguish them from dysplasia or neoplasia.
Dysplasia and Conventional Squamous Cell Carcinoma Squamous cell carcinoma (SCC) is the most common malignancy to arise in the head and neck.2,3 Invasive SCC tends to occur in the sixth decade of life or later and generally has a strong male predominance. Carcinogenesis is directly related to tobacco and/or alcohol in the vast majority of cases,4-6 although a viral etiology for specific tumor types is well recognized. Epstein-Barr virus (EBV) is related to Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma (see the “Nasopharynx” section), and even leiomyosarcoma. A strong association is evident between HPV and squamous papilloma, oropharyngeal carcinoma (tonsil specifically), and some oral carcinomas.7-13 In the uterine cervix, HPV-infected dysplastic cells tend to express p16ink4a as 245
246
Immunohistology of Head and Neck Lesions
TABLE 9-1 General List of Antibodies Used in the Evaluation of Head and Neck Specimens Antibody
Source
Dilution
34βE12 (HMWK; K903)
Sigma
1 : 20
α-Amylase (G-10)
Santa Cruz Biotechnology
1 : 250
Adrenocorticotropic hormone (ACTH) (02A3)
Dako
1 : 4000
Actin (HHF35; muscle specific)
Enzo Life Sciences
1 : 100
Androgen receptor
Biogenex
1 : 2000
Bcl-2 (124)
Dako
1 : 200
β-Catenin (14)
BD Transduction Laboratories
1 : 4000
Brachyury (C-19)
Santa Cruz Biotechnology
1 : 100
BRST-2 (GCDFP-15, D6)
Covance
1 : 50
Calcitonin (polyclonal)
Fisher/Biomedical
1 : 8
Calponin
Dako
1 : 200
Calretinin (polyclonal)
Zymed
1 : 750
CAM5.2 (CK7/8)
Covance
1 : 8
CD1a (O10)
Beckman-Coulter
1 : 4
CD31
Dako
1 : 40
CD34
Dako
1 : 800
CD45RB (leukocyte common antigen)
Dako
1 : 20
CD56 (AB-2, 123C3.D5)
Lab Vision/NeoMarkers
Neat
CD57 (Leu-7, HNK-1)
Pharmagen
1 : 600
CD68 (PG-M1)
Ventana
Neat
CD79a (HM57)
Dako
1 : 200
CD99 (O13)
Signet Laboratories
1 : 400
CD117 (c-Kit)
Dako
1 : 400
CD138 (BC/B-B4)
BioCare
1 : 800
CD163 (10D6)
Leica
1 : 800
CD207 (Langerin, 12D6)
Leica
1 : 200
CDX-2 (CDX-2/88)
Bio Genex
1 : 50
Carcinoembryonic antigen (CEA)
Boehringer-Mannheim
1 : 4000
Chromogranin-A (LK2H10)
Ventana
Neat
Pancytokeratin (AE1/AE3)
Dako
1 : 40
CK4
Novocastra
1 : 100
CK5/6 (D5/16 B4)
Dako
1 : 25
CK7 (OV-TL-12/30)
Dako
1 : 200
CK8
Novocastra
1 : 60
CK10
Novocastra
1 : 50
CK13
Dako
1 : 100
CK14
Novocastra
1 : 40
CK19
Novocastra
1 : 50
CK20 (KS20.8)
Ventana
Neat
Desmin
Biogenex
1 : 2000
EBV RNA in situ hybridization
Ventana
Neat
Squamoproliferative Lesions
247
TABLE 9-1 General List of Antibodies Used in the Evaluation of Head and Neck Specimens—cont’d Antibody
Source
Dilution
E-cadherin (36B5)
Leica
1 : 50
Epidermal growth factor receptor (31G7)
Invitrogen
1 : 100
Epithelial membrane antigen (EMA) (E29)
Ventana
Neat
Fli-1
Santa Cruz Biotechnology
1 : 40
Factor VIIIRAg (F8/86)
Dako
1 : 25
Follicle-stimulating hormone (FSH) (C10)
Dako
1 : 50
Glial fibrillary acidic protein (GFAP) (6F2)
Dako
1 : 300
Growth hormone (GH) (GH-45)
Novus Biologicals
1 : 2000
HER-2/neu
Dako
1 : 100
Human herpes virus 8 (HHV8)
Dako
1 : 50
Human melanoma black 45 (HMB-45)
Biogenex
1 : 60
Human papilloma virus (HPV) (ISH)
Inform HPV (Family 6 or 16 Probes)
Neat
Ki-67 (MIB1)
Dako
1 : 100
Laminin
Sigma
1 : 20
Luteinizing hormone (LH) (C93)
Dako
1 : 600
MCM2 (N-19; polyclonal)
Santa Cruz Biotechnology
1 : 400
Melan-A
Novocastra
1 : 40
Microphthalmic transcription factor (MIFT)
Neomarkers
1 : 40
Muscle-specific actin (HHF35)
Enzo
1 : 8000
MUC1 (VU-4-H5)
Invitrogen
1 : 4800
MUC2
Novocastra
1 : 100
MUC4
Novocastra
1 : 200
MUC5
Novocastra
1 : 150
MyoD1 (5.8A)
Dako
1 : 900
Myogenin
Novocastra
1 : 30
Neurofilament protein (NFP) (2F11)
Dako
Neat
Neuron-specific enolase (NSE) (BBS-NC-V1)
Ventana
Neat
NUTM1 antibody
Cell Signaling Technologies
Neat
p16 (3G5D5)
MTM Laboratories (Ventana, Roche)
Neat
p27 (SX53G8)
Dako
1 : 250
p53 (DO-7)
Dako
Neat
p63 (7jul)
Leica Microsystems
1 : 40
Pax-2
LifeSpan BioSciences
1 : 250
Pax-8
LifeSpan BioSciences
1 : 100
PiT-1
Santa Cruz Biotechnology
1 : 200
Prolactin
Thermolife Scientific
1 : 1500
Parathyroid hormone (PTH) (MRQ-31)
CellMarque
1 : 150
S-100 protein (polyclonal)
Dako
1 : 2000
Smooth muscle actin (sma-1)
Leica
1 : 200
Smooth-muscle myosin heavy chain (SMMHC) (SMMS-1)
Dako
1 : 100 Continued
248
Immunohistology of Head and Neck Lesions
TABLE 9-1 General List of Antibodies Used in the Evaluation of Head and Neck Specimens—cont’d Antibody
Source
Dilution
Synaptophysin
Ventana
Neat
Thyroglobulin (mono 2Hii/6EI)
Dako
1 : 32,000
Thyroid stimulating hormone (TSH) (QB2/6)
Leica Microsystems
1 : 400
TFE3 (MRQ-37)
Cell Marque
1 : 200
TLE1 (polyclonal)
Santa Cruz Biotechnology
1 : 50
Thyroid transcription factor 1 (TTF-1)
Neomarkers
1 : 50
Tyrosinase
Novocastra
1 : 20
Villin (1D2C3)
Bilcare
1 : 75
Vimentin
Biogenex
1 : 20
Wilms tumor 1 (WT-1) (6F-H2)
Dako
1 : 200
a surrogate marker for HPV presence in the genome,14-16 and this has also been seen in studies of HPV-related head and neck SCC.12,13,17-19 However, in the head and neck, the p16 gene may also function as a tumor suppressor gene, and therefore altered expression does not absolutely correlate with the presence of HPV.20-22 IHC stains for HPV are not very sensitive for detection of the virus. In situ hybridization (ISH) detects integrated HPV genomic material, which is considered to be important to suggest a causative relationship.8,21 Polymerase chain reaction (PCR)-based assays only detect the presence of the HPV DNA in general; this technique is highly sensitive (perhaps overly sensitive), but information about the cellular localization is lost when PCR is used.23 When patients come to medical attention with metastatic SCC in the neck, often a primary tumor cannot be found despite exhaustive clinical and imaging studies. In these cases, p16 or ISH for HPV can be useful, because so many of these tumors are from the tonsil and tongue-base area.11,12,19,24-26 When HPV is positive in the neck nodes from a patient with an unknown primary tumor, the clinical management can be directed toward these high-risk areas. Dysplastic and neoplastic transformation of the squamous mucosa is typically classified into four basic categories: 1) mild dysplasia, 2) moderate dysplasia, 3) severe dysplasia/carcinoma in situ, and 4) invasive carcinoma. The cytologic and architectural features of dysplasia are quite characteristic, although with high interobserver and intraobserver variability. In general, dysplasia shows architectural disorganization, thickening of the parabasal zones, lack of maturation with irregular perpendicular to parallel rotation, loss of polarity, bulbous or pointed rete, abnormal keratinization, even spongiosis, increased mitoses above the parabasal zone, and atypical mitoses. The cells show a similar size to the basal zone but have abnormal keratinization—glassy cytoplasm, dyskeratosis, karyorrhectic keratinization, surface keratinization, paradoxical keratinization—along with nuclear irregularities that include pleomorphism, hyperchromasia, and streaming. Classification into mild, moderate, and severe dysplasia/
carcinoma in situ (CIS) is based on progression of these features to involve the whole epithelium, although certain features—such as atypical mitoses, full-thickness pleomorphism, and absent maturation—may still qualify the lesion as high-grade dysplasia when present anywhere in the epithelium.2,27-32 Mutations and overexpression of the p53 gene are common in head and neck SCC, and approximately 50% to 60% of tumors show aberrant p53 expression,33-36 although prognostic significance is not proved.37 However, p53 status may be linked to response to chemoradiation and radiation therapy for SCC.38,39 The intensity and location of both p53 and Ki-67 immunoreactivity has shown promise in grading dysplasia (Fig. 9-1).40,41 However, p53 mutational analysis suggests that DNA alterations precede histologic alterations, but these studies have not yet gained widespread use.42-44 Cyclin D1 amplification and p16 deletion have
Figure 9-1 Squamous epithelium shows a strong and diffuse reaction in the nuclei for p53 (left) and Ki-67 (right). These features can be used to highlight dysplasia or squamous cell carcinoma.
R R
N
Synaptophysin
N
N
CD117
TTF-1
N
N
+ R
N
+ (~50%)
N
N
N
N
N N
N + (myoepithelial)
+
S
+
R
S
R (not peripheral)
N
+
N
N
NR
N +
NR
+ +
S
N
NR N
NR
+ (nuclear)
+ (50%)
N
N
N
N
High
+
NR
+
NR
R (focal)
NR
NR
NR
NR
NR
+
S
N
+
NUTM1 Midline Carcinoma
N
N
N
R
N
N
R
Low
+ (peripheral, myoepithelial)
S
+
+
N
NR
+
NR
NR
+
+
+
+
+
Adenoid Cystic Carcinoma
+
+
N N
N
R
N
N
N
R (focal)
S
S
S
NR
N
N
N N
N
N
High
R (weak)
+
N
+ (punctate/dot)
S
N
NR
NR
NR
+ (nonkeratinizing type)
N
High
+
+
R (focal)
+ (patchy)
N
+
+ (patchy)
N
+
NR
N
R (punctate/dot)
R
+ (punctate/dot)
Small Cell Neuroendocrine Carcinoma
Data from references 30, 36, 56 to 62, and 115. +, Almost always positive; N, negative; S, sometimes positive; R, rarely positive; NR, not reported. CK, Cytokeratin; EMA, epithelial membrane antigen; HLA, human leukocyte antigen; HPV, human papilloma virus; ISH, in situ hybridization; PanCK, pancytokeratin; SMA, smooth muscle actin; TTF-1, thyroid transcription factor 1. *If positive with p16, origin is much more likely to be oropharynx, with only rare hypopharynx and sinonasal tract cases positive.
N
R
N
SMA
S R
+
N
Vimentin
S
N
R
CD56
S-100 protein
R
NR
N
HLA-DR
Chromogranin-A
N N
N
N
CD34
NUTM1 antibody
N
N
+ (50%)
N
N
+
High
S
+ (50%)
+ (50%)
NR
N
+ (~50%)
NR
N
N
N
mCEA
S
N + (patchy)
+ (~50%) +
+
+
+
Nasopharyngeal Carcinoma (Nonkeratinizing Type)
N
N
+
Sinonasal Undifferentiated Carcinoma
N
N
N
HPV (ISH)
p16
EBER (ISH)
S*
+*
Ki-67
+ High
+
High
p63 (nuclear)
+ +
+
+
EMA
p53 (nuclear)
+
+
CAM5.2
N N
+
+
CK14
CK18
N
+ +
+
CK13
CK19
N
+
CK8
CK20
N +
N
+
CK7
+ +
+
+
PanCK (AE1/AE3)
34βE12 (K903)
+
+
Antibody Marker
CK5/6
Basaloid Squamous Cell Carcinoma
Squamous Cell Carcinoma (Usual Type)
Tumor Type
TABLE 9-2 Immunohistochemical Expression for Various Mucosal Upper Aerodigestive Tract Carcinomas
Squamoproliferative Lesions
249
250
Immunohistology of Head and Neck Lesions
been associated with a poor outcome45-47 but are not used in daily practice. Epidermal growth factor receptor (EGFR) is a unique biomarker, and several FDA-approved therapies use this receptor for targeted drug therapy (such as cetuximab).48-50 In head and neck SCC, EGFR overexpression has been identified by IHC,51 and anti-EGFR therapies have been used with limited success.51-54 However, the role for testing EGFR expression or DNA-level somatic mutational analysis before therapy is questionable, because protein overexpression does not yet correlate with response to therapy.55 In poorly differentiated tumors, particularly in metastatic sites, cytokeratin stains may be helpful, because conventional SCC can usually be diagnosed with H&E alone. Typically, head and neck SCCs are positive for the so-called cytokeratin cocktail, AE1/AE3. Cytoplasmic expression of cytokeratins 5, 5/6, 14, and 17 are also frequently found in SCCs, along with nuclear p63 expression.30,36,56-62 Table 9-2 highlights the patterns of CK reactions in various upper aerodigestive tract mucosal primary tumors. Detection of metastatic disease may occasionally require the use of IHC stains in challenging specimens, such as postradiation lymph nodes. Subtle, postradiation residual tumors (primary or in lymph nodes) may show only granulomatous or necrotic tissue without viable tumor. In these cases, CK stains can be helpful to identify tumor within the necrotic deposits. Sentinel lymph node examinations are only rarely performed in head and neck tumors, because lymphatic drainage for most primary mucosal sites is so complex that IHC evaluation of lymph nodes is unreliable.
KEY DIAGNOSTIC POINTS Squamous Cell Carcinoma • Squamous carcinomas are nearly always positive for CK. • Common CK expression in squamous carcinomas includes AE1/AE3 and CKs 5, 5/6, 14, and 17. • Nuclear p63 expression is common in squamous cell carcinomas but is not specific. • Cytokeratin stains may help detect subtle metastatic foci, especially in posttreatment lymph nodes. • Overexpression of p53 may be linked to response to radiation and/or chemotherapy.
Oropharyngeal Squamous Cell Carcinoma Oropharyngeal squamous cell carcinoma (OPSCC), also referred to as basaloid squamous cell carcinoma (BSCC), is an uncommon, histologically distinct variant of SCC. In the upper aerodigestive tract, it occurs frequently in Waldeyer’s ring—at the base of the tongue and in the tonsils, hypopharynx, palate,7,8,59,63-67 and larynx—but it has also been described in many other locations, such as the buccal mucosa, floor of the
mouth, nasopharynx, nasal cavity, and trachea.30,36,59,68-71 Recently, HPV-related SCCs of the oropharynx specifically have been separated from SCCs of other upper aerodigestive tract sites, because they have a unique clinical and prognostic profile. A strong male bias is reported, and it occurs in slightly younger patients than conventional SCC.66,67,71 Although many patients do not have alcohol or tobacco use histories, these etiologic agents are not excluded in this tumor type. OPSCC can be classified into nonkeratinizing, nonkeratinizing with maturation, and keratinizing histologic types,31,32 and the appearance is biphasic; the basaloid component is dominant with only focal or minor conventional SCC (Fig. 9-2).31,67,72 BSCC usually grows in smooth, contoured lobules; large nests; or trabecular cordlike arrangements of small clusters or single cells. The lobules frequently contain central comedonecrosis with a peripheral palisade of nuclei. Cystic spaces and abortive ducts can be seen, along with a few cases that show basement membrane– type material deposition and a cribriform growth pattern. The conventional SCC component can be focal dyskeratosis; keratin pearl formation; or focal areas of “maturation”; part of invasion of in situ tumor; it can be found in separate foci or may merge with the basaloid component (Fig. 9-3). Cytologically, the cells are round to oval and have hyperchromatic nuclei with a high nuclear/cytoplasmic ratio. Mitoses and apoptotic bodies are often prominent.31,32,67,73-76 The nonkeratinizing type has a very strong association with HPV no matter what detection method is used (Fig. 9-4), but slightly less so with the nonkeratinizing with maturation type and a weak association with the keratinizing type. Transcriptionally active HPV, detected by RNA ISH, is found in as much as 97% of the nonkeratinizing type of SCC (see Fig. 9-4).19,32,77 However, a diffuse (>75% of the cells) strong nuclear and cytoplasmic immunoreactivity in the neoplastic cells with p16 (see Fig. 9-4) or more than a 50% strong and diffuse staining combined with confluent staining (back-to-back cell staining of >25%) is an excellent surrogate marker with a strong predictive value of HPV status and, more specifically, an association with a good prognosis and response to intensity-modulated radiotherapy and/or chemotherapy.26,67,78-80 Interestingly, it seems that HPV-positive p16-positive tumors, no matter what the underlying histologic type, tend to have a better prognosis.26 When poorly differentiated SCC is found in a cervical lymph node in the setting of an unknown primary, detection of p16 and/or HPV with ISH may help direct the search for an oropharyngeal primary.67,81-83 By contrast, BSCC of the larynx and sinonasal tract has a male predominance (4 : 1), and the mean age at diagnosis is approximately 63 years (range 27 to 88 years).30,36,73,76 Several studies have suggested BSCC has a worse prognosis than conventional SCC, especially with a higher risk of lymph node metastases in BSCC.30,36,59,68,73,76,84 BSCCs are positive for AE1/AE3, epithelial membrane antigen (EMA), CK5/6, CAM5.2, and p63; they are sometimes positive with carcinoembryonic antigen (CEA; ~50%) and S-100 protein (~40%) but rarely with
Squamoproliferative Lesions
A
B
C
D
251
Figure 9-2 A panel of illustrations of oropharyngeal squamous cell carcinoma (SCC). A, No maturation, sheetlike growth of cells, high nuclear/cytoplasmic ratio. B, Invasive tumor with slightly more mature appearance on the right. C, A sheetlike distribution of cells with a basaloid phenotype. D, Focal area of keratinization (maturation) in an otherwise undifferentiated SCC.
Figure 9-3 Oropharyngeal squamous cell carcinoma (OPSCC) with areas of maturation (left). High-power magnification shows areas of dyskeratosis, keratin pearl formation, intercellular bridges, and nuclear pleomorphism in this keratinizing OPSCC.
Figure 9-4 Oropharyngeal carcinoma, nonkeratinizing type. Left, Staining with p16 shows a strong, diffuse nuclear and cytoplasmic reaction. Right, Human papilloma virus with dotlike nuclear reaction detected by RNA in situ hybridization.
252
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-5 Basaloid squamous cell carcinoma from the nasal cavity. A, Basaloid neoplastic cells with central comedonecrosis and abrupt keratinization. B, AE1/AE3 shows strong cytoplasmic reaction in the neoplastic cells. C, Cytokeratins 5 and 6 highlight the inner cells more strongly than the outer basaloid cells. D, In this example, p63 highlights the basaloid cells, but this is not a typical reaction because it is usually strong and diffuse in all cells. This shows an example of staining variability.
CD117 and neuroendocrine markers (Fig. 9-5).57,68,69,85-88 The pattern of distribution of p63 between the strong and diffuse reaction in BSCC versus the peripheral “myoepithelial/basal” staining in adenoid cystic carcinoma (ACC) may help distinguish between these tumors.56,70,89 p53 is often strongly positive in BSCC, whereas it is only occasionally seen in the solid type or in a high-grade transformation of ACC.30,36,63,86,90-93 The differential diagnosis for OPSCC is broad, especially in small biopsies, in which all of the histologic features are not appreciated. The major differential diagnosis includes ACC, sinonasal undifferentiated carcinoma, nasopharyngeal carcinoma (nonkeratinizing type), small cell neuroendocrine carcinoma (SCNEC), and NUTM1 midline carcinoma. IHC reaction differences between these tumors, combined with their unique anatomic sites and histologic features, are useful in resolving this differential diagnosis (see Table 9-2).
KEY DIAGNOSTIC POINTS Basaloid Squamous Cell Carcinoma • The major differential diagnosis for BSCC includes adenoid cystic carcinoma, sinonasal undifferentiated carcinoma, nasopharyngeal carcinoma, and small cell neuroendocrine carcinoma. • The best immunohistochemical markers include p63, p53, and neuroendocrine markers. BSCC will be positive for p63 and p53 but will be negative for most neuroendocrine markers.
Spindle Cell Squamous Cell Carcinoma Spindle cell “sarcomatoid” squamous cell carcinoma (SCSCC) is a rare variant of SCC, previously referred to as sarcomatoid carcinoma and carcinosarcoma. The
Squamoproliferative Lesions
mean age at presentation is 65 years (range, 30 to 95 years), with a strong male predominance (12 : 1). Common primary locations of tumors include the glottis (70%), supraglottis, and numerous other head and neck locations. Tobacco and alcohol are the leading risk factors.94-96 Grossly, the vast majority of tumors are polypoid (mean, 2 cm), often with an ulcerated surface. Histologically, SCSCC can be quite difficult to diagnose, particularly on small biopsies. The bulk of the tumor is composed of the spindled component, ranging from hypocellular to hypercellular and from bland to highly atypical (Fig. 9-6). An overlying dysplastic or, more rarely, an invasive SCC component can help suggest the true nature of the tumor. IHC may aid in confirming the diagnosis, but it is important to realize that as many as 30% of cases will be keratin negative.94,95 In general, if keratins are going to be positive in the spindle cells, the best result is with pankeratin (AE1/AE3 and CK1), although EMA, CK1, and CK18 are also variably positive (Figs. 9-7 and 9-8). Vimentin is always positive (see Fig. 9-7), but CK20 and HMB-45 are uniformly
253
Figure 9-6 Spindle cell squamous cell carcinoma. Left, The spindle cell population is immediately adjacent to an area of frank squamous cell carcinoma, showing a keratin pearl formation. Right, This spindle cell proliferation shows remarkable pleomorphism and a large number of mitoses that include many atypical forms.
A
B
C
D
Figure 9-7 The neoplastic spindle cells of a spindle cell squamous cell carcinoma are highlighted by a variety of different markers. A, AE1/ AE3. B, Vimentin. C, Epithelial membrane antigen. D, Cytokeratin 18.
254
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-8 The neoplastic spindle cells of a spindle cell squamous cell carcinoma highlighted by a variety of different markers. A, Cytokeratin 14. B, Cytokeratin 1. C, Cytokeratin 17. D, Smooth muscle actin.
negative. SCSCC can show variable expression with S-100 protein (5%), smooth-muscle actin (30%), actinHHF-35 (15%), CK7 (5%), CK5/6 (7%), CK14 (15%), CK17 (15%; see Fig. 9-8), and p63 (approximately 5%; nuclear reaction); therefore a positive result should not be used to exclude the diagnosis.94-96 The major differential diagnosis for SCSCC includes reactive and benign neoplastic stromal proliferations and rare primary sarcomas of mucosal sites. Benign entities include granulation tissue, lobular capillary hemangioma, contact ulcer, leiomyoma, fibromatosis, and nodular fasciitis. Malignant neoplasms include melanoma, fibrosarcoma, and synovial sarcoma. IHC can be helpful in differentiating SCSCC from some of these lesions, but because of overlapping histologic and immunophenotypes, the final diagnosis will rest on the histologic features and a compatible immunoprofile. Interestingly, SCSCCs that lack IHC staining for epithelial markers have a better prognosis.94
KEY DIAGNOSTIC POINTS Spindle Cell Squamous Cell Carcinoma • The diagnosis of SCSCC requires the identification of a component of squamous neoplasia or immunohistochemical epithelial differentiation of the spindle cells. • Despite the use of multiple cytokeratin and epithelial markers, as many as 30% of SCSCCs will be nonreactive in the spindle cells.
Nasal Cavity and Paranasal Sinuses Many of the tumors of the nasal cavity and paranasal sinuses fall under the category of small round blue cell neoplasms.97-107 Among these, the most important include olfactory neuroblastoma, sinonasal undifferentiated carcinoma, melanoma, SCNEC, lymphoma,
Nasal Cavity and Paranasal Sinuses
extramedullary plasmacytoma, ectopic pituitary adenoma, rhabdomyosarcoma, and Ewing sarcoma/ peripheral neuroectodermal tumor (ES/PNET); these neoplasms are discussed below. Other, less common lesions also occur in this region, but these will be discussed in more detail in other chapters.
Olfactory Neuroblastoma Olfactory neuroblastoma (ONB) is the prototypic small round blue cell tumor of the sinonasal tract. ONB comprises approximately 2% of all sinonasal tract tumors and is thought to arise from the specialized sensory neuroepithelial (neuroectodermal) olfactory cells usually found in the upper part of the nasal cavity, including the superior nasal concha, upper part of septum, roof of the nose, and the cribriform plate of the ethmoid sinus. The normal olfactory epithelium contains three cell types that can be histologically identified in the tumorous counterpart: 1) basal cells, 2) olfactory
255
neurosensory cells, and 3) supporting sustentacular cells. ONB has a bimodal age distribution in the second and sixth decades, although it can be seen over a broad age range (2 to 94 years) and affects both sexes equally. Patients come to medical attention with nonspecific symptoms, and anosmia occurs in less than 5% of cases. A dumbbell-shaped mass that extends across the cribriform plate to involve the intracranial fossa and nasal cavity is a classic imaging finding. Ectopic tumors within the paranasal sinuses are highly exceptional, except in recurrent lesions. The tumor is frequently polypoid.99,102,108,109 The tumor has a lobular architecture irrespective of tumor grade, and is comprised of “primitive” neuroblastoma cells. The circumscribed lobules or nests of tumor are below an intact mucosa and are separated by a vascularized fibrous stroma (Fig. 9-9). Rosettes of Homer Wright (~30% of cases) and Flexner-Wintersteiner types (5%) may be seen. Necrosis and mitoses are uncommon, except in high-grade tumors. The tumor cells are small round blue cells, slightly larger than
A
B
C
D
Figure 9-9 Olfactory neuroblastoma (ONB). A, The lobular architecture is maintained irrespective of the grade of tumor. B, Small pseudorosettes are noted in this lobule of ONB. C, The primitive cells show a high nuclear/cytoplasmic ratio. Note the overlying intact respiratory epithelium. D, Nuclear chromatin is heavy and coarse, but a lobular architecture is noted.
R
p63
R R
N + (dot/punctate)
+ (~50%)
N
R (focal only)
EMA
CAM5.2
R (dot/punctate) N
N + (~50%)
N
N
CK 5/6
CK7
S (20%)
S
R (weak)
N
N
N
N
R
N
N
N
N
N
N
S (up to 50%, focal, weak)
N
N
N
S (up to 50%; weak; punctate/dot)
S
R (focal to diffuse, 20%)
R (<20%)
N
N
R (< 30%)
N
+
N
N
N
+ (80%; dot/ punctate)
N
N
R
N
N
N
+ (dot/punctate)
+
R, focal and weak
PanCK
Large polygonal cells, isolated pleomorphism, inconspicuous mitoses, no necrosis, no neurofibrillary matrix
Small cells, no perineural or vascular invasion, may have pleomorphism, limited mitoses, necrosis can be seen, no neurofibrillar matrix Medium round cells, vacuolated cytoplasm, fine chromatin, scant pleomorphism, easily identified mitoses, necrosis, limited-toabsent lymphovascular invasion, no neurofibrillary matrix, rosettes present Round, strapped, spindled rhabdomyoblasts, primitive, pleomorphism present, variable mitoses, limited necrosis, rare lymphovascular invasion, no neurofibrillary matrix or rosettes
Polymorphous, small to large, folded, cleaved and grooved nuclei, pleomorphism, high mitotic count, necrosis, lymphovascular invasion, no neurofibrillary matrix or rosettes
Large polygonal, epithelioid, rhabdoid, plasmacytoid, spindled cells, pigmented, pleomorphism, high mitotic count, limited necrosis, rare vascular invasion, surface involvement, no neurofibrillary matrix
Small cells, with high nuclear/ cytoplasmic ratio, nuclear molding, nuclei crushed, moderate pleomorphism, inconspicuous nucleoli, high mitotic count, necrosis, no neurofibrillary matrix, pseudorosettes may be present
Medium cells, inconspicuous nucleoli, pleomorphism, high mitotic count, prominent necrosis, lymphovascular invasion, no neurofibrillary stroma, pseudorosettes usually absent
Salt-and-pepper chromatin, small nucleoli (grade dependent), limited mitoses, scant necrosis, neurofibrillary matrix present, pseudorosettes and true rosettes
Morphologic features
Nested, zellballen
Sheets, rosettes, trabecular
Sheets, nests
Paraganglioma
Sheets, alveolarped
Pituitary Adenoma
Diffuse
Ewing Sarcoma/ PNET
Protean
Rhabdomyosarcoma
Syncytial, islands, ribbons sheets
Extranodal NK/T-cell Lymphoma, Nasal Type
Sheets, nests
Lobular
Pattern
Mucosal Melanoma
Olfactory Neuroblastoma
Small Cell Neuroendocrine Carcinoma
Result
Sinonasal Undifferentiated Carcinoma
TABLE 9-3 Immunohistochemical Staining of Small Blue Round Cell Tumors of the Sinonasal Tract
N
EBER (ISH)
N
N
N
NR
N
N
N
R (hormones) N
N
S
N
P
N
N
N N N
N
N
N
N
S (~35%)
N
NR
+
N +
N
+ +
N +
R (up to 15%, focal)
R (up to 20%, focal)
N
S (up to 30%, focal)
N
+(~75%)
N N
N
N
+ N
S
N
+
N
R (weak and focal)
R (sustentacular only)
N
+ (sustentacular only)
N
NR
S
+
+ (membrane)
+
+
S
N
N
N
+
N
N
R (focal, weak)
S (20%)
NR
+ S (~40%)
+
+
+
+
+
R (10%, focal)
R (2%, focal)
S (focal)
+
NR
N
N
N
N
R (focal)
R
N
+
R (up to 20%, weak)
R (up to 30%, weak)
R
NR
N
N
+ N
N
+
N
+ N
R
N
+
N
N
N
N
N
N
N
+
N +
N +
S
N
S
N
N
R
R
R (focal)
N
+
+
+
+
Data from references 58, 98 to 101, 111, 141, 149, and 159. +, Almost always positive; N, negative; S, sometimes positive; R, rarely positive; NR, not reported. CK, Cytokeratin; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; ISH, in situ hybridization; NK, natural killer; NSE, neuron-specific enolase; PanCK, pancytokeratin; PNET, peripheral neuroectodermal tumor; TTF-1, thyroid transcription factor 1. *Pituitary hormones and/or pituitary transcription factors, but may include or peptides and hormones (ADH, oxytocin)
N
N
Pituitary*
TTF-1
N
N
Myogenin
CD117
N
N
N
N
+
Calretinin
N
N
+ (sustentacular only)
GFAP
Vimentin
N
N
HMB-45
CD45RB
S (<15%)
+ (sustentacular only)
S-100 protein
NR N
R
N
50% S (<10%)
+
N
NSE
CD99
FLI-1
S (<10%) R
+ (may be weak
+ (membrane)
Chromogranin
CD56
Calcitonin
S (<15%)
+ (may be weak)
Synaptophysin
258
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-10 A variety of neuroendocrine markers are positive in olfactory neuroblastoma. A, Synaptophysin. B, Neuron-specific enolase. C, CD56. D, Chromogranin.
Figure 9-11 The sustentacular supporting framework Schwannian cells are positive with S-100 protein (left) or glial fibrillary acidic protein (upper right). The neoplastic cells are reactive with calretinin (lower right).
mature lymphocytes, arranged in a syncytium with a high nuclear/cytoplasmic ratio. The nuclei are homogeneously uniform, with hyperchromatic, delicate, uniform “salt-and-pepper” chromatin. The background is formed from a tangle of neuronal processes, giving it a fibrillar quality. As the grade of the neoplasm increases (from grade I to IV), the nuclear features become more pleomorphic, and less stroma is evident.
IHC is more helpful when the tumor is of a higher grade, because the differential diagnosis is more broad (Table 9-3). The tumor cells are positive for neuroendocrine markers that include synaptophysin, neuronspecific enolase (NSE), CD56, neurofilament protein (NFP), and chromogranin (Fig. 9-10).99,105 Reactivity with calretinin is often strong and focal to diffuse (>75% of cells; Fig. 9-11).101 Isolated cells may be positive with
Nasal Cavity and Paranasal Sinuses
AE1/AE3 or CAM5.2 (as many as 30% of cases) but are negative with EMA, muscle markers, CD 99 (MIC2), HMB-45, CD117, and Epstein Barr virus–encoded RNA (EBER).110 Rare cells may be positive with p63.111 The sustentacular support (Schwannian) cells at the periphery of the lobules are reactive with S-100 protein and/or glial fibrillary acidic protein (GFAP; see Fig. 9-11).99,103,104,109 Rare cases may show divergent differentiation that includes ganglion cells, melanin-containing cells, and rhabdomyoblasts; these component cells stain accordingly.112,113 The small round blue cell tumor differential is considered within this spectrum and is discussed with each of the subsequent entities. Although lymphoma of the sinonasal tract is not covered here, natural killer (NK) T-cell and B-cell lymphomas are considered within the differential diagnosis of ONB.
KEY DIAGNOSTIC POINTS Olfactory Neuroblastoma • ONB is composed of small round blue cells that grow in a lobular to diffuse pattern. • The tumor cells are positive for synaptophysin, CD56, NSE, and calretinin; isolated cells may be positive for AE1/AE3. • Supporting peripheral sustentacular cells stain for S-100 protein and GFAP.
259
Sinonasal Undifferentiated Carcinoma Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignant epithelial neoplasm without evidence of squamous or glandular differentiation. It is locally destructive, associated with necrosis, and must be separated from ONB and lymphoepithelial carcinoma. SNUC is more common in men than women (3 : 1) and occurs over a broad age range (mean, sixth decade). The tumor arises within the nasal cavity but quickly spreads to the paranasal sinuses, orbits, and skull base. The tumors are large (mean, >4 cm) fungating masses with ill-defined margins.114-119 Microscopically, SNUC shows several patterns of growth that include lobular, trabecular, and sheetlike growth and also ribbons and solid islands. Confluent tumor necrosis and comedonecrosis are prominent, and bone destruction, lymphovascular invasion, and perineural invasion are easily identified (Fig. 9-12). Surface ulceration often obscures any possible surface involvement. The polygonal cells are medium to large with a high nuclear/cytoplasmic ratio, hyperchromatic to vesicular nuclear chromatin, inconspicuous nucleoli, and usually a syncytial appearance (see Fig. 9-12). Most cases fit within the Western type, but Asian and large cell types are also recognized. No neurofibrillary matrix or true rosettes are present. In some cases, rare isolated areas of abrupt keratinization may be seen, which also raises the differential diagnosis of NUTM1 midline carcinoma.
Figure 9-12 Upper left, Sinonasal undifferentiated carcinoma shows a solid, ribbon, and glandlike pattern of infiltration with an area of central comedonecrosis. Lower left, CAM5.2 highlights the neoplastic cells. Right, Bone destruction is seen in cells with a high nuclear/ cytoplasmic ratio, vesicular nuclear chromatin, and a syncytial appearance.
260
Immunohistology of Head and Neck Lesions
SNUCs are almost always strongly and diffusely positive for AE1/AE3, CK8, CAM5.2 (see Fig. 9-12), and p16 (see Tables 9-2 and 9-3) and are positive with CK7, CK19, EMA, and p53 in approximately 50% of cases (see Fig. 9-12).114,115,117,120 Also, p63 is positive in some cases.117,120 A few cases may also show neuroendocrine marker reactivity, along with CD99 and S-100 protein (<15%), although purists argue that the SNUC designation should only be applied to undifferentiated tumors.117 CD117 is positive, and calretinin and Her-2/neu are negative.121 The Asian type of SNUC may show EBER reactivity,122 but in this setting, separation from secondary sinonasal tract involvement by nasopharyngeal carcinoma (NPC) may be challenging, although NPC is negative for CD117. Curiously, most cases are strongly positive with p16 but lack any HPV DNA expression.120 It may be difficult to separate SNUC from NPC, SCNEC, and NUTM1 midline carcinoma based on histology alone. In most cases, SNUC is positive with AE1/ AE3, CK7, CD117, and p16 and is usually negative with CK5/6, p63, and 34Eβ12; NPC is strongly positive with CK5/6, 34βE12, p63, and EBER, and it is negative with CK7, neuroendocrine markers, p16, and CD117.58,111,114-116,120,123 NUTM1 midline carcinoma is positive with the NUTM1 antibody (nuclear), CD34 (~50% of cases), CK7, and p63, and it is rarely positive for synaptophysin but is negative with CK5/6.61,62 SCNEC tends to show a punctate or dotlike AE1/AE3 and CAM5.2 reaction in addition to neuroendocrine marker reactivity, CD117 reaction, and rare p63 positivity, but it is negative with EBER, EMA, and CK7.
Figure 9-13 One of the most characteristic patterns of melanoma is a peritheliomatous growth (left). A solid, sheetlike appearance that shows many histiocytes raises a broad differential diagnosis in this melanoma (right).
KEY DIAGNOSTIC POINTS Sinonasal Undifferentiated Carcinoma • SNUC is a high-grade tumor with prominent necrosis and mitoses, made up of small- to medium-sized cells. • The tumor cells are positive for AE1/AE3, CD117, and p16, sometimes with CK7 and EMA, and they may show focal staining with neuroendocrine markers.
Figure 9-14 Melanoma has a variety of cell types. Left, An epithelioid appearance with prominent nucleoli and eosinophilic cytoplasm. Right, A plasmacytoid growth of this melanoma, still with prominent nucleoli and several mitoses.
Mucosal Melanoma Mucosal melanomas (MMs) develop from neural crest melanocytes found within the sinonasal tract, eye, oral cavity, and larynx. Within the sinonasal tract, they are uncommon, typically presenting in the fifth to eighth decades without any gender predilection. Most tumors arise on the nasal septum but expand into the paranasal sinuses early, with a mean size of approximately 3 cm.124-126 Surface ulceration may obscure junctional melanocytes, whereas amelanotic, spindled, epithelioid, or solid tumors require a high index of suspicion for diagnosis. The tumor may be solid, organoid, nested, storiform, papillary, fascicular, meningothelial, or distinctly peritheliomatous (Fig. 9-13). The morphologic appearance of the cells is protean and ranges from undifferentiated,
polygonal, epithelioid, small cell, and giant cell to plasmacytoid and rhabdoid; cells will frequently have prominent, brightly eosinophilic nucleoli, intranuclear cytoplasmic inclusions, and opacified cytoplasm (Fig. 9-14). Melanin is helpful when present, and mitoses are usually easily identified, as is tumor necrosis.102,124,125 In general, S-100 protein, HMB-45, tyrosinase, melan-A, microphthalmia transcription factor (MiTF), and KBA are variably positive in MM, although S-100 protein is the most sensitive, and HMB-45 is the most specific (Figs. 9-15 and 9-16; see Table 9-3). In addition, vimentin (100%), FLi-1 (as many as 100% of cases), NSE (focal in <50% of cells), CD117 (~33% of cases), CD99 (~25% of cases), and even CD56 (<10% of cases) are variably positive.119,124,125,127-130 Rarely,
Nasal Cavity and Paranasal Sinuses
A
B
C
D
261
Figure 9-15 Mucosal melanoma is positive with several melanocytic markers, shown here highlighting a spindle cell melanoma. A, S-100 protein. B, HMB-45. C, Tyrosinase. D, Melan-A.
Figure 9-16 A mucosal melanoma with an epithelioid appearance, highlighted with S-100 protein (left) and KBA (right). (Courtesy Dr. M. Miettinen.)
262
Immunohistology of Head and Neck Lesions
CAM5.2 and EMA may show focal positivity.131 In general, two positive melanocytic stains are more reassuring than only one positive reaction, depending on the morphology and clinical setting. The differential diagnosis for MM is quite broad, depending on pattern and cytologic features, and
includes ONB, SNUC, rhabdomyosarcoma, peripheral nerve sheath tumor (PNST; benign or malignant), leiomyosarcoma, melanotic neuroectodermal tumor of infancy, meningioma, plasmacytoma, and mesenchymal or myxoid chondrosarcoma, among others.102,129,132-134 In general, a pertinent IHC panel can be ordered based on the anatomic site, age of the patient, and pattern of growth, and it may be further honed by the specific location of the IHC reaction. As an example, ONB shows a sustentacular S-100 protein reaction that would not be seen in MM.99 A PNST would be positive with S-100 protein but would not show HMB-45, melan-A, or tyrosinase reactions (Fig. 9-17).129 Mesenchymal chondrosarcoma also shows S-100 reactivity but does not show HMB-45, melan-A, or tyrosinase reactions (Fig. 9-18).132
KEY DIAGNOSTIC POINTS Mucosal Melanoma
Figure 9-17 A malignant peripheral nerve sheath tumor shows a spindle cell pattern with focal necrosis (left side of image). The neoplastic cells in this case show a strong and diffuse S-100 protein nuclear and cytoplasmic positive reaction, although they are usually not this strong.
• MM shows a wide variety of patterns and cytomorphologic features, often with prominent nucleoli, intranuclear inclusions, and occasional pigmentation. • MMs react with an array of melanocytic markers—S-100 protein, human melanoma black 45 (HMB-45), melan-A, and tyrosinase—whereas CD117 and CD56 are infrequently positive, and CAM5.2 is rarely positive.
Figure 9-18 A mesenchymal chondrosarcoma shows a small round blue cell appearance set within a hemangiopericytoma like vascular pattern (left). Upper right, Note the juxtaposition of the primitive cells to the mature cartilage. Lower right, The primitive cells have a slightly spindled appearance as they merge with the cartilage.
Nasal Cavity and Paranasal Sinuses
263
Small Cell Neuroendocrine Carcinoma Several tumors in the sinonasal tract have “neuroendocrine” differentiation (SNUC, ONB, pituitary adenoma, paraganglioma, and NEC), but the diagnosis of a small cell NEC (SCNEC) should only be rendered when these other tumor types are actively excluded. SCNEC is histologically similar to lung small cell carcinoma. The tumor develops over a broad age range and affects men and women equally; it involves the nasal cavity and/or the paranasal sinuses and frequently shows extension into adjacent structures.56,102,105,118,135-139 Microscopically, SCNEC shows sheets, ribbons, and nests of small cells with a high nuclear/cytoplasmic ratio. Usually, no overlying dysplasia or carcinoma is evident. The cells may be round to spindled, with scant cytoplasm surrounding hyperchromatic nuclei with inconspicuous nucleoli. Nuclear molding, frequent mitoses, necrosis, and single-cell apoptosis are common (Fig. 9-19). SCNECs show a characteristically strong reaction for AE1/AE3 and CAM5.2, usually in a punctate or dotlike pattern (Fig. 9-20; see Tables 9-2 and 9-3). Rare cases
Figure 9-19 Neuroendocrine carcinoma is arranged in sheets, nests, and ribbons and focally with pseudorosettes (left). The cells have a high nuclear/cytoplasmic ratio with delicate, even nuclear chromatin distribution. Mitoses are seen (right).
A
B
C
D
Figure 9-20 Neuroendocrine carcinoma reacts with both epithelial and neuroendocrine markers. A, AE1/AE3. B, Synaptophysin. C, CD56. D, Chromogranin. Note the different staining patterns and dotlike (Golgi), cytoplasmic, membranous, and focal reactions.
264
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-21 Neuroendocrine carcinoma reacts with a variety of other markers. A, p63. B, Thyroid transcription factor 1. C, FLi-1. D, Ki-67 shows a high proliferation index.
may show CK20, CK5/6, 34βE12, p63, and FLi-1 reactions (Fig. 9-21). The cells show variable reactions with neuroendocrine markers, including synaptophysin, CD56, CD57, chromogranin, and NSE (see Fig. 9-20). In some cases, the cells may be positive with S-100 protein, CD117, and thyroid transcription factor 1 (TTF-1; see Fig. 9-21).97,101-105,111,137,139 In rare cases, ectopic hormone expression (calcitonin, adrenocortical hormone, β-melanocyte–stimulating hormone, serotonin, parathyroid hormone) can be detected,140 and the Ki-67 proliferation index is usually high (see Fig. 9-21).
KEY DIAGNOSTIC POINTS Small Cell Neuroendocrine Carcinoma • SCNEC shows sheets, ribbons, and nests of small cells with molding, high nuclear/cytoplasmic ratio, hyperchromatic nuclei, necrosis, and high numbers of mitoses. • SCNEC is positive for AE1/AE3 and CAM5.2 (dotlike) and for a variety of neuroendocrine markers, including synaptophysin, CD56, CD57, chromogranin, and NSE.
Metastatic disease to the sinonasal tract from a pulmonary primary must always be considered, along with SNUC, ES/PNET, pituitary adenoma, and NUTM1 midline carcinoma. Pituitary adenoma does not have vascular invasion, tends to lack mitotic figures, and will be reactive with various pituitary hormones or transcription factors.100 ES usually shows FLi-1 and CD99 but shows weak to focal keratin and neuroendocrine reactions.105,141
Pituitary Adenoma Pituitary adenoma may develop in the sinonasal tract by direct extension from an intracranial tumor (most common, in as many as 3% of cranial primaries) or as an ectopic tumor (normal sella with tumor in the sphenoid sinus, followed by nasopharynx and then nasal cavity). These tumors are frequently misdiagnosed as other neoplasms in these sites and usually present in the sixth decade (mean, 54 years) with a slight female/male bias (1.3 : 1). Hormone production may result in clinical symptoms or serologic hormone elevation in several cases, and tumors are often large (mean, 3 cm).100,102,103,142,143
Nasal Cavity and Paranasal Sinuses
265
Tumors are identified beneath an intact respiratory epithelium, arranged in many different patterns that include solid, organoid/insular, glandular, trabecular, packets, rosettes, and single file (Fig. 9-22); they frequently show bone invasion but lack lymphovascular invasion. Secretions are common, and necrosis is infrequently noted. Mitoses are inconspicuous. The tumors show a variable cellularity with polygonal, plasmacytoid,
granular, and oncocytic tumor cells. Severe pleomorphism is uncommon. The nuclear chromatin is delicate and has a salt-and-pepper distribution (see Fig. 9-22) that shows intranuclear cytoplasmic inclusions and multinucleated tumor cells. The cells are supported in a vascularized to sclerotic or calcified stroma. IHC highlights the neuroendocrine nature of the tumors, with synaptophysin (>95%), CD56 (>90%),
Figure 9-22 A pituitary adenoma identified below an intact respiratory epithelium. Note the syncytial appearance of the small cells and ample cytoplasm. Pleomorphism is limited, with no mitoses.
Figure 9-23 A pituitary adenoma will be positive, with AE1/AE3 (left) and CAM5.2 (right) to a variable degree.
A
B
C
D
Figure 9-24 A variety of neuroendocrine markers will be positive in the neoplastic cells of a pituitary adenoma. A, Chromogranin (dotlike). B, Synaptophysin (membrane and cytoplasm). C, CD56 (membrane). D, Neuron-specific enolase (granular cytoplasm).
266
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-25 Prolactin shows a strong cytoplasmic reaction (A), although not every cell is positive (B). Other hormones may be positive, including follicle-stimulating hormone (C) and adrenocorticotropic hormone (rare cells; D).
NSE (>75%), chromogranin (>70%), and CD99 (~40%) with AE1/AE3 (80%) or CAM5.2 (60%) confirming the epithelial component, frequently in a dotlike Golgi accentuation (Figs. 9-23 and 9-24, see Table 9-3). As many as 80% of cases are positive for pituitary hormones such as prolactin (most common), folliclestimulating hormone (FSH), luteinizing hormone, adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), and growth hormone, and they are frequently plurihormonal (Fig. 9-25). If required, pituitary transcription factors such as Pit-1, T-pit, SF-1, ER-α, and GATA-2 will help to confirm the diagnosis in hormone-silent cases. Calcitonin may be positive in a few cases, whereas CK7, CK5/6, and S-100 protein are almost always negative.100,142-144 The differential diagnosis includes ONB, NEC, SNUC, paraganglioma, ES/PNET, and mucosal melanoma (MM). The relative monotony of the cells, lack of lymphovascular invasion, inconspicuous mitoses, and tumor location help to guide the IHC evaluation (i.e., pituitary hormones would be performed after
lymphoma, rhabdomyosarcoma, or melanoma were excluded). KEY DIAGNOSTIC POINTS Pituitary Adenoma • Pituitary adenoma may directly invade into or be ectopic within the sinonasal tract and nasopharynx. It shows a variety of patterns of growth, bone invasion, and necrosis but lacks lymphovascular invasion and significant mitoses. • Tumors generally express pancytokeratin and CAM5.2, with neuroendocrine markers (synaptophysin, CD56, NSE, chromogranin, CD99), and a variety of pituitary hormones or transcription factors.
Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and is also the most common soft tissue sarcoma in the head and neck. Within the
Nasal Cavity and Paranasal Sinuses
head and neck, RMS primarily involves the orbit, ear, and temporal bone in addition to the sinonasal tract, oropharynx, and nasopharynx.103,107,145,146 Embryonal RMS (E-RMS) tends to develop in the ear and mastoid, whereas alveolar RMS (A-RMS) is more common in the sinonasal tract (Figs. 9-26 and 9-27). Specifically, in the sinonasal tract, A-RMS develops in adults, with a slight male predominance (1.2 : 1).119,146,147 In the sinonasal tract, the tumors are below an intact or partially ulcerated epithelium. The primitive mesenchymal cells are arranged in loose alveolar patterns; cells are loosely attached to the periphery of the nest and show central degeneration or dilapidation (see Fig. 9-26). Focal tumor-cell spindling (see Fig. 9-27) or a plasmacytoid appearance can be seen. The cells have a high nuclear/cytoplasmic ratio and slightly eccentric, darkly eosinophilic cytoplasm surrounding slightly pleomorphic, hyperchromatic nuclei. Multinucleation is common, as are mitoses; cross-striations are rare, and necrosis is typically present.102-104,107,119,145,146 RMS is positive with vimentin, desmin, actins, myoglobin, and myosin, with strong nuclear reactions for myogenin, MiTF, and MyoD1 (Figs. 9-28 and 9-29; see also Fig. 9-27).102,103,107,145,146,148 Myogenin tends to be
267
Figure 9-26 Left, Alveolar rhabdomyosarcoma (A-RMS) shows a solid to alveolar pattern composed of slightly enlarged epithelioid cells with a plasmacytoid appearance. Right, A-RMS with an area of central necrosis/degeneration, which creates a dilapidated appearance. The cells have a high nuclear/cytoplasmic ratio, but isolated cells have a plasmacytoid appearance.
A
B
C
D
Figure 9-27 Rhabdomyosarcoma (RMS). A, An embryonal RMS with a spindle cell population that abuts cartilage. The neoplastic cells react with a variety of markers. B, Myogenin. C, Desmin. D, CD56.
268
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-28 Rhabdomyosarcoma (RMS) is highlighted by a variety of markers. A, Vimentin. B, Desmin. C, MyoD1. D, Myogenin. Note the diffuse, strong reaction compared with the embryonal RMS in Fig. 9-27.
A
B
C
D
Figure 9-29 Rhabdomyosarcoma also shows reactivity with several other markers. A, Smooth muscle actin. B, CD56. C, AE1/AE3. D, Neuron-specific enolase.
Nasal Cavity and Paranasal Sinuses
269
Figure 9-30 Teratocarcinosarcoma is a malignant sinonasal tract tumor that shows carcinomatous elements admixed with sarcomatous elements, including rhabdomyosarcoma (RMS), in a teratoma like pattern. Left, Primitive RMS cells juxtaposed to epithelial islands. Right, Primitive cells show focal rosette formation next to a squamous-lined cyst.
stronger in A-RMS than in E-RMS, especially in cells adjacent to fibrous septa. Cytoplasmic MyoD1 is noninformative, because only a nuclear reaction is considered positive. The tumor cells are also positive with CD56, whereas as many as 50% of cases will show a weak, focal, punctate, or dotlike AE1/AE3 and/or CAM5.2 immunoreactivity (see Fig. 9-29).149 S-100 protein and HMB-45 are negative, but there may be weak to focal reactivity with synaptophysin, chromogranin, NSE, CD99, Pax-2, and FLi-1.150-152 A-RMS has a characteristic translocation between the FKHR (13q14) forkhead region and either PAX3 (2q35) or PAX7 (1p36), which can be confirmed by a break-apart fluorescence in situ hybridization (FISH) probe for the FKHR gene fusion with PAX3 or PAX7 or by reverse transcription polymerase chain reaction (RT-PCR).146,153 Although the other small round blue cell tumors of the sinonasal tract are included in the differential diagnosis, as presented in Table 9-3, it is important to remember several unique sinonasal tract tumors in the differential diagnosis that may have rhabdoid or rhabdomyoblastic features. ONB may have rhabdoid differentiation,112,113 and teratocarcinosarcoma is a unique sinonasal tract malignancy that shows a teratoma like distribution of carcinoma and sarcoma, and the latter is frequently an RMS (Fig. 9-30).154,155 Malignant peripheral nerve sheath tumors (MPNSTs) may show rhabdomyoblastic differentiation, frequently referred to as a triton tumor (Fig. 9-31).156 Desmoplastic round cell tumor is a unique entity that shows desmin, keratin, neuroendocrine marker, and Wilms tumor 1 (WT1) reactivity, but it is negative with myogenin, MyoD1, and S-100 protein (Fig. 9-32); a FISH or RT-PCR for
Figure 9-31 A triton tumor shows rhabdomyoblastic differentiation within a malignant peripheral nerve sheath tumor. Both S-100 protein and muscle markers would be positive but differentially expressed in each compartment. Desmin is shown in this case. (Inset courtesy Dr. Roman Carlos.)
the EWSR1/WT1 t(11;22)(p13;q12), the classic translocation, is confirmatory.157 Although myogenin is sensitive for RMS, it can be seen in infantile fibrosarcoma and synovial sarcoma, although these tumors are not usually in the sinonasal tract differential.148 Caution is advised when entrapped or atrophic muscle fibers may be within a tumor, because they may react with myoid markers.
270
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-32 A, A desmoplastic round cell tumor shows heavy sclerosing fibrosis with primitive cells associated with areas of glandular differentiation. The neoplastic cells have a polyphenotypic IHC expression with various patterns. B, AE1/AE3 (dotlike). C, Desmin (dotlike). D, CD56 (cytoplasmic and membranous).
KEY DIAGNOSTIC POINTS Rhabdomyosarcoma • A-RMS may arise in the sinonasal tract, showing an alveolar to nested arrangement with cells that have eccentric eosinophilic cytoplasm. • A variety of myoid markers are positive (desmin, myogenin, MyoD1, myoglobin, actins), but it is important to remember that AE1/AE3, CAM5.2, and CD56, along with synaptophysin, may be focally positive in some cases.
Ewing Sarcoma/Primitive Neuroectodermal Tumor Ewing sarcoma/primitive neuroectodermal tumor (ES/ PNET) is a high-grade malignancy composed of primitive small round tumor cells of an undifferentiated or neuroectodermal phenotype. The Ewing family of
tumors represents a spectrum of lesions that may be found in bone, soft tissue, and various parenchymal organs (lung, pancreas, kidney); approximately 20% develop in the head and neck. The Ewing family of tumors show several molecular alterations, but the majority of cases show either t(11;22)(q24;q12) translocation (85% to 90%) or t(21;22)(q22;q12) (5% to 10%), or they show a fusion of the EWSR1 gene with the FLI1 or ERG genes, respectively, which creates a chimeric gene product.158 Approximately 80% of patients are younger than 20 years at presentation, and adults are uncommonly affected. In all sites, there is a slight male predilection (1.4 : 1), but this is not as prominent in sinonasal tract tumors. Extension by bone destruction beyond the sinonasal tract to adjacent organs (orbit, brain) is common and results in a large tumor size at presentation (as large as 6 cm).102,141,159,160 ES/PNETs are composed of uniform round cells that often grow in a lobular or nested configuration
Nasal Cavity and Paranasal Sinuses
(Fig. 9-33), although sheets of tumor cells can be seen. Coagulative and geographic necrosis is easily identified, and mitoses are increased. The cells have a high nuclear/ cytoplasmic ratio with finely dispersed chromatin. The cytoplasm is scant, poorly defined, and frequently pale or clear owing to an abundance of glycogen (Fig. 9-34; see Fig. 9-33). Homer Wright pseudorosettes are found in as many as 10% of cases, and rarely, FlexnerWintersteiner rosettes may be present. Rare cases may also show atypical features such as extracellular matrix formation, pleomorphism, and tumor-cell spindling. ES/PNETs are positive for FLi-1, CD 99 (membranous; see Fig. 9-34), SNF5, vimentin, p16, and β-catenin (cytoplasmic).107,141,161 It is important to note that FLi-1 protein nuclear expression is not specific for ES, although the EWSR1/FLI1 fusion gene is specific (as detected by FISH or RT-PCR).151,152 Tumor cells may also react with protein gene product 9.5 (PGP9.5; 75%), NSE (50%), synaptophysin (~35%), claudin-1 (~40%), CD117 (~35%), S-100 protein (30%), GFAP (20%), calretinin (15%), and CD56 (10%).102,103,119,141 Up to 30% may
271
Figure 9-33 Ewing sarcoma/peripheral neuroectodermal tumors have small- to medium-sized uniform cells with a high nuclear/ cytoplasmic ratio and delicate nuclear chromatin. Coagulative necrosis is frequently present (right side of panel).
A
B
C
D
Figure 9-34 Ewing sarcoma/peripheral neuroectodermal tumor cells are reactive with several studies. A, Periodic acid–Schiff stain without diastase highlights the glycogen. B, FLi-1 yields a strong nuclear reaction (endothelial nuclei serve as an internal control). C, CD99 has a delicate membrane reaction. D, Staining with AE1/AE3 shows a very faint, delicate cytoplasmic reaction.
272
Immunohistology of Head and Neck Lesions
also be positive, either focally or diffusely, for AE1/AE3 (see Fig. 9-34), CAM5.2, and/or EMA, possibly related to expression of abnormal tight junctions.162,163 Rarely, desmin and chromogranin (2%) may be focally positive, whereas WT1 and myogenin are negative. The principal differential diagnoses include olfactory neuroblastoma, lymphoma, RMS, mesenchymal chondrosarcoma, small cell osteosarcoma, pituitary adenoma, and sinonasal undifferentiated carcinoma. A pertinent and targeted panel will often resolve these cases to the correct diagnosis. However, it is critical to remember that significant overlap in IHC reactions can be seen, not only in positive versus negative but also in location and character of the reaction.
KEY DIAGNOSTIC POINTS Ewing Sarcoma/Primitive Neuroectodermal Tumor • ES/PNET is composed of uniform round cells with scant cytoplasm arranged in a lobular, nested, or diffuse configuration, often with necrosis and increased mitoses. • Tumor cells are positive for CD99 and FLi-1, along with variable reactions with epithelial, neuroendocrine, and other mesenchymal markers.
Two additional tumors of the sinonasal tract deserve consideration, specifically because IHC assists with their accurate classification and diagnosis. They are sinonasal tract adenocarcinomas, specifically not of salivary gland origin, and glomangiopericytoma.
Sinonasal Intestinal-Type Adenocarcinoma Adenocarcinomas of the sinonasal tract are classified into three main types by the World Health Organization (WHO): salivary gland type, intestinal type, and nonintestinal type adenocarcinomas.164 Salivary gland type tumors are much more common but are similar to primary tumors in major or minor glands.165 The nonintestinal type and intestinal-type adenocarcinomas (ITACs) are more frequently misinterpreted.166 The ITAC group represents rare tumors that recapitulate intestinal adenocarcinoma or even normal small intestinal mucosa.167,168 A very strong epidemiologic link to occupational exposure has been reported, specifically in the hardwood and shoe industries, usually after a very prolonged exposure history, often to wood dust particles.169 ITAC is significantly more common in men (4 : 1 prevalence), and it peaks in the fifth to seventh decades. In nonexposure cases, a slight female predominance has been reported.170 If detected as part of a screening program, tumors tend to be much smaller than those encountered in non–exposure-related patients.167 One of the major dilemmas in ITAC is in differentiating this type of adenocarcinoma from
metastatic adenocarcinoma of the colon, for which accurate clinical history is critical.171-175 The Barnes classification is more widely used than the Kleinsasser,168,170 and it separates the tumors into five morphologic categories based on pattern and cytologic features: colonic (40%), solid (20%), papillary (18%), mucinous (8%), and mixed (12%; Fig. 9-35).167 In general, the colonic type has a tubuloglandular architecture, only rare papillae, and an increased nuclear pleomorphism and nuclear stratification with mitoses. The solid type is solid to trabecular with rare tubules, smaller cuboidal cells, vesicular nuclei, prominent nucleoli, and nuclear pleomorphism. The papillary type has a dominant papillary architecture, much like a tubular-villous adenoma of the colon, and it shows limited pleomorphism and rare mitoses. The mucinous type shows mucin, either intracellularly or in extracellular pools. Large glands may be distended by mucin, or there may be signet-ring–type cells floating in mucin lakes. Mixed types have an admixture of any of these aforementioned types. But no matter which type is present, villi, Paneth cells, enterochromaffin cells, and even a muscularis mucosa can be seen. ITACs show both sinonasal and intestinal IHC reactions and will give a strong reaction for CK7 (Table 9-4), similar to the Schneiderian membrane,175-179 but they will also be strongly positive for CK20, CDX-2, and MUC2, typically seen in intestinal-derived malignancies (Fig. 9-36).174,175,177-180 Further, they will react with villin and MUC5, polyclonal CEA (pCEA; see Fig. 9-36), and a wide variety of neuroendocrine markers (synaptophysin, chromogranin, CD56) or even hormone peptides such as serotonin, gastric, or somatostatin.181 Usually, p53 is overexpressed, although to a lesser degree in the mucinous type.182 Several other epithelial markers— such as EMA, B72.3, BerEP4, BRST-1, and human milk fat globulin 2 (HMFG-2)—show variable reactivity.171 Tumors are negative with vimentin, actins, p63, and K903.176 Interestingly, KRAS and HRAS mutations are seen in as many as 25% of tumors by molecular methods, which further highlights the similarity between ITAC and intestinal tumors.173,183 Nuclear β-catenin is present in approximately 30% of papillary and colonic types, a much higher presence than in solid or mucinous types; membranous staining can be seen, but this is not considered meaningful.184,185 In general, without a good clinical history, imaging studies, or colonoscopy, it may be impossible to separate a metastatic tumor from the GI tract to the sinonasal tract based on histology or IHC alone. Colon carcinomas tend not to be CK7 positive and may show variable reactivity with chromogranin and MUC5. Separation from sinonasal nonintestinal adenocarcinomas (SNACs) may be a little easier, because they are nonreactive for CK20, CDX-2, villin, and MUC2, although CEA and MUC5 may be positive.166,186 Other lesions in the differential diagnosis may include malignancies (salivary gland adenocarcinomas, nasopharyngeal papillary adenocarcinoma) and benign conditions such as papillary rhinosinusitis, respiratory epithelial adenomatoid hamartoma (Table 9-5), and serous adenoma.166,187,188
Nasal Cavity and Paranasal Sinuses
A
B
C
D
273
Figure 9-35 Intestinal-type adencarcinomas are separated into a variety of different types based on pattern of growth and cellular components. A, Colonic type with stratified nuclei and Paneth cells. B, Papillary type looks identical to a tubular adenoma of the colon, including a muscularis mucosae. C, Solid type shows stratified cells within a glandular architecture. D, Mucinous type shows neoplastic cells suspended in lakes of mucin.
TABLE 9-4 Sinonasal Tract Adenocarcinoma Immunohistochemistry Versus Metastatic Colon Adenocarcinoma AE1/AE3 (panCK)
CK7
CK20
CDX-2
ITAC
+
+
+
+
+ (usually weak)
+
Non-ITAC
+
+
N
N
+ (50%)
Colon adenocarcinoma
+
R
+
+
+ (strong)
CEA
NSE
S-100 Protein
MUC2
MUC5AC
+ (often numerous)
S
N
+
+*
N
N
+
S
N
+
+
R (isolated)
N
N
+
S
Villin
Chromogranin
Data from references 166, 171, 174, 177 to 180. +, Almost always positive; N, negative; S, sometimes positive; R, rarely positive. CEA, Carcinoembryonic antigen; CK, cytokeratin; ITAC, intestinal-type adenocarcinoma; NSE, neuron-specific enolase; panCK, pancytokeratin. *The MUC5AC is seen specifically in mucinous or signet-ring types.181
274
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-36 Intestinal-type adenocarcinomas yield reactions similar to their intestinal counterparts but also show reactions unique to the sinonasal tract site. A, Cytokeratin 20 (cytoplasmic). B, CDX-2 (nuclear). C, CK7 (cytoplasmic). D, Polyclonal carcinoembryogenic antigen (luminal and cytoplasmic).
TABLE 9-5 Selected Immunohistochemistry Results in Sinonasal Tract Lesions Diagnosis
p63
S-100 Protein
N
+ (basal)
S
N
+
+
+
N
+
N
CK7
CK20
CEA
CDX-2
34βE12
SMA
Calponin
REAH
+
N
N
+
+
N
+ (basal)
Schneiderian papilloma, inverted type
+
N
+ (epithelial)
N
+
N
N
Sinonasal adenocarcinoma, intestinal type
+
+
S (epithelial)
S
+
N
N
Nonintestinal type sinonasal tract adenocarcinoma
+
N
N
+ (basal)
N
N
N
Chronic sinusitis
N
N
+ (basal)
N
+
N
N
Data from Jo VY, Mills SE, Cathro HP, Carlson DL, Stelow EB. Low-grade sinonasal adenocarcinomas: the association with and distinction from respiratory epithelial adenomatoid hamartomas and other glandular lesions. Am J Surg Pathol 2009;33:401-408. +, Almost always positive; N, negative; S, sometimes positive. CEA, Carcinoembryonic antigen; CK, cytokeratin; REAH, respiratory epithelial adenomatoid hamartoma; SMA, smooth muscle actin.
Nasal Cavity and Paranasal Sinuses
KEY DIAGNOSTIC POINTS Sinonasal Intestinal-Type Adenocarcinoma • Sinonasal ITACs very closely resemble intestinal adenocarcinomas; they are separated into four major histologic types: colonic, solid, papillary, and mucinous. • Sinonasal ITACs are usually positive with AE1/AE3, CK7, CK20, CDX-2, villin, and CEA and also show neuroendocrine differentiation in some cases.
Glomangiopericytoma (Sinonasal-Type Hemangiopericytoma) Glomangiopericytoma was originally designated sinonasal type hemangiopericytoma, but the perivascular myoid nature is more closely related to glomus tumor.124,189-191 Glomangiopericytoma is an uncommon soft tissue tumor that affects the nasal cavity more often than it does the paranasal sinuses. The tumor usually develops in the seventh decade of life but it shows a broad age range at presentation and slight female predominance (1.2 : 1).124 The tumors present with a polypoid mass and have a mean size of approximately 3 cm. Microscopically, there is a characteristic subepithelial proliferation that appears separate from a usually intact
275
surface epithelium. The tumor is arranged in short, compact to whorled or palisaded fascicles composed of spindled to epithelioid cells with indistinct cell borders (Fig. 9-37). The cytoplasm is amphophilic, and it surrounds nuclei with coarse nuclear chromatin and is without pleomorphism. There is a well-developed, branching vascular stroma. A peritheliomatous heavy hyalinization is characteristic, and extravasated erythrocytes, mast cells, and eosinophils are noted throughout (see Fig. 9-37), whereas mitoses are inconspicuous. Tumor giant cells, lipomatous change, and hematopoiesis can be present, along with solitary fibrous tumor. Rare malignant cases are also recognized.124,191,192 The neoplastic cells are reactive with vimentin, actin (smooth muscle more than muscle specific), β-catenin (nuclear and cytoplasmic; Fig. 9-38), and factor XIIIa (FXIIIa),124,193 whereas laminin highlights the matrix. Isolated tumor cells in a few cases are positive with CD34, S-100 protein, Bcl-2, CD68, and GFAP. The tumor cells are negative with CD31, factor VIIIRAg (FVIIIRAg; see Fig. 9-38), desmin, keratin, NSE, EMA, EBER, and CD117, although the mast cells will be positive with CD117.124,191,194 The differential diagnosis includes lobular capillary hemangioma (LCH), solitary fibrous tumor (SFT), nasopharyngeal angiofibroma (NPAF), fibrosarcoma, PNST, and meningioma. In general, the heavy inflammatory infiltrate and granulation tissue–like appearance in LCH
Figure 9-37 Glomangiopericytoma shows a patternless spindled to oval cell population below an intact respiratory epithelium (left). Usually a very prominent to heavy perivascular hyalinization is apparent (upper right). Extravasated erythrocytes, eosinophils, and mast cells are usually easily seen throughout the tumor (lower right).
276
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-38 The neoplastic cells are highlighted by smooth muscle actin (A) and a strong and diffuse nuclear and cytoplasmic β-catenin reaction (B). There is no reaction with vascular markers such as CD31 (C) or factor VIIIRAg (D).
Figure 9-39 Left, A lobular capillary hemangioma is a so-called reactive lesion and shows tight “cuffing” of endothelial cells around a central capillary (metaplastic squamous epithelium is present in the upper left corner). Upper right, CD31 highlights the endothelial cells. Lower right, Factor VIIIRAg is noted within the endothelial cells.
Nasal Cavity and Paranasal Sinuses
A
B
C
D
277
Figure 9-40 Solitary fibrous tumor. A, A spindle cell proliferation is set within a heavily collagenized stroma. B, CD34 shows a very strong and diffuse cytoplasmic reaction in the neoplastic cells. C, Bcl-2 highlights the neoplastic cells. D, CD99 shows a variable reaction in the cytoplasm of the lesional cells.
helps make the separation. LCH arises in the Kiesselbach triangle area, develops in younger patients, and would be CD31, CD34, and FVIIIRAg positive (Fig. 9-39). An SFT can be seen at the same time as a glomangiopericytoma but is usually easy to separate, because a lower cellularity, higher deposition of wiry collagen, and strong CD34 and Bcl-2 reaction are evident, along with a variable CD99 reaction (Fig. 9-40). NPAF occurs in a different location and exclusively in males, usually before age 20 years and it shows a spectrum of vessels within the lesion. PNSTs may also have peritheliomatous hyalinization but tend to have areas of Antoni B myxoid degeneration and show S-100 protein immunoreactivity. Meningiomas are usually meningothelial in this site; they may have psammoma bodies and tend to lack the rich vascularity of a glomangiopericytoma. Meningioma may be EMA and CK7 positive but does not usually react with smooth muscle actin (SMA), muscle-specific actin (MSA), or FXIIIa.
KEY DIAGNOSTIC POINTS Glomangiopericytoma • Glomangiopericytomas are unique spindle-cell tumors arranged in short, cellular fascicles. Cells show a syncytial architecture, limited pleomorphism, peritheliomatous vascular hyalinization, extravasated red blood cells, and mast cells. • Glomangiopericytomas show a perivascular myoid phenotype and are strongly positive for SMA, vimentin, and MSA but are negative with CD34, CD31, factor VIIIRAg, desmin, and AE1/AE3.
Theranostic Applications Two molecular markers have diagnostic, prognostic, and theranostic value for neoplasms of the head and neck NUTM1 midline carcinoma, and melanoma. A small but well-defined group of undifferentiated tumors of the upper aerodigestive tract are positive for
278
Immunohistology of Head and Neck Lesions
the NUTM1-BRD4 t(15;19)(q14;p13.1), or, less commonly, NUTM1 is fused to a variant (BRD3, 9q34.2).61,62,195 These tumors show a relatively poorly differentiated-to undifferentiated growth with isolated areas of focal or abrupt keratinization. Such masses can be highlighted with AE1/AE3, p63, and NUTM1 protein and may also show CD34 positivity. Targeted molecular therapy may help to treat this tumor, which is otherwise nearly always lethal. Mucosal melanomas (MMs) do not harbor a significant number of BRAF mutations, as seen in their cutaneous counterparts, where targeted therapy is used for metastatic disease.196,197 However, approximately one third of MM will have mutations, similar to those seen in gastrointestinal stromal tumors (GISTs),198 and the CD117 immunohistochemistry overexpression seems to correlate with mutation status.127,199 Therefore, the potential for molecular targeted therapy exists. In contrast, sinonasal undifferentiated carcinoma frequently shows CD117 immunohistochemistry overexpression, but neither activating mutations nor gene amplifications for c-Kit are detected.121
Nasopharynx Nasopharyngeal Carcinoma The most common type of nasopharyngeal tumor is nasopharyngeal carcinoma (NPC). The etiology is multifactorial, with race, genetics, environment, and EpsteinBarr virus (EBV) all play a role. Although rare in Caucasian populations, NPC is one of the most frequent cancers in southeast Asians and North Africans, and it has endemic clusters in Indians and native Alaskans. NPC is primarily a tumor of adults with a peak occurrence between 40 to 60 years, although the tumor can occur in children. There is a strong male/female prevalence of approximately 3 : 1, irrespective of geographic location. There is an association with dermatomyositis.
EBV is almost always present in NPC and indicates an oncogenic role.123,200-203 Nasopharyngeal carcinoma was defined most recently by the WHO as “a carcinoma arising in the nasopharyngeal mucosa that shows light or ultrastructural evidence of squamous differentiation.”204 This broad definition encompasses SCC, nonkeratinizing carcinoma (both differentiated and undifferentiated types), and basaloid squamous cell carcinoma (BSCC).204 Tumors usually arise on the lateral wall of the nasopharynx and in the fossa of Rosenmüller, and are often exophytic and ulcerated, although a submucosal nodule can also be seen. The boundaries between the categories and subtypes is not always clear, sampling error can be a significant problem with small biopsies, and intraobserver and interobserver reproducibility variability is considerable. With these limitations in mind, the term squamous cell carcinoma is used for tumors that show definite evidence of squamous differentiation—intercellular bridges, keratinization, and distinct cell borders—in the majority of the tumor, and it is often associated with desmoplasia. The tumor is graded into well, moderately, and poorly differentiated categories. This type has a weak relationship to EBV. Nonkeratinizing carcinoma is the classic NPC (lymphoepithelial carcinoma), and there are two types: differentiated and undifferentiated.204,205 Solid sheets of syncytial-appearing large tumor cells are arranged in irregular islands and trabeculae that are intimately associated and intermingled with inflammatory elements (Fig. 9-41). Cellular overlapping is also apparent. The nuclear chromatin is cleared or vesicular, with prominent nucleoli, and the nuclear/cytoplasmic ratio is high, with amphophilic cytoplasm (Fig. 9-42). Tumors may have areas reminiscent of transitional epithelium of bladder (differentiated). A desmoplastic stroma is uncommon. When the lymphoid component is dominant, the “lymphoepithelioma” concept is brought to mind; the lymphoid component of the tumor is benign.
Figure 9-41 Left, Nonkeratinizing carcinoma shows a syncytial architecture of the epithelial cells, arranged in islands with a rich inflammatory investment around the periphery. Right, Higher power magnification shows individual neoplastic cells set within a heavy inflammatory infiltrate. This nasopharyngeal carcinoma type is difficult to diagnose.
Nasopharynx
Figure 9-42 Left, Nasopharyngeal carcinoma cells have a high nuclear/cytoplasmic ratio. Vesicular nuclei chromatin distribution and prominent, brightly eosinophilic nucleoli are evident. Right, Amyloid is present within the cytoplasm of the tumor cells in a few cases.
279
Granulomatous response to the tumor may be the dominant finding in a few cases, whereas a heavy eosinophilic infiltrate can simulate Hodgkin lymphoma. Occasionally, amyloid globules can be seen within the tumor intracellularly, derived from intermediate filaments (see Fig. 9-42). The lymphoepithelial pattern can be seen in nonnasopharyngeal locations. It is histologically identical but arises in the sinonasal tract, oropharynx, salivary gland, and larynx.116,206 These tumors are only diagnosed accurately when the nasopharynx is free of tumor, because the histology and IHC are identical.207 BSCCs of the nasopharynx are exceptionally rare and are composed of a basaloid neoplasm with focal areas of squamous differentiation, SCC in situ, or invasive SCC. An EBV association is seen, although it is not as well developed in comparison to undifferentiated NPC; reactivity for p16, a surrogate marker for HPV, may also be seen in a few cases. Nonkeratinizing NPC is strongly and diffusely positive for pancytokeratin, CK5/6, 34βE12, CK8 (patchy), CK13, and CK19, and it also shows strong p63 and p53 nuclear reactions (Fig. 9-43). The neoplastic cells are
A
B
C
D
Figure 9-43 The neoplastic cells of nasopharyngeal carcinoma can be highlighted with several epithelial and basal cell markers. A, Staining with AE1/AE3 shows a meshwork reaction. B, CK5/6. C, 34βE12 (K903). D, p63 (nuclear positive).
280
Immunohistology of Head and Neck Lesions
Figure 9-44 Nasopharyngeal carcinoma is strongly associated with Epstein-Barr virus and specifically yields a strong, diffuse, nuclear reaction with EBER (left). No reaction occurs with cytokeratin 7 (upper right), whereas S-100 protein will highlight dendritic cells (lower right).
negative for CK4, CK7 (Fig. 9-44), CK10, CK14, CK20, p16, and HPV (see Table 9-2).115 The nuclear reaction for EBER is strong and diffuse (see Fig. 9-44), but it is variably reactive with EBV latent membrane protein (EBV-LMP; the latter IHC is not recommended).208 The tumor cells are also positive with human leukocyte antigen DR (HLA-DR) but are generally negative with CD117, HER-2/neu, and CD45RB.209,210 S-100 protein– positive dendritic cells may be identified in the network of lymphoid elements (see Fig. 9-44). Diagnostic problems may arise in the differential diagnosis, specifically for nonkeratinizing carcinoma. Crush artifacts are common, which mandates careful evaluation of better preserved areas. Keratin staining will help in uncertain cases. In radiation cases, the stromal and cytologic atypia within the epithelium may make separation of reactive versus neoplastic cells more difficult. The differential diagnosis includes sinonasal undifferentiated carcinoma, melanoma, and lymphoma. Some benign conditions can mimic NPC: Floridly reactive germinal centers can sometimes contain large vesicular nuclei and lack a well-defined mantle zone. Plump endothelial cells in reactive lymphoid aggregates can also have vesicular nuclei and can be confused with NPC. Both of these cell types usually do not contain nucleoli and are negative for keratin. Sinonasal undifferentiated carcinoma (SNUC) is a completely separate tumor, the diagnosis of which is based on location and pattern of growth (see Tables 9-2 and 9-3).116 The differential diagnostic considerations can often be confirmed with a pertinent IHC panel.
KEY DIAGNOSTIC POINTS Nasopharyngeal Carcinoma • Nonkeratinizing carcinoma is the classic type of NPC showing a syncytial architecture of cells with vesicular nuclei and an intimate relationship with lymphoid elements. • The neoplastic cells are strongly and diffusely positive with EBER, AE1/AE3, CK5/6, and p63 but are nonreactive with CK7, desmin, and S-100 protein.
Nasopharyngeal Angiofibroma Nasopharyngeal angiofibroma (NPAF) is a benign, highly cellular, and richly vascularized mesenchymal neoplasm that arises in the nasopharynx in males. The tumor has a testosterone-dependent and pubertyinduced growth, and most patients come to medical attention before 20 years of age. An association with familial adenomatous polyposis (FAP) has also been reported. The tumor arises from the pterygoid region of the nasopharynx, but it is not uncommon to have a large tumor (as large as 22 cm) with extensive expansion into adjacent organs that simulates a malignancy clinically. Imaging studies are critical to assess extent of disease, and angiography has been used for presurgical embolization.211,212 Histologically, the tumor shows a submucosal vascular proliferation within a fibrous stroma and many variably sized and disorganized vessels. The vessel walls
Nasopharynx
Figure 9-45 Nasopharyngeal angiofibroma shows a proliferation of various sizes and shapes of vessels that include arteries, capillaries, and veins set within a variably collagenized stroma. The vessels may or may not contain smooth muscle, which may be either circumferential or padlike.
281
show varying thickness with a patchy to circumferential smooth muscle content (Fig. 9-45). The vessels range from slitlike capillaries to large, dilated, patulous vessels. A “staghorn” appearance to the vessels is quite characteristic (see Fig. 9-45), but the endothelial cells are unremarkable. The fibrous stroma has fine to coarse collagen fibers with plump, spindled, angular stellate cells; the stroma may completely overgrow the vascular component. Mitoses are scant, but mast cells are easily identified. If embolic material was used, a foreign body giant cell reaction may be noted.133,211 The stromal cells stain strongly positive for vimentin, nuclear β-catenin, androgen receptors, and estrogen receptor–β, and they stain variably with SMA (Fig. 9-46), CD117, and nerve growth factor (NGF).193,211,213-216 The stromal cells are negative with desmin, CD34, estrogen receptor–α, progesterone receptors, EBER, and human herpes virus 8 (HHV8).194,213-215,217 It is important to note that these results are in the stromal cells and not in the endothelial cells, which may have a different reactivity pattern. Both the CD117 (c-Kit) and β-catenin signaling pathways may
A
B
C
D
Figure 9-46 Nasopharyngeal angiofibroma shows variable staining with markers in the stromal cells or in the vessels. A, Nuclear and cytoplasmic β-catenin. B, Muscle-specific actin. C, CD34. D, CD31.
282
Immunohistology of Head and Neck Lesions
Figure 9-47 An antrochoanal polyp shows a welldeveloped fibrotic stroma without any minor mucoserous glands (left). Atypical stromal cells (right) should not be mistaken for rhabdomyoblasts or sarcoma cells because they are considered a degenerative phenomenon.
be exploited therapeutically, because antiandrogenic therapy in pubescent males is usually not accepted clinically. The differential diagnosis includes lobular capillary hemangioma (LCH), inflammatory polyp, and antrochoanal polyp. LCH arises in a different anatomic site, usually has surface ulceration and extensive granulationtype tissue, and the vessels appear more organized or are perpendicular to the surface (see Fig. 9-39). Usually CD34 and CD31 reactivity of the vascular proliferation is apparent. Inflammatory polyps may have atypical stroma cells, but they are set within an edematous stroma and lack the variety of vascular elements. Antrochoanal polyp arises specifically from the maxillary antrum, shows heavy stromal fibrosis, and lacks a vascular proliferation (Fig. 9-47). This lesion can have stromal atypia and mast cells, but it does not have β-catenin– positive stromal cells.
KEY DIAGNOSTIC POINTS Nasopharyngeal Angiofibroma • NPAF is a unique fibrovascular neoplasm that develops in males only. An association with FAP has been established. • The stromal cells are immunoreactive with nuclear β-catenin, vimentin, androgen receptors, and variably with SMA and CD117.
nasopharynx.218 This rare tumor tends to develop in younger patients (mean, 37 years) without a gender predilection. The tumors measure as large as 4 cm and show a papillary, polypoid, or cauliflower gross appearance.218-221 NPA is a surface-derived tumor that shows a complex, arborizing pattern of papillae and crowded/overlapping glands with invasion into the stroma and hyalinized fibrovascular cores (Fig. 9-48). The nuclei are columnar to cuboidal and show pseudostratification and overlapping with vesicular to optically clear nuclear chromatin and small nucleoli (see Fig. 9-48). Psammoma bodies and necrosis may be seen.218 Diastase-resistant, periodic acid–Schiff (PAS) intracytoplasmic material is present. The neoplastic cells are positive with AE1/AE3, EMA, CK7, CK19, TTF-1 (Fig. 9-49), and vimentin but are negative with CK5/6, CK20, CD15, S-100 protein, GFAP, EBER, and thyroglobulin.218-223 The TTF-1 immunoreactivity has led some to refer to this condition as thyroidlike lowgrade nasopharyngeal papillary adenocarcinoma.219,222,223 The major differential diagnosis is with metastatic thyroid papillary carcinoma and salivary gland neoplasms of the minor mucoserous glands. The lack of thyroglobulin helps, although to date, no galectin-3, HBME-1, or molecular studies have been done to exclude BRAF, RAS, or RET/PTC mutations. KEY DIAGNOSTIC POINTS
Nasopharyngeal Papillary Adenocarcinoma Nasopharyngeal papillary adenocarcinoma (NPA) is a low-grade adenocarcinoma that shows an exophytic growth of papillary fronds and glandular structures that arise within the lateral and posterior walls of the
Nasopharyngeal Papillary Adenocarcinoma • NPA is a surface epithelium–derived papillary and glandular tumor that mimics thyroid papillary carcinoma. • The neoplastic cells are immunoreactive with AE1/AE3, cytokeratins 7 and 19, and TTF-1 but are nonreactive with thyroglobulin and EBER.
Nasopharynx
283
Figure 9-48 Left, A nasopharyngeal papillary adenocarcinoma shows connection to the metaplastic squamous surface epithelium. Invasion into the stroma by a remarkably complex papillary proliferation shows hyalinized papillary cores. Right, The columnar cells that line the papillary cores contain cuboidal to elongated nuclei with nuclear chromatin clearing and subnuclear vacuolization. (Courtesy Dr. Bruce M. Wenig.)
A
B
C
D
Figure 9-49 A nasopharyngeal papillary adenocarcinoma reacts with a variety of epithelial markers. A, AE1/AE3. B, CK7. C, Epithelial membrane antigen. The neoplastic cells show a strong thyroid transcription factor 1 nuclear reaction (D) but do not react with thyroglobulin. (Courtesy Dr. Bruce M. Wenig.)
284
Immunohistology of Head and Neck Lesions
Oral Cavity Granular Cell Tumor Granular cell tumor (GCT) is an uncommon tumor composed of poorly demarcated granular cells, thought to be Schwann-cell derived, that frequently arise below a mucosa, the latter often showing pseudoepitheliomatous hyperplasia. Granular cell tumor tends to affect the oral cavity (tongue most commonly) and larynx (posterior) in addition to the usual presentation in skin. Patients of all ages are affected, but are usually between 40 and 60 years at the time of clinical presentation. The tumors develop more commonly in females than in males (2 : 1) and in blacks more often than
whites. Tumors are usually smooth surfaced, poorly demarcated, and are often polypoid, and measure from 1 to 2 cm.224-228 Pseudoepitheliomatous hyperplasia (PEH) is often extensive but is usually limited to the extent of the tumor (Fig. 9-50). The unencapsulated neoplastic cells are polygonal, large, eosinophilic cells with indistinct cell membranes. Granular, eosinophilic cytoplasm (filled with lysosomes) is abundant and surrounds small dark to vesicular nuclei (Fig. 9-51). Stromal desmoplasia is uncommon. Malignant tumors are recognized by pleomorphism, increased mitoses, and necrosis, but these are exceedingly rare in mucosal sites. The neoplastic cells yield a strong and diffuse nuclear and cytoplasmic S-100 protein reaction (see Fig. 9-51) and are also positive for CD68, NSE, α-1–antitrypsin,
Figure 9-50 Pseudoepitheliomatous hyperplasia (PEH) is often quite extensive but is usually limited to the area immediately overlying the tumor (left). At times the PEH is so remarkable as to simulate a squamous cell carcinoma; granular cells can be seen in the stroma (right).
Figure 9-51 Granular cell tumor is composed of large, polygonal to spindle cells that contain abundant granular material (top). A nerve is noted, highlighted with S-100 protein, which also stains the neoplastic cells (bottom).
Figure 9-52 Rhabdomyoma is composed of large, polygonal cells that show a slight separation artifact between cells (spiderweb cells, left). On high-power magnification, cross-striations can usually be seen (right).
Oral Cavity
A
B
C
D
285
Figure 9-53 Alveolar soft-part sarcoma shows a nested alveolar pattern with cells that have eosinophilic cytoplasm that surrounds round to regular nuclei with prominent nucleoli. The neoplastic cells are nonreactive with vimentin (B) and show a cytoplasmic reaction with MyoD1; (C) and a strong, diffuse nuclear reaction with TFE3 (D).
vimentin, inhibin-α, protein gene product 9.5 (PGP9.5), and calretinin.224-229 The tumor cells are negative for keratin, SMA, MSA, myoglobin, and desmin. The major differential considerations include rhabdomyoma, schwannoma, congenital epulis of the newborn, leiomyoma, paraganglioma, alveolar soft-part sarcoma, and SCC. The latter is only a consideration when PEH is dominant; PEH lacks p53 overexpression. Rhabdomyoma generally shows so-called spiderweb cells, more opaque cytoplasm, cross-striations, and reactivity with muscle markers (Fig. 9-52). Schwannoma will be positive with S-100 protein, but the tumor is arranged in fascicles, has Antoni A and B areas, shows perivascular hyalinization, and generally lacks CD68 and inhibin. Congenital epulis of the newborn is histologically identical but is only seen in newborns and infants; it lacks S-100 protein but still shows vimentin and NSE reactivity. Leiomyoma is also spindled but shows muscle markers; paraganglioma can be nested with “granular” cytoplasm, but the cytoplasm tends to be basophilic; a more zellballen architecture is present,
and although S-100 reactivity is evident, it is sustentacular. Strong reactivity with neuroendocrine markers will help with the separation. Alveolar soft-part sarcoma is very rare in the oral cavity, tends to show an alveolar to nested pattern, has more prominent nucleoli, and lacks S-100, vimentin, CD68, and SMA but reacts with NSE, MyoD1 (cytoplasmic), and transcription factor E3 (TFE3; Fig. 9-53).
KEY DIAGNOSTIC POINTS Granular Cell Tumor • GCT develops in the oral cavity (tongue) and larynx with pseudoepitheliomatous hyperplasia and large, polygonal cells with abundant granular cytoplasm that shows limited pleomorphism. • The neoplastic cells are immunoreactive with S-100 protein, CD68, vimentin, NSE, and calretinin but are nonreactive with keratins, muscle markers, HMB-45, and TFE3.
286
Immunohistology of Head and Neck Lesions
Ectomesenchymal Chondromyxoid Tumor Ectomesenchymal chondromyxoid tumor (ECT) is a benign intraoral tumor presumed to arise from an undifferentiated ectomesenchymal stem cell that migrates from neural crest, histologically equivalent to a soft tissue myoepithelioma. A rare tumor, nearly always in the anterior dorsal tongue, it affects predominantly younger patients (mean, 37 years). The tumor is a circumscribed gelatinous to tan tumor that may grow to approximately 2 cm.230,231 An unencapsulated but well-circumscribed tumor is usually separate from the adjacent skeletal muscle, although it can entrap the muscle fibers and nerve twigs. The cells are arranged in cords, strands, or netlike sheets set within a myxoid-chondroid matrix that may also be hyalinized. Neural features are suggested by tumor cell swirling. The uniform proliferation is composed of small round, oval, spindle, or stellate cells that have basophilic to clear cytoplasm (Fig. 9-54). Multinucleation and mitoses are usually not seen, although isolated pleomorphic cells can be identified. Glands, myoepithelial cells,
Figure 9-54 Left, The surface is uninvolved, separated from the circumscribed but unencapsulated neoplastic proliferation of an ectomesenchymal chondromyxoid tumor. Right, The cells are arranged in a netlike architecture within a myxoid-chondroid matrix material. The cells are uniform, small, and round to oval with lightly basophilic cytoplasm.
A
B
C
D
Figure 9-55 Ectomesenchymal chondromyxoid tumor has a strong stromal reaction with Alcian blue (A). The neoplastic cells show variable reactivity with glial fibrillary acidic protein (B) and S-100 protein (C), and they stain focally with desmin (D). Note the entrapped skeletal muscle fibers.
Larynx/Hypopharynx
necrosis, and atypical mitoses are not seen. The stromal matrix is Alcian blue (Fig. 9-55) and mucicarmine positive, but PAS is negative.230-232 This tumor demonstrates a multilineage phenotype; the neoplastic cells stain for GFAP, AE1/AE3, S-100 protein (see Fig. 9-55), SMA, and vimentin. However, the cells are usually negative with desmin, p63, calponin, EMA, CK7, CK8/18, and CK20.230-235 The differential diagnosis includes myoepithelioma, pleomorphic adenoma, myxoid neurofibroma, neurothekeoma, and extraskeletal myxoid chondrosarcoma or mesenchymal chondrosarcoma.132,230,236,237 Myoepithelioma usually does not develop in the anterior tongue; it shows surrounding minor salivary gland tissue and has both spindled and plasmacytoid myoepithelial cells. The neoplastic cells are positive with p63, calponin, and SMA. Likewise, pleomorphic adenoma is rare in the anterior tongue, shows a tubular/glandular appearance, and is immunoreactive with myoepithelial markers such as p63 and calponin. A myxoid neurofibroma lacks chondroid areas, has nuclei that are more wavy, and shows only a strong and diffuse S-100 protein reaction. A neurothekeoma (nerve sheath myxoma) is also rare in mucosal sites and shows only S-100 protein and PGP9.5. The tongue is a very uncommon location for extraskeletal mesenchymal chondrosarcoma, which tends to show undifferentiated sheets of round to spindle-shaped cells with an abrupt transition to nodules of benignappearing hyaline cartilage. The cartilage may show calcification or ossification, and there may be a hemangiopericytoma-like vascular pattern (see Fig. 9-18). The immature cells are positive with NSE, Leu-7, and CD99 (membranous) but are negative with keratins and GFAP (the cartilage will be positive with S-100 protein). Focal, oral, mucinous (gingiva, myxoid tissue with stellate fibroblasts) and mucocele (extravasated mucinous material, histiocytes, inflammatory cells) may rarely be raised in the differential diagnosis.
287
groups the neuroendocrine tumors (NETs) into four categories: 1) typical carcinoid, 2) atypical carcinoid, 3) SCNEC, and 4) paraganglioma,238 the latter of which will not be further discussed here because of its rarity.239
Typical Carcinoid Typical carcinoid makes up only approximately 3% to 5% of all NECs of the larynx and develops most commonly in the supraglottic larynx. The tumors are often polypoid and measure between 1 and 2 cm.238,240 Histologically, the tumors are arranged in nests, trabeculae, sheets, glands, or even rosettes. The cells are somewhat monotonous; eosinophilic cytoplasm surrounds nuclei with finely stippled to more dense chromatin (Fig. 9-56), and nucleoli are small to inconspicuous, with isolated or limited mitoses. Usually a vascularized stroma is apparent, although it can sometimes be hyalinized. Oncocytic and mucinous variants have also been described.
Atypical Carcinoid Atypical carcinoid is the most common neuroendocrine neoplasm of the larynx (~55%) and shows a strong male predilection (3 : 1), with patients on average 60 years old.238,241,242 The vast majority of atypical carcinoids are found in the supraglottis and come to medical attention as a polypoid to hemorrhagic submucosal mass on average slightly larger than typical carcinoids. Histologically, these are infiltrative tumors that show a spectrum of growth patterns similar to typical carcinoid. The cells are larger than the cells in typical carcinoid and show vesicular chromatin with more prominent nucleoli. Cellular pleomorphism is more easily identified, and tumor cells can be spindled to plasmacytoid and cuboidal (Fig. 9-57). Mitoses (2 to 10/10 high power fields) and necrosis are seen, along with lymph, vascular, and/or perineural invasion.
KEY DIAGNOSTIC POINTS Ectomesenchymal Chondromyxoid Tumor • A rare tumor, usually of the dorsal anterior tongue, that shows a spindled and epithelial proliferation with an associated myxoid-chondroid matrix. There is no glandular/ tubular component. • The neoplastic cells are immunoreactive with GFAP, AE1/ AE3, S-100 protein, and SMA, but they are nonreactive with p63, calponin, and EMA.
Larynx/Hypopharynx A wide variety of tumors arise within the confines of the larynx/hypopharynx, and SCC and its variants account for the vast majority. However, the neuroendocrine neoplasms are somewhat unique and often require IHC to aid in the diagnosis. The WHO classification
Figure 9-56 Typical carcinoid of the larynx shows a trabecular, insular, and organoid architecture (left). The neoplastic cells are monotonous, have a low to medium nuclear/cytoplasmic ratio, and demonstrate finely stippled “salt and pepper” nuclear chromatin distribution (right).
288
Immunohistology of Head and Neck Lesions
Figure 9-57 Atypical carcinoid of the larynx may involve the surface epithelium (left), and cells are arranged in a solid, trabecular, and insular architecture. The cells have easily identified pleomorphism, more prominent nucleoli, and easily identified mitoses. A glandular appearance may be seen (right).
Figure 9-58 A laryngeal small cell carcinoma shows sheets of cells that are crushed and smudged (left) as well as nuclear molding (right). The chromatin is more dense and coarse, with many mitoses and areas of necrosis (right).
Small Cell Carcinoma, Neuroendocrine Type Small cell carcinoma, neuroendocrine type, is rare (~1%) and develops more often in men, who are usually older at initial presentation and report a heavy tobacco use history.238,243 These tumors are identified in the supraglottis as an ulcerated mass, and many patients come to medical attention with lymph node metastases. Paraneoplastic syndromes have been documented. The tumors are composed of sheets or ribbons of closely packed cells arranged in a syncytium. The cells are crushed and show molding of nuclei that have dense chromatin and absent
nucleoli (Fig. 9-58). Mitoses, necrosis, and perineural, lymph, and vascular invasion are almost always identified. Rosettes may also be seen. Similar to their lung counterparts, intermediate- and large-cell types are rarely identified. Combined carcinoma shows a NEC combined with SCC or adenocarcinoma. In general, no difference is apparent in the epithelial and neuroendocrine immunoreactivity among these tumors, with a few exceptions. They are all positive with chromogranin, synaptophysin, CD56, and NSE along with AE1/AE3, carcinoembryonic antigen (CEA), or EMA (Fig. 9-59).138,238 Atypical carcinoid and small cell carcinoma frequently react with calcitonin (see
Larynx/Hypopharynx
A
B
C
D
289
Figure 9-59 Atypical carcinoids are reactive with a variety of epithelial and neuroendocrine markers. A, AE1/AE3 shows a strong cytoplasmic reaction (surface epithelium is an internal control). B, Chromogranin with a strong cytoplasmic reaction (epithelium is negative). C, Synaptophysin (granular cytoplasmic reaction). D, Calcitonin (cytoplasmic; strong but focal reaction).
TABLE 9-6 Immunohistochemical Staining Pattern for Tumors in the Differential Diagnosis for Larynx Neuroendocrine Neoplasms Carcinoid Tumor
Paraganglioma
Medullary Carcinoma
Melanoma
Metastatic (Thyroid or Lung Carcinoma)
Chromogranin
+
+
+
N
+
Synaptophysin
+
+
S
N
+ +
Antibody
CD56
+
+
+
R
Cytokeratin 7
S
N
N
N
+
Cytokeratin 20
S
N
N
N
N
Polyclonal CEA
S
NR
+
N
+
S-100 protein
N
+ (sustentacular)
N
+
N
Calcitonin
N
N
+
N
S
TTF-1
N
N
+
N
+
+, Almost always positive; N, negative; S, sometimes positive; R, rarely positive; NR, not reported CEA, Carcinoembryonic antigen; TTF-1, thyroid transcription factor 1.
Fig. 9-59), serotonin, and/or somatostatin.238,242 TTF-1 is also seen in these tumors in as many as 50% of cases.238,244 The differential diagnosis of carcinoid and atypical carcinoid tumors of the larynx includes paraganglioma, melanoma, and direct extension or metastasis from thyroid medullary carcinoma (Table 9-6). Paraganglioma shows a more characteristic zellballen and isolated
pleomorphic nuclei and will demonstrate a sustentacular S-100 protein reaction with the neuroendocrine markers, and it will be negative for cytokeratins. Mucosal melanoma may exhibit surface (pagetoid) origin and more opacified cytoplasm; it may have cytoplasmic melanin pigment, it tends to show intranuclear cytoplasmic inclusions, and it would be positive with
290
Immunohistology of Head and Neck Lesions
melanoma markers (S-100 protein, HMB-45, tyrosinase, melan-A). Thyroid medullary carcinoma will be positive with calcitonin, chromogranin, and CEA and will also express TTF-1. If required, calcitonin gene-related peptide may also help with the separation.245 However, clinical history and endoscopic and/or imaging studies may be required to definitively separate these lesions, although serum calcitonin tends to be more frequently seen in thyroid than in larynx tumors. SCNEC must also be differentiated from BSCC, lymphoma, and metastatic lung carcinoma. BSCC does not usually exhibit neuroendocrine staining, and lymphoma can be detected by using typical markers for hematopoietic cells such as CD20, CD3, CD43, and CD45RB. Primary head and neck NETs and metastatic disease from the lung can be difficult to separate, because the staining patterns for CK7, CK20, and rarely TTF-1 can overlap; clinical and radiographic correlation is essential.
KEY DIAGNOSTIC POINTS Neuroendocrine Tumors of the Larynx • The three categories of neuroendocrine tumors—carcinoid, atypical carcinoid, and small cell neuroendocrine carcinoma—are distinguished from one another mainly on the basis of histologic appearance. • The immunophenotype of these tumors includes positivity for neuroendocrine markers and for cytokeratins. • TTF-1 may be positive in these extrapulmonary neuroendocrine and small cell carcinomas.
Salivary Glands The majority of salivary gland tumors can be diagnosed by using routine H&E-stained slides. Many salivary gland tumors show remarkable IHC overlap, such that a basaloid/myoepithelial phenotype is seen in many different tumors. However, IHC may play a role in the diagnosis of some tumors, especially to identify myoepithelial cells, highlight perineural invasion, or assess the proliferative rate of a tumor. Further, several tumors are now recognized by a specific molecular alteration, which may be needed for diagnostic confirmation.
Pleomorphic Adenoma Pleomorphic adenoma (PA) is the most common salivary gland tumor and is also the most common benign salivary gland tumor, also known as benign mixed tumor. It occurs in all age groups, although it is uncommon in pediatric patients; PA affects females more often than males and may arise in major or minor salivary glands. When it originates in a major salivary gland, it is always encapsulated, although bosselations or multinodular tumor growth may make assessment more challenging. Tumors of minor salivary gland origin are not encapsulated and include sinonasal tract, larynx, trachea, and external auditory canal, among other sites.246,247 PA is a benign epithelial tumor that shows both epithelial and modified myoepithelial elements mixed with mesenchymal myxoid-, mucoid-, or chondroid-appearing material. The tumor shows a wide architectural diversity but is bland cytologically. Tumors may be solid, tubular,
Figure 9-60 Left, Cellular pleomorphic adenoma shows a mixture of myoepithelial cells and isolated duct-tubular structures. Upper right, The myxoid matrix material is easily seen, along with areas of metaplastic squamous epithelium. Lower right, A cellular pleomorphic adenoma that lacks welldeveloped stroma or tubules. A case such as this one may require immunohistochemistry confirmation.
Salivary Glands
trabecular, or cystic, and cells may be set within a variable stroma of chondroid, myxoid, or hyaline matrix (Fig. 9-60). Bone or fat may occasionally be seen. The epithelial-myoepithelial cells are spindled, epithelioid, plasmacytoid, and basaloid and show squamous, mucinous, and sebaceous differentiation focally. Clear cell change can be seen. Small duct structures are set within the epithelial component, lined by cuboidal to columnar cells. Tyrosine and collagenous or oxalate-type crystals can be found. When the tumor is removed intact, the diagnosis is not usually a challenge. However, in core or limited biopsies, the features may not be as easily recognized. Helpful features are shown in Table 9-7.88,248-253 IHC will help to highlight the epithelial and myoepithelial nature of the proliferation (Fig. 9-61). The neoplastic cells will be strongly positive with AE1/AE3, CK7, CK14, and CK5/6, among other epithelial markers.
291
S-100 protein will highlight both the spindled cells and ductal structures, which are also highlighted by CK14 and p63. Calponin will highlight the myoepithelial cells, especially around ductlike structures, whereas SMA will stain spindle cells in cords and strands; SMA is nonreactive in plasmacytoid cells. GFAP yields a variable reaction but is usually positive in a pattern similar to S-100 protein.254-257 CD117 is positive but not as strongly as is seen in adenoid cystic carcinoma.88,258 CD10 is positive in about 30% of cases,256 and E-cadherin is positive in the peripheral cells and in plasmacytoid cells.259 Usually, the proliferation index as measured by Ki-67 or minichromosome maintenance complex component 2 (MCM2) is low.251,260 Presently, five PA tumor-specific fusion genes are recognized to involve the pleomorphic adenoma gene 1 (PLAG1) and part of the zinc finger transcription factor group, and several translocations
TABLE 9-7 Features of Basaloid Salivary Gland Tumors
Feature
Pleomorphic Adenoma
Basal Cell Adenoma
Canalicular Adenoma
Polymorphous Low-Grade Adenocarcinoma
Site
Any location
Major > minor
Upper lip, other
Minor salivary gland only
Any location
Encapsulated
Y
Y
Y
N
N
Chondroid
Y
N
N
N
N
Perineural invasion
N
N
N
Y (targetoid)
Y (prominent)
Vimentin
+
+
+
+
+
Pancytokeratin (AE1/AE3)
+
+
+
+
+
CK7
+
+
+
+
+
CK18
+
+
N
+
+
CK5/6
+
+
NA
+
+
34βE12
+
NA
NA
+
+
S-100 protein
+
+
+
+
+
GFAP
+
N
+ (Linear)
R (weak, few cells)
R (isolated cells)
p63
+
+
N
+
+
Smooth muscle actin
+ (abluminal)
+
N
S (abluminal)
+ (abluminal)
Muscle specific actin
+ (abluminal)
+
N
+
+ (abluminal, ~50%)
SMMHC
+ (abluminal)
+
N
+ (50%)
+ (abluminal)
Calponin
+
+
N
S (20%)
+ (abluminal)
Maspin
NA
NA
NA
+
+
CD117
S
+
S (weak)
+ (weak, ~60% of cases)
+ (inner cell layer)
Ki-67
<5%
<2%
<2%
<5%
>20%
MCM2
R (limited cells)
R (limited cells)
NA
S (<9%)
+ (nuclear >10%)
Bcl-2
+ (weak)
NA
N
S
+ (strong)
E-cadherin
+ (− in plasmacytoid cells)
NA
+
+
+
Data from references 88, 249, 252-255, 293, 296, 303, and 304. +, Almost always positive; N, negative; S, sometimes positive; R, rarely positive; NA, not applicable. CK, Cytokeratin; GFAP, glial fibrillary acidic protein.
Adenoid Cystic Carcinoma
292
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-61 The immunohistochemistry evaluation of a pleomorphic adenoma shows an epithelial and myoepithelial phenotype. A, Triple cocktail of epithelial membrane angtigen (red-membrane positive), CK5/6 (brown cytoplasmic reaction), and p63 (brown nuclear stain) highlights both epithelial and myoepithelial/basal proliferation. B, Glial fibrillary acidic protein stains many of the tumor cells, although it usually stains more of the myoepithelial component. C, S-100 protein yields a strong, diffuse nuclear and cytoplasmic reaction that is stronger in the myoepithelial component. D, Calponin highlights the myoepithelial cells rather than the epithelial component.
involve the 8q12 region. The high-mobility group protein gene HMGA2 (formerly HMGIC) is the target gene for the second most common translocation, involving several partners within the 12q13-15 region.261,262 The differential diagnosis includes myoepithelioma, basal cell adenoma, adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, and carcinoma expleomorphic adenoma. By definition, myoepithelioma has no glandular component and lacks a chondromyxoid stroma. Basal cell adenoma is a monomorphic adenoma that shows a proliferation of basaloid cells encircled by a prominent basal lamina without a chondromyxoid stroma. Adenoid cystic carcinoma (ACC) is invasive, shows perineural invasion, and has a glycosaminoglycan material and a reduplicated basement membrane surrounding small, uniform cells with an angulated shape and hyperchromatic nuclei. Small ductlike structures can be seen in both tumors. GFAP tends to be absent, whereas CD117 highlights the central cells more strongly.
Polymorphic low-grade adenocarcinoma (PLGA) develops only in the minor salivary glands. It shows prominent perineural invasion and more uniform, oval nuclei with delicate, fine, vesicular nuclear chromatin; it lacks GFAP and p63. A carcinoma expleomorphic adenoma shows infiltration, marked pleomorphism, increased mitoses, and necrosis.
KEY DIAGNOSTIC POINTS Pleomorphic Adenoma • Tumors are benign epithelial and myoepithelial masses with a large number of patterns but relatively bland cytology, set within a myxoid-chondroid matrix material. • The cells are highlighted by a mix of epithelial and myoepithelial markers that include AE1/AE3, CK5/6, CK7, and CK14; S-100 protein; p63; SMA; calponin; and GFAP. • The major differential diagnosis is with myoepithelioma, basal cell adenoma, ACC, and PLGA.
Salivary Glands
Myoepithelioma A myoepithelioma is a benign salivary gland neoplasm composed entirely of myoepithelial differentiated cells without any ductal elements present. A rare tumor, it develops in the third to fifth decades of life without a gender predilection. The majority of tumors occur in the parotid, followed by the palate. The tumors are usually well demarcated but not entirely encapsulated, and they are usually small (<3 cm).89,263-268 The tumors appear in a broad range of architectures with solid, mucoid, reticular, nested, and cordlike patterns; morphology is often mixed, but a dominant cell type is usually present that is primarily spindled to plasmacytoid (hyaline cells; Fig. 9-62). The plasmacytoid cells have hyperchromatic nuclei set eccentrically in an eosinophilic cytoplasm. Clear, polygonal,
Figure 9-62 A myoepithelioma may show an oncocytic appearance of epithelioid cells (left) or a spindle cell morphology (right), both of which lack any ductlike structures.
Figure 9-63 A myoepithelioma shows reactions with S-100 protein (left) and a muscle marker (right), helping confirm the diagnosis.
293
oncocytic, and stellate cells are less frequently noted. An acellular stroma may be seen, but it is mucoid rather than myxoid or chondroid. Many antibodies highlight myoepithelial cells, including AE1/AE3, CAM5.2, CK5/6, 34βE12, CD10, S-100 protein, GFAP, p63, calponin, SMA, smooth muscle myosin heavy chain (SMMHC) maspin, metallothionein, and CD109; all show different sensitivities and specificities. In general, it is unwise to rely on a single marker; rather it is best to utilize a panel that includes calponin, p63, CK5/6, S-100 protein, and/or SMMHC to help yield the best sensitivity and specificity (Fig. 9-63). Myoepithelioma is reactive with AE1/ AE3, CK7, CK14, p63, GFAP, and S-100 protein but shows more variable reactivity with SMA, MSA (HHF35), SMMHC, CD109, and calponin. The plasmacytoid cells are not reactive with actins.89,256,265-270
294
Immunohistology of Head and Neck Lesions
The differential diagnosis is with pleomorphic adenoma or myoepithelial carcinoma but also with leiomyoma, schwannoma, and other nonepithelial spindle cell lesions. Plasmacytoma is included in the differential for plasmacytoid lesions (shows a Hofzone and plasmacytic IHC: CD79a, CD138). Clear cell myoepithelioma is included in another differential diagnostic group of lesions. Myoepithelial carcinoma may develop as either a de novo lesion or as part of a carcinoma expleomorphic adenoma. In either case, the typical features of malignancy—invasion, pleomorphism, necrosis, atypical mitoses, and increased mitoses—are present.270,271
KEY DIAGNOSTIC POINTS Myoepithelioma • A benign tumor of myoepithelial cells exclusively, lacking ductal elements and usually identified in the parotid or palate. • Solid, myxoid, reticular to cordlike proliferation of either spindled or plasmacytoid cells, possibly showing clear cells. • Variable reactivity with a variety of myoepithelial markers that include AE1/AE3, CK5/6, 34βE12, S-100 protein, p63, calponin, SMA, and SMMHC.
Mucoepidermoid Carcinoma Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy; it represents between 2% and 16% of all salivary gland tumors and up to one third of malignant salivary gland tumors.272 About 50% of MECs occur in the major salivary glands, and the minor salivary glands in the palate are the most common secondary site.273 Other affected sites include the retromolar area, floor of the mouth, buccal mucosa, lip, and tongue.274,275 MEC is the most common malignant salivary gland tumor in children,276-278 and rare tumors are thought to arise primarily within the bone.279 The tumors range from less than 1 cm up to large, disfiguring masses. They vary from circumscribed and encapsulated to poorly defined and widely invasive. Cysts can be seen macroscopically. Histologically, MEC is a malignant epithelial tumor with variable components of mucous, epidermoid, and intermediate cells. Cystic spaces (usually >10 cell widths across to qualify as a cyst) vary within each tumor and are used in the grading systems. They may be filled with mucus, debris, or blood. Papillary projections into the spaces are uncommon. Mucous cells can be either clear cells that contain glycogen or mucin,272 or they may be columnar gobletlike cells that contain abundant mucin; these can be prominent in low-grade tumors (Fig. 9-64).
A
B
C
D
Figure 9-64 Mucoepidermoid carcinoma (MEC) may show a wide range of histologic appearances. A,The clear cells are seen within an intermediate cell proliferation. These cells are not mucicarmine positive. B, The glandular component dominates in this MEC, with only limited epidermoid growth. C, A high-grade MEC shows nuclear pleomorphism, increased mitoses, and apoptosis. Cyst formation is limited. D, Only isolated cells have mucicarmine-positive mucin.
Salivary Glands
The mucin must be intracytoplasmic to qualify as true mucinous differentiation. When mucin is extravasated, an inflammatory or foreign-body giant cell reaction may be seen. Epidermoid cells are polygonal, large cells that have eosinophilic cytoplasm and well-defined borders, but frank keratinization is vanishingly rare, except in tumors previously subjected to needle-aspiration biopsy. Intermediate cells can be either basal or larger cells that have a transitional appearance between squamous and mucin-secreting cells. Intermediate cells are often in sheets or nests. Clear, columnar, and reserve cells make up minor components of MEC.273 Occasionally, MECs can be oncocytic, referred to as the oncocytic variant of MEC.280,281 Tumor-associated lymphoid proliferation (TALP) is often seen at the leading edge of the tumor, even forming germinal centers, and must not be confused with a lymph node. The grade of the tumor is based on the amount of cyst formation and presence or absence of necrosis, perineural invasion, increased mitoses (including atypical forms), and profound pleomorphism. Vascular invasion can also be seen but is not included in the grading system. A three-tier grading system is used that is strongly correlated to prognosis.272,282-284 Sclerosing, clear cell, and sarcomatoid types can also be seen. MEC reacts with various epithelial and basal markers, whereas p63 highlights the basal cells but can also react with the intermediate and epidermoid cells, and tumors that show a strong, diffuse nuclear reaction have been
295
associated with a poor prognosis; CK5/6 preferentially highlights the epidermoid cells and is frequently negative in transitional cells (Fig. 9-65). AE1/AE3 and CK7 usually yield a strong reaction in all of the neoplastic cells, although less so in mucocytes. Her-2/neu may be positive, often with a strong reaction in higher grade tumors, correlated to a poor prognosis.285,286 Ki-67 can be used to highlight proliferation and to more accurately assess tumor grade and prognosis.286 The differential diagnosis includes necrotizing sialometaplasia, mucocele (extravasation type), SCC, salivary duct carcinoma, and several clear cell tumors (Table 9-8). Necrotizing sialometaplasia usually has a lobular architecture, no cystic appearance, and lacks intermediate cells. Residual mucocytes should not be misinterpreted to be part of a tumor.287,288 Mucus extravasation reaction usually has mucus within macrophages and an inflammatory reaction that lacks epithelial or intermediate cells. SCC has well-developed intercellular bridges (spinous or prickle cells), areas of keratinization, dyskeratosis or keratin pearl formation, and more opacified cytoplasm but lacks intermediate cells. Metastatic SCC to intraparotid or salivary gland lymph nodes is much more common than a primary SCC of salivary glands. In minor glands within the oral cavity, a mucosalbased primary SCC growing into the minor mucoserous gland should be excluded. Clear cell malignancies, such as clear cell carcinoma (no mucocytes or intermediate cells), epithelial-myoepithelial carcinoma (no
Figure 9-65 Mucoepidermoid carcinoma will react with markers of squamoid differentiation. Left, CK5/6 highlight the epidermoid and intermediate cells. Upper right, Staining with p63 highlights the intermediate and squamoid cells. Lower right, Cytokeratin highlights the neoplastic cells, although not usually the mucocytes.
296
Immunohistology of Head and Neck Lesions
TABLE 9-8 Differential Diagnosis for Clear Cell Salivary Gland Tumors With Immunohistochemical Results Stain
Oncocytoma
Clear Cell Myoepithelioma
Hyalinizing Clear Cell Carcinoma
Renal Cell Carcinoma
EMA
N
+
+
+
CD10
N
S
NR
+
Vimentin
+
+
NR
+
Pax-2
NR
NR
NR
+
CA9
NR
NR
NR
+
RCC
N
NR
NR
+
CEA
NR
N
+
N
N
+
N
S
SMA
NR
+
N
N
GFAP
NR
+
N
N
+
+
NR
N
S-100 protein
p63
+, Almost always positive; N, negative; S, sometimes positive; R, rarely positive; NR, not reported. CEA, Carcinoembryonic antigen; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; RCC, renal cell carcinoma; SMA, smooth muscle actin.
mucocytes or intermediate cells, distinct biphasic appearance, supported immunophenotypically), and clear cell myoepithelioma (no mucocytes or epidermoid cells) can usually be separated by morphology and pertinent IHC.289 Primary intraosseous MEC must be separated from glandular odontogenic tumor, clear cell odontogenic carcinoma, and ghost cell odontogenic tumors. KEY DIAGNOSTIC POINTS Mucoepidermoid Carcinoma • Three cell populations can generally be seen in MEC— epidermoid cells, mucous cells, and intermediate cells— variably set within a cystic background. • CK5/6, Ki-67, and p63 nuclear expression may help with the differential diagnosis.
Polymorphous Low-Grade Adenocarcinoma Polymorphous low-grade adenocarcinoma (PLGA) is an uncommon, low-grade, slowly growing tumor that exclusively affects the minor salivary glands, most often of the palate.249,290,291 Females are affected twice as often as males, with a broad age range (16 to 95 years), but the mean age at presentation is 60 years. Most patients will have a slowly growing, firm, nontender mass, often discovered incidentally. The tumor is usually about 2 cm, solid to firm, and is covered by an intact mucosa. Histologically, PLGA is a malignant epithelial tumor characterized by infiltrative growth of cytologically uniform cells arranged in architecturally diverse patterns set within a characteristic stroma. Tumors are unencapsulated but well circumscribed. They incarcerate or entomb minor mucoserous glands and invade into
the adjacent soft tissues. Perineural invasion is prominent and yields a “targetoid” appearance with the nerve that forms the nidus (Fig. 9-66). The striking variety of growth patterns gives a low-power “eye of the storm,” or “streaming” appearance. Lobules, tubules, trabeculae, interconnecting cords or fascicles, and linear, single-cell infiltration is common (see Fig. 9-66). Rare cribriform areas may also be present. The tumor comprises isomorphic, small to medium cells with indistinct cell borders and pale to eosinophilic cytoplasm, surrounding round to oval nuclei with pale, open, vesicular nuclear chromatin (ground-glass like). Nucleoli are small to inconspicuous (Fig. 9-67), and mitoses are rare. Hyalinized, slate colored gray-blue stroma is seen between the cells, although collagenized stroma can also be seen. Chondroid matrix is absent, but tyrosine like crystals are occasionally seen. Metaplastic changes (squamous, sebaceous, mucous, clear, oncocytic) are uncommon.247,249,292 All tumor cells will be positive with AE1/AE3 and vimentin, and nearly all will be positive with S-100 protein, EMA, CK5/6, CK14, CAM5.2, and CK7. Myoepithelial markers such as SMA, MSA, and p63 will highlight selected cells; p63 is localized to peripheral tumor cells at the stromal junction (Fig. 9-68). Calponin is usually nonreactive, and GFAP is only focally positive, with a faint or weak stain. CD117 is positive in most tumor cells, as is Bcl-2. Ki-67 yields a low proliferation index, further supported by a less than 10% MCM2 reaction.60,247,249,255,258,267,292-294 The differential diagnosis is with pleomorphic adenoma (PA), adenoid cystic carcinoma (ACC), and adenocarcinoma not otherwise specified (NOS; see Table 9-7). Separation can be very difficult on small, core, or incisional biopsies. PA lacks perineural invasion and is often plasmacytoid in the palate. The chondromyxoid matrix material can help with identification. GFAP is usually strong and diffuse in PA, whereas PLGA shows a faint, focal, and weak reaction in luminal epithelial
Salivary Glands
Figure 9-66 Left, A polymorphous low-grade adenocarcinoma arranged in a swirling or whorling appearance (“eye of the stroma”). Note the single-cell infiltration at the periphery. Right, Neoplastic cells show a very prominent targetoid pattern of perineural invasion (top) and encasement of the minor mucoserous glands (bottom).
297
Figure 9-67 Left, The nuclei of a polymorphous low-grade adenocarcinoma are round to oval with delicate, very light nuclear chromatin and small, inconspicuous nucleoli. Right, The slate-gray stroma between the epithelial units is characteristic of this tumor.
A
B
C
D
Figure 9-68 Polymorphous low-grade adenocarcinoma shows a wide range of immunohistochemical (IHC) reactions. A, S-100 protein highlights the myoepithelial/basal cells (note the nerve at the center of the target). B, Epithelial membrane antigen preferentially highlights the luminal, glandular, or ductal cells. C, A triple-stained sample highlights both compartments in a single slide; p63 (brown nuclear stain) stains the basal cells, and CAM5.2 (red-membrane positive) and CK5/6 (brown cytoplasmic reaction) highlight the epithelial cells. D, Note the variability of reactivity with CK7, showing a prominent central reaction (top) and a more diffuse reaction (bottom), which underscores the remarkable variability that can be seen in these IHC findings.
298
Immunohistology of Head and Neck Lesions
cells.249,295 ACC lacks a streaming architecture, shows many more cribriform areas, and has peg- or carrotshaped, angular, hyperchromatic nuclei. The glycosaminoglycans and reduplicated basement membrane material are not seen in PLGA. ACC tends to have peripheral myoepithelial staining with S-100 protein, whereas EMA tends to stain only the true luminal cells in ACC but shows a strong and diffuse reaction in PLGA. ACC has a higher proliferation index with a much stronger and higher MCM2 reaction (>10%).247,258,295-297 KEY DIAGNOSTIC POINTS Polymorphous Low-Grade Adenocarcinoma • A malignant tumor characterized by infiltrative growth of cytologically uniform cells arranged in architecturally diverse patterns that develop exclusively in minor salivary glands. • Reacts with EMA, S-100 protein, and Bcl-2, findings that can help with the separation from PA and ACC.
Adenoid Cystic Carcinoma Adenoid cystic carcinoma (ACC) is a tumor with a relentless clinical course, characterized by repeated local recurrences, late metastasis, and ultimate death over a course of 15 to 20 years. It is more common in females (3 : 2) and occurs over a broad age range (mean, sixth
decade), although it is uncommon in individuals younger than 20 years of age. The tumor is found with equal frequency in major and minor salivary glands, and the palate is the most common minor salivary gland location. Patients have a mass, frequently associated with pain or nerve involvement. Tumors are poorly circumscribed and unencapsulated with a white-gray cut surface.298-301 Histologically, the tumor shows myoepithelial and ductal differentiation; tumors are widely infiltrative and show a predilection for perineural invasion. Many patterns can be seen, and cribriform (“Swiss cheese”), tubular, trabecular, and solid patterns are variably seen within each tumor (one usually predominates). The cribriform pattern is formed when pseudocysts that contain either amorphous glycosaminoglycans or hyalinized basal lamina material surround or engulf the epithelial cells (Fig. 9-69). Small, true ductal lumina are seen more often in the tubular pattern, where myoepithelial cells form the basal layer. Eosinophilic, hyalinized stroma separate the tumor nests. At least 30% of the tumor must have a solid pattern to be called a solid type. The mitotic rate is usually increased, and tumor necrosis is present in the solid pattern. All of the patterns are comprised of small to medium tumor cells with a high nuclear/cytoplasmic ratio. The nuclei are angulated and peg- or carrot-shaped with very heavy and coarse basophilic chromatin.
Figure 9-69 Left, Adenoid cystic carcinoma (ACC) arranged in a glandular pattern with the mucopolysaccharide material (“blue goo”) intimately admixed with the neoplastic cells. Upper right, A loose stroma surrounds the tubular to canalicular ACC cells. This pattern mimics canalicular adenoma. Lower right, A tubular pattern is prominent with the background reduplicated basement membrane material in this ACC.
Salivary Glands
A
B
C
D
299
Figure 9-70 A, The small “tubules” seen in adenoid cystic carcinoma can be highlighted with an epithelial membrane antigen, whereas the remaining myoepithelial cells are not highlighted. B, Cytokeratin 5/6 also highlights the inner luminal or epithelial cells. C, A monoclonal carcinoembryonic antigen selectively highlights just the luminal aspect. D, CD117 highlights the epithelial cells, shown here in a tubular pattern.
Figure 9-71 Upper left, Abluminal basal cells are highlighted by p63; the luminal cells are negative. Lower left, S-100 protein also highlights the myoepithelial cells. Right, MCM2 is a proliferation marker that shows a much higher percentage of positive cells than pleomorphic adenoma or polymorphous low-grade carcinoma, a finding that may be helpful in differential diagnosis with adenoid cystic carcinoma.
300
Immunohistology of Head and Neck Lesions
The neoplastic cells show a biphasic pattern of immunoreactivity with epithelial and myoepithelial markers. AE1/AE3, CK7, CAM5.2, CK5/6, CK14, CK19, and monoclonal CEA (mCEA) highlights the tumor cells, often differentially expressed between the luminal and abluminal cells (Fig. 9-70). CD117 is seen more often in the solid than in the tubular type, highlighting the epithelial cells (see Fig. 9-70). SMA, SMMHC, p63, and S-100 protein highlight the basal or abluminal cells (Fig. 9-71).57,87,90,250,260,266,293,294,296,302-304 Calponin also stains the abluminal cells but is not as constant a finding.88,93,250 MCM2 is expressed in G1/ G2/S and labels cells that are enabled to proliferate. It shows the highest expression (>10%) in ACC (see Fig. 9-71) but is low or absent in PLGA and PA.260,293 There is a balanced translocation between MYB and NFIB in up to 50% of ACC. An antibody to MYB can be detected by a strong IHC staining, specifically identified in the basal cells, even though all cells have the translocation. MYB has not yet been detected in other salivary gland neoplasms.305-309 ACC is usually straightforward to recognize when it has been totally excised. However, small core or incisional biopsies may pose difficulties in diagnosis. The major differential diagnoses are PA, PLGA, basal cell adenoma/adenocarcinoma, epithelial-myoepithelial carcinoma, and NEC. PA is not infiltrative, it lacks perineural invasion and glycosaminoglycan material, shows a chondromyxoid matrix, and often has plasmacytoid cells; the cells are usually reactive with S-100 protein
and GFAP in a different pattern than ACC. MCM2 is also differentially expressed. PLGA only develops in minor salivary glands, shows a targetoid or whorled growth, and has bland cells with pale, vesicular nuclear chromatin. EMA reacts in only the true lumen of ACC but reacts diffusely in PLGA, and MCM2 is differentially expressed. Basal cell adenoma and adenocarcinoma tend to show peripheral palisading and lack pleomorphism, tend to lack mitoses, and do not have glycosaminoglycan material. Basal cell adenocarcinoma is infiltrative by definition. Epithelial-myoepithelial carcinoma has large, clear myoepithelial cells in a periductal distribution and usually lacks mitoses. NEC does not have a cribriform pattern and is reactive with neuroendocrine markers, and the latter are absent in ACC. In the rare ACC that undergoes high-grade transformation, the areas of transformation tend to be only epithelial, losing the myoepithelial phenotype; there is strong overexpression of p53.92,93 KEY DIAGNOSTIC POINTS Adenoid Cystic Carcinoma • ACC has three prominent growth patterns: cribriform, tubular, and solid, and it is composed of epithelial and myoepithelial cells. • Differential immunohistochemical stains highlight each cell compartment and may be used in separation from other tumors in the differential.
Figure 9-72 Epithelial-myoepithelial carcinoma (EMC) can show many different patterns. Left, The classic “ductal” structure is surrounded by the myoepithelial proliferation, showing cleared cytoplasm. Upper right, An oncocytic variant shows compressed “ducts” in the center, whereas the myoepithelial cells are more oncocytic in appearance. Lower right, A spindle cell morphology can be seen in EMC, although if it is exclusively spindled without ducts, myoepithelial carcinoma would be the more appropriate category.
Salivary Glands
Epithelial-Myoepithelial Carcinoma Epithelial-myoepithelial carcinoma (EMC) is a lowgrade, malignant, biphasic salivary tumor that comprises 1% to 2% of all salivary neoplasms, the majority of which develop in the parotid gland.310,311 It is more common in women (2 : 1) and occurs in patients in the sixth and seventh decades.311,312 Patients come to medical attention with a slow-growing, painless mass that has often been present for years. Minor salivary gland tumors may be less well defined and show ulceration in comparison to their major-gland counterparts. Most tumors are about 2.5 cm in size, with a well-defined but unencapsulated appearance. The tumor is nodular or multinodular with a bosselated surface, and perineural, lymph, and vascular invasion are common. Tumors are partially encapsulated to unencapsulated and are arranged in variable proportions of a biphasic pattern of inner luminal epithelial ductlike cells and an outer abluminal layer of myoepithelial-like cells; the inner layer is formed by a single row of cuboidal
301
to columnar epithelial cells with dense eosinophilic cytoplasm that surrounds an oval-round nucleus. Secretion may be seen in the lumen, but it is not mucicarmine positive. The duct cells are surrounded by one or more layers of large, polygonal myoepithelial cells with clear cytoplasm (Fig. 9-72), indistinct cell borders, and eccentric vesicular nuclei. The myoepithelial cells have abundant glycogen (diastase-sensitive PAS+). Variable pattern mixtures can be seen that include organoid, thèque like, solid, glandular, tubular, papillary, and cystic patterns. Likewise, the tumor cells may be spindled, clear, oncocytic,311 or granular (see Fig. 9-72). In general, pleomorphism is mild. A hyalinized basement membrane like material may separate the ducts, and mitoses are sparse.313-315 On immunostaining, the ductal cells are strongly positive for cytokeratins such as AE1/AE3, CAM5.2, CK5/6, and CK7 (Fig. 9-73). The myoepithelial component is usually strongly positive for typical myoepithelial cell markers such as p63, SMA, S-100 protein, and calponin (Fig. 9-74). CD117 is also positive. Her-2/
A
B
C
D
Figure 9-73 Epithelial-myoepithelial carcinoma shows a well-developed differential staining with epithelial and myoepithelial markers. A, AE1/AE3 accentuates the inner ductal cells but also highlights the basaloid cells. B, AE1/AE3 highlights the luminal cells but does not react with the basaloid cells in this example. C, Cytokeratin 7 highlights the ductal cells. D, Cytokeratin 5/6 preferentially highlights the basal/myoepithelial cells.
302
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-74 Epithelial-myoepithelial carcinoma shows a well-developed differential staining with epithelial and myoepithelial markers. A, Smooth muscle actin highlights the myoepithelial compartments, as does the p63 (B). C, S-100 protein also highlights the myoepithelial compartment. D, CD117 is reactive in the neoplastic cells and highlights the ductal/tubular cells, but it can also be seen in the myoepithelial cells.
neu is not overexpressed, and the Ki-67 index is usually low, preferentially highlighting the myoepithelial cell compartment.89,256,263,267,312,313,316-318 The differential diagnosis is with PA, ACC, myoepithelioma or myoepithelial carcinoma, and clear cell tumors (MEC, clear cell adenocarcinoma, acinic cell carcinoma, oncocytoma; see Table 9-8). PA has a bosselated growth of a biphasic epithelial-myoepithelial population but is set in a myxochondroid matrix material, generally does not show a bilayered tubule formation, and lacks a prominent clear-cell population. ACC has a cribriform pattern, which is not seen in EMC, and the cells are peg- or carrot-shaped with hyperchromatic nuclei. Myoepithelioma and myoepithelial carcinoma lack ducts or tubules and may have areas of tumor-cell spindling. Clear cell MEC usually has cyst formation, mucocytes, and intermediate cells without a well-developed biphasic appearance. Clear cell acinic cell carcinoma usually shows other areas that are more characteristic, but it lacks a biphasic
appearance and generally lacks myoepithelial immunoreactivity. Clear cell adenocarcinoma is usually of minor salivary glands, lacks myoepithelial differentiation, and has very prominent hyalinization. Usually, several foci are evident within a clear cell oncocytoma that show granular cytoplasm. Oncocytoma are positive with p63 but generally not with other myoepithelial markers.
KEY DIAGNOSTIC POINTS Epithelial-Myoepithelial Carcinoma • EMC is a biphasic tumor that shows ductlike spaces surrounded by clear myoepithelial cells, often separated by dense hyalinized stroma. • The epithelial and the myoepithelial cell components can be distinctly separated with immunohistochemical stains.
Salivary Glands
Hyalinizing Clear Cell Carcinoma Many salivary and nonsalivary tumors contain clear cells. Among these are mucoepidermoid carcinoma, acinic cell carcinoma, oncocytoma, renal cell carcinoma, myoepithelioma, and clear cell odontogenic carcinoma.269,289,318,319 One rare tumor that shows clear cells is known as hyalinizing clear cell carcinoma. Patients come to medical attention in the sixth decade of life, and women are affected slightly more often than men (1.6 : 1). The vast majority of tumors arise primarily from minor salivary glands in the oral cavity (tongue, palate, floor of the mouth) and oropharynx but can occasionally affect the parotid and submandibular glands. The tumors are usually smaller than 3 cm, poorly circumscribed, and infiltrate into the adjacent tissues. This epithelial malignant salivary gland neoplasm is composed of neoplastic clear cells set within a loose to densely hyalinized stroma and must not have features of any other salivary gland tumor. The cells infiltrate into the surrounding tissues in sheets, cords, nests, or trabeculae, and perineural invasion is common. The monotonous cells are round to polygonal with sharply defined cell borders and variably clear cytoplasm, and they contain glycogen but not mucin
303
(Fig. 9-75). The nuclei are usually eccentric and not atypical, and a few cells may show a more eosinophilic cytoplasm. Gland or duct formation is not seen, and mitoses are rare. The cells are separated by a dense hyalinized stroma, although it may sometimes be loose to myxoid.246,294,318,319 The cells are positive for AE1/AE3, 34βE12, CAM5.2, CK7, EMA, p63, and occasionally for pCEA (Fig. 9-76).320 They are usually negative for myoepithelial markers that include S-100 protein, MSA, SMA, SMMHC, calponin, and GFAP and are also negative for CD10, CK20, vimentin, desmin, RCC, CA9, and Pax-2 (see Fig. 9-76).320-323 A consistent and novel EWSR1/ATF1 fusion is evident in hyalinizing clear cell carcinoma, which is not identified in the usual differential diagnosis of mucoepidermoid carcinoma and epithelial-myoepithelial carcinoma (by RT-PCR or FISH techniques).321 Although the differential diagnosis is quite broad, the lack of myoepithelial cells in hyalinizing clear cell carcinoma helps to remove several tumors from further consideration (see Table 9-8).269,318 Renal cell carcinoma metastatic to head and neck tends to have sinusoidal vessels and extravasated erythrocytes and demonstrates a reaction for RCC, vimentin, CD10, and Pax-2 among others, and it is negative with 34βE12, CK1, and p63.324
Figure 9-75 Left, A hyalinizing clear cell carcinoma shows a very heavy background of sclerosing fibrosis, in which the clear neoplastic cells are set. The clear cells may be single or focally clustered and show well-developed perineural infiltration. Upper right, Periodic acid– Schiff stain strongly highlights the cytoplasmic glycogen. Lower right, Negative mucicarmine stain (minor salivary glands are positive in the background).
304
Immunohistology of Head and Neck Lesions
A
B
C
D
Figure 9-76 Reaction pattern for hyalinizing clear cell carcinoma. A, Epithelial membrane antigen highlights the neoplastic cells. B, Staining with p63 shows a strong and diffuse nuclear reaction. C, Clear cell carcinoma is negative with S-100 protein, although the nerves and native minor mucoserous glands show a positive reaction. D, Note how the neoplastic cells are negative with vimentin, although the remaining tissues, including nerves, are strongly positive.
KEY DIAGNOSTIC POINTS Hyalinizing Clear Cell Carcinoma • Hyalinizing clear cell carcinoma is an infiltrative tumor composed of monotonous clear cells with prominent cell borders and abundant glycogen; they lack any myoepithelial differentiation. • The neoplastic cells are positive with AE1/AE3, CAM5.2, CK7, EMA, and p63; cells are negative with S-100 protein, calponin, actins, and GFAP. • The tumor must be separated from other tumors that have a clear cell component, including metastatic clear cell RCC.
Salivary Duct Carcinoma Salivary duct carcinoma (SDC) is a relatively uncommon high-grade adenocarcinoma that resembles highgrade breast ductal carcinoma. The tumor is clinically
aggressive and has a high rate of metastatic disease (30% of patients have metastases at presentation), and it is frequently the carcinoma component of a carcinoma ex-pleomorphic adenoma. Men are affected more commonly than women (3 : 1), and tumors develop most commonly in the seventh decade of life.325 The vast majority are identified in the parotid (90%), but rare masses may be found in the minor salivary glands. Rapid growth of a mass associated with nerve paresthesia, pain, or paralysis is common. Surface ulceration can be seen, and tumors are large (mean, 3.5 cm), poorly circumscribed, widely infiltrative, and show a multinodular appearance with a solid, cystic, and necrotic cut surface.325-330 Microscopically, SDCs are characterized by both intraductal and infiltrating ductal carcinoma. The tumor grows in rounded, solid, cystic, ductal, cordlike, papillary, and cribriform patterns with central comedonecrosis (Fig. 9-77). “Roman bridge” architecture is quite characteristic. Perineural, lymph, and vascular
Salivary Glands
305
Figure 9-77 Salivary duct carcinoma has a histologic appearance similar to breast ductal carcinoma. Left, A pleomorphic neoplastic proliferation with central comedonecrosis. Right, The cells are arranged in a solid to ductal pattern that shows pleomorphism and increased mitoses.
Figure 9-78 Salivary duct carcinoma (SDC). Left, SDC shows more of a squamoid appearance but also has ductal structures and heavy sclerosis. Upper right, The epidermoid or even squamous appearance of this SDC can cause confusion with metastatic squamous cell carcinoma. Note the central comedonecrosis. Lower right, An infiltrate tumor shows a keratin pearl or dyskeratosis focally within the proliferation.
invasion are easily identified, as are mitoses, including atypical forms. A desmoplastic fibrosis is usually conspicuous along with a heavy inflammatory infiltrate (Fig. 9-78), usually with a lack of nonneoplastic glandular tissue between tumor nodules. The tumor cells have moderate to marked pleomorphism, and cells can be cuboidal, columnar, polygonal, or apocrine to spindled.
The cytoplasm ranges from amphophilic to eosinophilic and granular and surrounds large, pleomorphic, hyperchromatic to vesicular nuclei with very prominent, irregular nucleoli (see Fig. 9-78). Dystrophic calcification may be seen and is often part of the residual pleomorphic adenoma in carcinoma ex-pleomorphic adenoma.
306
Immunohistology of Head and Neck Lesions
Several variants are recognized, although in general, typical features of SDC are still identified. Variants include sarcomatoid (SDC and spindle cell elements; spindled cells will be positive with EMA and p53), micropapillary (morulalike small epithelial cell clusters without fibrovascular cores, positive with CK7 and EMA, with a distinctive “inside-out” pattern), mucin rich, and osteoclast-type giant cell.328,329,331,332 The tumor cells are positive with AE1/AE3, CK7, CK5/6, and EMA (Fig. 9-79), and pCEA is expressed in most cases. More than 90% of SDCs are positive for Her-2/neu (membrane reaction), androgen receptor (AR; nuclear reaction), and E-cadherin (Fig. 9-80) but are nearly always negative for estrogen and progesterone receptors.60,325-327,329,332-335 Studies to evaluate prognostic significance and therapeutic significance are ongoing, and Herceptin therapy has shown promise. Isolated tumor cells will be positive with gross cystic disease fluid protein 15 (GCDFP-15) and BRST-2 (see Fig. 9-79). p63 may highlight an in situ proliferation because it lines the lobules, but it can also be present in the
neoplastic population.329,335 Rare cases may also stain with prostate-specific antigen (PSA) and/or prostatic acid phosphatase (PAP), which together with a positive androgen receptor (AR) may result in confusion with metastatic prostatic carcinoma.333 There is usually a very high Ki-67 proliferation index, and p53 is found in as many as 60% of cases.334 The major differential diagnostic considerations include metastatic SCC, metastatic breast and prostate carcinoma, cystadenocarcinoma, oncocytic carcinoma, and high-grade mucoepidermoid carcinoma. Metastatic SCC will usually have keratinization, well-developed cell bridges, and dyskeratosis. A primary skin lesion is most likely, but mucosal tumors are also in the differential diagnosis. SCCs are not Her-2/neu or AR positive, although there is overlap with several other antigens. Metastatic breast carcinoma generally presents after a known primary. If there is concurrent sialodochodysplasia, metastatic disease could be excluded. Depending on the primary tumor, metastatic tumor cells would be positive with estrogen receptor (ER) and
A
B
C
D
Figure 9-79 Salivary duct carcinoma shows a variety of epithelial and basal markers but also shows expression with breast and prostate markers. A, Epithelial membrane antigen. B, Staining with p63. C, CK5/6 (note the perineural invasion). D, BRST-2 highlights isolated cells.
Salivary Glands
A
B
C
D
307
Figure 9-80 Salivary duct carcinoma is positive with a number of prognostic markers or potentially therapeutic markers. A, Her-2/neu. B, Androgen receptor. C, E-cadherin. D, Ki-67 (note the very high proliferation index and a small area of comedonecrosis).
progesterone receptor (PR) but negative with AR. Metastatic prostatic carcinoma does not usually have a Roman-bridge pattern. The rare PSA reactivity may cause difficulty with diagnosis, although Her-2/neu, BRST-2, and GCDFP-15 may help.336 Cystadenocarcinoma is a papillary and cystic tumor that shows lowgrade cytologic features and generally lacks infiltration and comedonecrosis. Oncocytic carcinoma has very large tumor cells with abundant granular cytoplasm. Cystic, papillary, and cribriform patterns are usually lacking, and comedonecrosis is rare. High-grade MEC tends to lack a prominent papillary or cribriform pattern and will have mucocytes and transitional cells. KEY DIAGNOSTIC POINTS Salivary Duct Carcinoma • SDC resembles high-grade breast carcinoma. • SDC is positive for EMA, GCDFP-15, BRST-2, androgen receptor, and Her-2/neu.
Low-Grade Cribriform Cystadenocarcinoma Still in taxonomic limbo, the terms low-grade cribriform cystadenocarcinoma, low-grade salivary duct carcinoma, and low-grade intraductal carcinoma337-339 have all been applied to this tumor. The tumor develops predominantly in patients in the seventh decade of life and most commonly affects the parotid gland. Tumors are usually composed of single or multiple large cysts with an adjacent epithelial proliferation that may have a cribriform, papillary, or micropapillary architecture with a Roman-bridge formation (Fig. 9-81). The tumor cells are small, bland-appearing ductal cells with fine chromatin and small nucleoli. Apocrine features, including snouts, can be seen. Adjacent to the cysts are areas of intraductal papillary proliferation architecturally and cytologically similar to atypical ductal hyperplasia or low-grade ductal carcinoma in situ. Distinctive microvesicles that contain refractile pigment have also been described. Invasion is occasionally seen, as are rare mitoses and focal necrosis.
308
Immunohistology of Head and Neck Lesions
Figure 9-81 Low-grade cribriform cystadenocarcinoma demonstrates several different morphologic patterns.
The neoplastic cells are strongly and diffusely immunoreactive for S-100 protein (Fig. 9-82), and myoepithelial studies (calponin, p63) stain the supporting peripheral myoepithelial cells. If there is an intact myoepithelial layer, the tumor may be considered in situ or intraductal. Her-2/neu is negative. The primary differential is with acinic cell carcinoma, mammary analogue secretory carcinoma, cystadenocarcinoma, and salivary duct carcinoma. Acinic cell carcinoma does not have this architectural pattern and will have secretory granules and secretions in the background. Mammary analogue secretory carcinoma is also S-100 protein positive, but coexpression of mammaglobin, BRST-2, MUC1, and GCFDP-15 should help with the separation. Cystadenocarcinoma is a higher grade tumor. Salivary duct carcinoma is also a high-grade tumor and shows Her-2/neu and AR positivity. KEY DIAGNOSTIC POINTS Low-Grade Cribriform Cystadenocarcinoma • Low-grade cribriform cystadenocarcinoma is a rare tumor that grows in a variety of patterns. • This tumor is positive for S-100 protein in a strong and diffuse manner, supported by a layer of myoepithelial cells at the periphery.
Mammary Analogue Secretory Carcinoma A newly described entity reminiscent of secretory carcinoma of the breast, previously considered to be within the “nongranular” acinic cell carcinoma group, is now referred to as mammary analogue secretory carcinoma.340-342 The tumor resembles acinic cell carcinoma and lowgrade cystadenocarcinoma but lacks well-developed acinar differentiation. The tumor develops in middleaged patients (mean, 46 years) with a slight male predominance, mostly in major salivary glands. Most tumors are an intermediate size (mean, 2 cm).338-342
Figure 9-82 Low-grade cribriform cystadenocarcinoma shows typical strong and diffuse immunostaining with S-100 protein (left); a delicate layer of p63-positive myoepithelial cells is noted at the periphery of the tumor lobules (right).
Histologically, tumor growth is lobulated with evidence of invasion in some tumors; tumors have a microcystic to glandular appearance with eosinophilic, homogenous, or bubbly secretory material within the lumen, and several areas show thyroid colloidlike material (Fig. 9-83). The nuclei are vesicular with finely granular chromatin but distinct centrally placed nucleoli and ample pale pink, granular, or vacuolated cytoplasm. Pleomorphism is limited, mitoses are rare, and serous acinar differentiation is not seen.340-344 The lesional cells are positive with CK7, CK8, CK18, S-100 protein, and vimentin with strong reaction with mammaglobin STAT5a, MUC1, and MUC4; staining with GCDFP-15, EMA, and CD117 is variable (Fig. 9-84).344 The cells are negative with p63, calponin, CK14, SMA, and CK5/6. The Ki-67 proliferation index is quite variable, from 5% to 30%.340-344 This tumor has a specific balanced chromosomal translocation detected by RT-PCR or FISH with an ETV6-NTRK3 fusion transcript at t(12;15)(p13;q25), identical to secretory breast carcinoma.340 The major differential is with acinic cell carcinoma and papillary cystadenocarcinoma. This tumor lacks any granules typical of acinic cell carcinoma and shows a homogenous appearance of microcystic and dilated glandular spaces with secretory material present in the lumen. Papillary cystadenocarcinoma tends to lack mammaglobin, MUC1, and MUC4 expression.340
KEY DIAGNOSTIC POINTS Mammary Analogue Secretory Carcinoma • A lobular tumor with microcystic and glandular spaces filled with eosinophilic homogenous or bubbly secretions but lacking acinar granules. • This tumor is positive with AE1/AE3, mammaglobin, MUC1, MUC4, S-100 protein, STAT5a, GCDFP-15, and EMA.
Salivary Glands
309
Figure 9-83 Left, Mammary analogue secretory carcinoma shows a microcystic and glandular appearance with colloidlike material within the lumen. Right, The nuclei are vesicular with a distinct nucleolus. Note the colloidlike material.
A
B
C
D
Figure 9-84 Mammary analogue secretory carcinoma demonstrates features similar to breast tumor. A, S-100 protein staining in most of the tumor cells. B, Mammaglobin highlights most of the neoplastic cells, with the secretions highlighted. C, MUC1 shows a membraneluminal reactivity. D, Gross cystic disease fluid protein 15 highlights isolated cells but is also present in the secretions.
310
Immunohistology of Head and Neck Lesions
Theranostic Applications Although CD117 is typically positive in ACC, PLGA, and PA, these tumors do not harbor mutations in the c-Kit gene, and therefore Gleevec has shown little effectiveness against ACC.87,345,346
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications The molecular events that contribute to the development of salivary gland tumors such as MEC, ACC, hyalinizing clear cell carcinoma, and mammary analogue secretory carcinoma are beginning to be identified. An interesting discovery in MEC was the identification of t(11;19)(q21-22;p13) between WAMTP1(MECT1) and MAML2, which disrupts the notch signaling pathway.347-350 Recent evidence suggests that this translocation may have prognostic relevance, because it is seen almost exclusively in low- and intermediategrade MEC and not in high-grade tumors.348 There is a balanced translocation between MYB and NFIB in as much as 50% of ACC, which can be detected by an antibody to MYB; this antibody has not yet been detected in other salivary gland neoplasms.305-309 A consistent and novel EWSR1-ATF1 fusion is found in hyalinizing clear cell carcinoma that is not seen in other tumors in the differential diagnosis (MEC, epithelial-myoepithelial carcinoma). Most certainly an antibody will be available soon.321 Finally, mammary analogue secretory carcinoma has been defined by the balanced translocation and creation of a fusion product, ETV6-NTRK3.340-344
Figure 9-85 The ectopic glial tissue (lighter pink) is noted within a background of heavy fibrosis (darker pink). A peripheral nerve (upper left) is entrapped within the fibrosis.
Ear and Temporal Bone Glial Heterotopia Heterotopic neuroglial tissue is defined as an ectopic mass of mature brain tissue isolated from the cranial cavity.351 Described in several anatomic sites, middle ear/temporal bone and nasal sites are the most common.352,353 Glial heterotopia (GH) must be separated from an encephalocele, which maintains a CNS connection.354 Patients of all ages can be affected. However, patients without previous surgery, trauma, or infectious etiologies tend to be much younger. GH is often completely unexpected in the specimen and may be part of another lesion (cholesteatoma, granulation tissue). The tissue is often quite fibrotic, and the fibrosis partially obscures the true lesional cells. Gliosis often mimics fibrosis (Fig. 9-85). Chronic inflammation is frequently seen, but leptomeninges, ependyma, and choroid plexus are not identified.352,353 The lesional cells are positive with GFAP, S-100 protein (glial tissue; Fig. 9-86), and NFP, which highlights the neuronal tissue. Cytokeratins are negative. Mature glial tissue is often seen in a teratoma, but all three germ cell primordia are present. A true astrocytoma has an increased cellularity, disorganized growth, and lacks fibrosis. A meningoma shows whorled or
Figure 9-86 Glial heterotopia can be highlighted with either an S-100 protein (left) or a glial fibrillary acidic protein (right) immunohistochemical reaction. Note the peripheral nerve (left) as an internal control.
syncytial cells, intranuclear cytoplasmic inclusions, and sometimes psammoma bodies, and it is immunoreactive with EMA.
KEY DIAGNOSTIC POINTS Glial Heterotopia • Glial heterotopia represents glial tissue in an abnormal location; middle ear, temporal bone, and nasal sites are the most common. • The lesional cells are positive with GFAP and S-100 protein but are negative with keratin.
Ear and Temporal Bone
Ceruminous Adenoma An uncommon benign, glandular neoplasm of ceruminous glands (modified apocrine sweat glands), ceruminous adenoma arises solely from the external auditory canal, specifically the outer half.355 Most tumors are small (mean, 1.2 cm), largely a function of the anatomic confines of the region. Grossly polypoid, these masses are usually fragmented during removal. The tumors are grouped histologically into three types: 1) ceruminous adenoma, 2) ceruminous pleomorphic adenoma, and 3) ceruminous syringocystadenoma papilliferum.355 The tumors are well circumscribed but not encapsulated, lacking surface origin, and they are moderately cellular, arranged as glandular profiles with a dual cell population; inner luminal secretory cells are apparent with apocrine decapitation secretions that occasionally show lipofuscinlike pigment granules (cerumen; Fig. 9-87). These cells are surrounded by a basal myoepithelial cell layer. Pleomorphism is limited, and mitoses are lacking. Ceruminous pleomorphic adenoma shows a classic pleomorphic adenoma appearance with ceruminous differentiation within the epithelial cells. Ceruminous syringocystadenoma papilliferum has papillary projections lined by epithelial cells and a dense plasmacytic infiltrate.355,356 By IHC there is an accentuation of the biphasic appearance. The luminal cells are positive with AE1/ AE3, EMA, and CK7 (see Fig. 9-87), whereas only AE1/ AE3 and EMA are positive in the basal cells. The basal
311
cells are also positive with CK5/6, p63, and S-100 protein (Fig. 9-88). CD117 is usually seen in the luminal cells but can also be seen in the basal cells. There is no reactivity with neuroendocrine markers.355,357 The tumor must be separated from ceruminous adenocarcinoma, typically by observing an infiltrative/ destructive growth, increased pleomorphism, prominent nucleoli, lack of cerumen pigment granules, and the presence of necrosis and mitoses (Fig. 9-89). Ki-67 may help to highlight increased mitoses. A neuroendocrine adenoma of the middle ear arises in a different anatomic site but shows plasmacytoid cells, neuroendocrine nuclear chromatin distribution, and no decapitation secretions. The cells are positive with chromogranin, synaptophysin, and CD56. Paraganglioma has a zellballen architecture and isolated pleomorphic cells, and the cells will be reactive with chromogranin, synaptophysin, and CD56.
KEY DIAGNOSTIC POINTS Ceruminous Adenoma • Ceruminous adenoma is an unencapsulated benign tumor of ceruminous apocrine glands of the outer half of the external auditory canal. • Luminal cells are positive with pancytokeratin, EMA, and CK7, whereas basal cells are also positive with CK5/6, p63, and S-100 protein.
Figure 9-87 Ceruminous adenoma. Left, The inner luminal cells show decapitation secretions along with cerumen pigment (yellow cytoplasmic granules). Differential staining with CK7 (upper right) stains the luminal cells, and CK5/6 (lower right) highlights the basal myoepithelial cells.
312
Immunohistology of Head and Neck Lesions
Figure 9-88 S-100 protein reacts with the basal cells of a ceruminous adenoma (left). Upper right, Staining with p63 highlights the basal cells. Lower right, CD117 predominantly highlights the luminal secretory cells.
Figure 9-89 Ceruminous adenocarcinoma is a widely invasive tumor, with heavy sclerosing fibrosis (left) and central comedonecrosis (right). Profound nuclear pleomorphism and numerous mitoses, including atypical forms, are apparent.
Paraganglioma Paragangliomas of the head and neck can occur in many locations, most commonly in the neck (carotid or vagal bodies), middle ear (jugulotympanic), and larynx358-363 with exceptional cases occuring in the nasal or oral
cavity.241,242,359 The tumors arise from paraganglia, which are chemoreceptor cells derived from neural crest that respond to changes in blood oxygen and carbon dioxide levels. This tumor follows the “rule of 10” in that it is 10% multicentric, 10% bilateral, 10% familial, 10% pediatric, and 10% malignant. Patients come to medical attention over a wide age range, with a mean in the sixth decade of life. Sporadic tumors show a strong female predilection (5 : 1), whereas syndrome-associated tumors are more common in males.358,361 Angiography and octreotide or metaiodobenzylguanidine (MIBG) scintigraphy may aid in identifying occult or multifocal tumors, and presurgical embolization will reduce bleeding.364 Tumors are irregular, firm, reddish masses from 0.3 up to 6 cm. Histologically, tumors are usually infiltrative, lack encapsulation or circumscription,365 and are arranged in a characteristic clustered, nested, or zellballen architecture. These balls of paraganglia are surrounded by a layer of supporting or sustentacular cells, not readily appreciable by H&E alone, and are invested by a richly vascularized stroma (Fig. 9-90). Tumors are cellular and composed of intermediate cells that contain ample granular to basophilic cytoplasm. The nuclei are rather monotonous, although isolated marked pleomorphism can be seen. Nuclear chromatin is usually delicate to coarse, and mitoses are inconspicuous.361,362 Malignancy in extraadrenal paraganglioma rests nearly exclusively on finding metastatic deposits.366 Necrosis is seen after embolization, and capsular, vascular, and perineural invasion are not indicators of malignancy.367
Ear and Temporal Bone
Figure 9-90 Left, A paraganglioma is identified immediately below an intact squamous epithelium. A rich vascular plexus surrounds the zellballen alveolar architecture. Right, Sometimes the paraganglia cells are difficult to see within the richly vascularized stroma; cases such as this can be augmented with immunohistochemistry.
313
The paraganglia cells (chromaffin cells) are positive with a variety of neuroendocrine markers that include synaptophysin (delicate cytoplasmic), chromogranin (granular cytoplasmic), NSE (diffuse cytoplasmic), and CD56 (membrane; Fig. 9-91).368,369 The peripherally located sustentacular supporting cells are positive for S-100 protein and GFAP (see Fig. 9-91 and Table 9-6).370 However, the S-100 protein may be lost in malignant tumors.371,372 The tumor cells are negative for cytokeratins, EMA, CEA, and human pancreatic polypeptide. Germline mutations in several genes that encode various subunits of the succinate-ubiquinone oxidoreductase gene (SDH) can be identified. These enzymes are in the mitochondrial respiratory chain complex II. Specifically, PGL1 to PGL4 encodes SDH subunits A through D on chromosome 11q, 1q, and 1p. Inactivating mutations in SDHB, SDHC, and SDHD genes cause hereditary paraganglioma.373,374 The differential diagnosis of paraganglioma will vary by anatomic location and by the size of the biopsy (often small from the middle ear and sinonasal tract vs. neck). The differential diagnosis of jugulotympanic paraganglioma will include neuroendocrine adenoma of
A
B
C
D
Figure 9-91 The cells of a paraganglioma may be highlighted differentially. A, Chromogranin. B, S-100 protein stains the sustentacular cells (note the negative surface epithelium at the top). C, CD56 highlights the paraganglia cells. D, S-100 protein shows a more delicate reaction of the sustentacular cells.
314
Immunohistology of Head and Neck Lesions
the middle ear (NAME), schwannoma, and meningioma.375,376 NAME is a neuroendocrine lesion but shows a different pattern of growth, has a glandular appearance and salt-and-pepper chromatin, and demonstrates strong keratin reaction but is negative for S-100 protein. Meningiomas are often EMA positive and are negative for neuroendocrine markers.376 Schwannoma tends to be spindled, has peritheliomatous hyalinization, and will be diffusely reactive with S-100 protein only. Paraganglioma in other sites must be separated from other epithelial NETs such as carcinoids, atypical carcinoids, SCNEC, and medullary thyroid carcinoma (see Table 9-6). KEY DIAGNOSTIC POINTS Paraganglioma • Paraganglioma develops most commonly in the neck, middle ear, and larynx. • Tumor cells are reactive with chromogranin, synaptophysin, and CD56 with sustentacular reaction for S-100 protein or GFAP. • Malignancy is based only on metastatic disease in head and neck tumors.
Ectopic Meningioma Meningioma is a benign neoplasm of meningothelial cells frequently identified outside the cranial cavity, such as within the ear and temporal bone or sinonasal
tract, among other sites. No matter the site, it is important to exclude an intracranial tumor with extraneuraxial extension before yielding an “ectopic” diagnosis.133,376-379 Meningiomas of the temporal bone/mastoid or sinonasal tract are more common in females than males (2 : 1), and most patients are middle aged (average 50 years). Most tumors involve the middle ear followed by the external auditory canal, whereas sinonasal tract tumors involve the nasal cavity most frequently. The tumors are often sizeable, and imaging studies are required to exclude a possible intracranial component.133,376,379 Histologically, ectopic meningiomas resemble their intracranial counterparts, and meningothelial and psammomatous meningiomas are the most common types. Syncytial, whorled, cohesive epithelial cell clusters are set within a fibrous stroma; the cells have a bland appearance, a low nuclear/cytoplasmic ratio, delicate nuclear chromatin, and intranuclear cytoplasmic inclusions (Fig. 9-92). Psammoma bodies and prepsammoma bodies are often seen, but necrosis, pleomorphism, and atypical mitoses are uncommon. The neoplastic cells are uniformly positive with vimentin and show variable reactivity with EMA and PR; cells may show a strong CK8/18 and CK7 reaction in a peripsammomatous location (Fig. 9-93), and pCEA may also give a similar appearance. The differential diagnosis includes schwannoma, paraganglioma, NAME, and meningocele. The strong S-100 protein immunoreactivity in a schwannoma along
Figure 9-92 Meningioma. Left, The slightly whorled arrangement is seen within this meningioma, with focal concretions or “prepsammoma” bodies. Upper right, A vague nested to whorled architecture is noted. Lower right, The concretions within this meningioma are a mimic of a neuroendocrine adenoma of the middle ear. Note the infiltrative pattern within the fibrous connective tissue stroma.
Ear and Temporal Bone
A
B
C
D
315
Figure 9-93 Various immunohistochemistries will help confirm the diagnosis of a meningioma. A, Epithelial membrane antigen stains a few cells but also highlights the prepsammoma body–like structures. B, Vimentin shows a strong cytoplasmic reaction. C, CAM5.2 can be seen in a few cells and in a peripsammoma body–like reaction. D, Polyclonal carcinoembryonic antigen (pCEA) strongly highlights the psammomalike concretions.
with spindled, alternating cellular and hypocellular areas and perivascular hyalinization should help with the separation. Paraganglioma will have more of a nested architecture, basophilic cytoplasm, and isolated nuclear pleomorphism. The chromaffin cells will be positive with chromogranin, synaptophysin, and/or CD56. The cells of a NAME are infiltrative, show a glandular architecture, and will have salt-and-pepper nuclear chromatin distribution. The cells are immunoreactive with chromogranin, synaptophysin, and/or CD56. A meningocele is an acquired cystic lesion, usually associated with a previous surgery or an infectious or traumatic setting; it will have an IHC profile similar to meningioma.
KEY DIAGNOSTIC POINTS Ectopic Meningioma • Meningioma may develop in an ectopic location or may be a direct extension from an intracranial site. • A meningothelial or psammomatous pattern is present with a whorled architecture, bland cytology, and intranuclear cytoplasmic inclusions. • Lesional cells will be positive with vimentin and EMA; CK8/18 or CK7 may show a peripsammomatous reactivity.
Middle Ear Adenoma Neuroendocrine adenoma of the middle ear (NAME), synonymously referred to as middle ear adenoma or middle ear adenomatous tumor (MEAT), is a benign glandular neoplasm of the middle ear that shows both cytomorphologic and IHC evidence of neuroendocrine and mucinous differentiation.375,380-382 This uncommon tumor presents in the fifth decade of life with an equal sex distribution. Symptoms are nonspecific, although hearing loss is common. The tumor frequently encases or entraps the ossicular chain, which must be removed if the patient is to be disease free. The unencapsulated neoplastic cells are found below the surface epithelium, arranged in a variety of different patterns, from glandular to trabecular, cordlike, solid, and single cell (Fig. 9-94). The tumor cells are juxtaposed to one another, frequently back-to-back, and secretions are occasionally noted. A dual cell population shows inner, luminal, flattened eosinophilic cells surrounded by a basal, cuboidal-columnar cell layer with granular cytoplasm and eccentric nuclei (Fig. 9-95). The chromatin is delicate and fine, mitoses are inconspicuous, and myoepithelial cells are not present. Desmoplasia contributes to the “infiltrative” sense of the tumor. Lymph and vascular invasion, necrosis, and pleomorphism are absent. Concurrent cholesteatoma may be seen.375,380-383
316
Immunohistology of Head and Neck Lesions
Figure 9-94 A neuroendocrine adenoma of the middle ear characteristically shows a wide variety of patterns that include single, glandular, solid, and trabecular cells.
Figure 9-95 Left, The glandular cells show an “inner” lining by a second population. Upper right, AE1/AE3 reacts with all of the tumor cells, but it reacts more strongly with the inner luminal cells. Lower right, Cytokeratin 7 highlights only the inner lining cells.
Differential IHC shows the inner luminal cells to be positive with CK7, whereas the outer cells are positive with chromogranin, synaptophysin, CD56, and human pancreatic polypeptide (Fig. 9-96). Both cell types will be reactive with AE1/AE3, CAM5.2, and vimentin. The cells are negative with S-100 protein, EMA, GFAP, and TTF-1.375,381,383,384 The differential diagnosis includes otitis media with glandular differentiation (OMGD), ceruminous
adenoma, jugulotympanic paraganglioma, endolymphatic sac tumor (ELST), and a metastasis to the temporal bone. In contrast to middle ear adenoma (MEA), the glands in OMGD are not densely concentrated but rather are loosely arranged in an inflammatory background (Fig. 9-97). These glands are negative for neuroendocrine markers. A ceruminous adenoma involves the external auditory canal, is circumscribed, lacks atypia, shows apocrine snouts, and stains with CK5/6,
Ear and Temporal Bone
A
B
C
D
317
Figure 9-96 Neuroendocrine adenoma of the middle ear is usually positive with a variety of neuroendocrine markers. A, Chromogranin. B, Synaptophysin. C, Neuron-specific enolase. D, Human pancreatic polypeptide.
Figure 9-97 Otitis media with glandular differentiation or tunnel clusters can sometimes simulate a neuroendocrine adenoma of the middle ear (left). Histiocytes, calcifications, and inflammatory cells are often present (right), whereas neuroendocrine markers would be lacking.
318
Immunohistology of Head and Neck Lesions
p63, and S-100 protein but not with neuroendocrine markers. A paraganglioma lacks glands, has a nested growth, and is negative with cytokeratin but positive with neuroendocrine markers and S-100 protein (sustentacular). An ELST develops in a specific site, has a characteristic papillary architecture, and shows keratin immunoreactivity only. Metastatic tumors generally show cellular pleomorphism, abnormal mitoses, and infiltrative growth with specific markers unique to the underlying tumor type. KEY DIAGNOSTIC POINTS Neuroendocrine Adenoma of the Middle Ear • Middle ear adenoma is an infiltrative tumor that arises within the middle ear, often encasing the ossicular chain. • The neoplastic cells are arranged in glands, nests, and single cells with a biphasic appearance of small cells that have a “salt-and-pepper” nuclear chromatin distribution and scant mitoses. • The neoplastic cells are reactive with AE1/AE3, CAM5.2, CK7, chromogranin, synaptophysin, CD56, and human pancreatic polypeptide.
Endolymphatic Sac Tumor Endolymphatic sac tumor (ELST) is a slow-growing, locally aggressive, papillary tumor of endolymphatic sac
origin that characteristically involves the middle ear/ temporal bone and endolymphatic sac specifically, and it has a high association with von Hippel-Lindau (VHL) syndrome.385-388 Approximately 10% to 15% of VHL patients will have endolymphatic sac tumors.389 Patients come to medical attention with hearing loss, tinnitus, vertigo, and vestibular dysfunction, which, if bilateral, must raise the suspicion of VHL. Symptoms referable to other organs affected by VHL may also be present. Imaging studies are required to show the often large and destructive, hypervascular, lytic lesions within the endolymphatic sac. Tumors are often large, as much as 10 cm in greatest dimension.386,390-396 These unencapsulated, destructive lesions result in bone invasion and remodeling. The tumor is arranged in simple, coarse, broad papillary projections within cystic spaces. Fibrovascular cores are usually seen within the papillary structures (Fig. 9-98). The cystic spaces may contain serum, secretions, or erythrocytes, and cuboidal cells that line the projections have indistinct cell borders and surround small, round, hyperchromatic nuclei. The neoplastic cells are immunoreactive with AE1/ AE3, CAM5.2, vimentin (see Fig. 9-98), and EMA and stain focally or weakly with NSE, S-100 protein, and GFAP, whereas they are negative with TTF-1, thyroglobulin, and transthyretin.384,387,392,397-399 ELST is most often confused with metastatic thyroid papillary carcinoma (TPC), choroid plexus papilloma (CPP), and metastatic carcinoma. Metastatic carcinoma tends to show a more destructive pattern, has
Figure 9-98 Left, An endolymphatic sac tumor shows a broad papillary architecture with fibrovascular cores. The cells are simple cuboidal to columnar cells. Note the secretions or colloidlike material. AE1/AE3 (upper right) and cytokeratin 7 (lower right) are strongly and diffusely positive in the neoplastic cells. Thyroid transcription factor 1 and thyroglobulin are negative.
Metastatic Tumors
marked pleomorphism, increased mitoses, and often necrosis. Metastatic TPC has nuclear contour irregularities, chromatin clearing, and overlapping, and it will be positive with thyroglobulin and TTF-1. CPP usually occurs in the midline with no bone destruction and shows a positive reaction for transthyretin and GFAP, whereas ELST is usually negative for both.398,400 KEY DIAGNOSTIC POINTS
319
Metastatic Tumors Almost every malignancy can result in metastases to the head and neck, whether to lymph nodes, organs, or mucosal sites. Although there are differences in frequency and primary site based on lymph node versus organ or mucosal site, the following are some that should be considered.
Endolymphatic Sac Tumor
Lung Carcinoma
• Endolymphatic sac tumor is a low-grade papillary epithelial neoplasm that arises from the endolymphatic sac and shows a well-developed association with von Hippel-Lindau syndrome. • The tumors are unencapsulated, destructive lesions that show simple, coarse, papillary cells within cystic spaces lined by cells that are low cuboidal to columnar. • The neoplastic cells are positive with cytokeratin 7 and CAM5.2 and are focally positive with S-100 protein, EMA, and GFAP. The cells are negative with thyroglobulin, TTF-1, and transthyretin.
Primary lung carcinomas may result in metastases to lymph nodes (most commonly lower cervical) or to various organs of the head and neck. The major types are adenocarcinoma, SCC, and NEC (whether atypical carcinoid or small cell carcinoma/large cell carcinoma). Although each tumor has a specific IHC profile, considerable overlap may be found between a primary and metastatic tumor. For example, a lung adenocarcinoma may be TTF-1, CK7, and napsin positive. This profile is identical for a tall cell variant of thyroid papillary carcinoma (Fig. 9-99).401 Lung SCCs show a similar
A
B
C
D
Figure 9-99 Metastatic lung adenocarcinoma to the thyroid gland. A, Note the difference in the metastatic tumor set within the colloidfilled spaces of the thyroid gland. B,Thyroglobulin is positive in the native tissue but not in the lung tumor. C, Thyroid transcription factor 1 is positive in both components but in a different pattern for each cell type. D, Napsin is shown here in a tall-cell variant of papillary thyroid carcinoma. It is important to know that napsin is not unique to the lung.
320
Immunohistology of Head and Neck Lesions
immunophenotype as SCCs in other sites, although they are not as frequently p16 positive as primary oropharyngeal carcinoma. Small cell carcinomas show AE1/AE3, TTF-1, and various neuroendocrine markers in most cases. However, this same profile can be seen in medullary carcinoma and in small cell carcinomas of the sinonasal tract and larynx. Clinical and imaging correlation is mandatory in settings with the potential for a metastatic tumor.
Metastatic Renal Cell Carcinoma Metastatic renal cell carcinoma (RCC) to the head and neck area may be seen as the sentinel event, or it may be part of known metastatic disease.402 Histologically, these tumors usually exhibit clear cells set within a richly vascularized stroma, showing extravasated erythrocytes. The differential diagnosis is dependent on the site affected but can range from disease of the primary salivary glands, sinonasal tract, larynx, oral cavity, temporal bone, or endocrine organs besides lymph node disease. Renal neoplasms express a wide variety of markers that include AE1/AE3, vimentin, Pax-2, Pax-8, RCC marker, CD10, CA9, E-cadherin, claudin-7, CD117, TFE3, thrombomodulin, uroplakin III, p63, and CD57, although each specific marker is uniquely expressed in various renal tumors and their subtypes.403,404 Pax-2 can be helpful, but it is important to remember it is positive in renal tumors, Wilms tumor, RMS, thyroid tumors, and B-cell acute lymphocytic
leukemia, among other tumors. In this setting, an IHC panel that includes CD10, Pax-2, and Pax-8 is recommended to exclude RCC.150
Metastatic Breast or Prostate Adenocarcinoma Breast and prostate carcinomas may result in metastatic disease to the cervical lymph nodes and less commonly to head and neck organ sites.336,405-407 Both of these tumors can have a variety of histologic appearances. A glandular or single-cell pattern can be seen, and prostatic adenocarcinoma may have prominent nucleoli. The immunostaining pattern of these tumors is similar to that of primary prostatic adenocarcinoma, although
KEY DIAGNOSTIC POINTS Metastatic Tumors • Metastatic tumors are most commonly attributed to head and neck primary tumors, but sites below the clavicle are also possible. • An IHC staining panel can often help to identify the tumor type. • Determining the source of a metastatic carcinoma in the head and neck can be difficult or impossible even with IHC stains; often it will require clinical and radiologic correlation.
Figure 9-100 Metastatic breast carcinoma to the ear and temporal bone (left). Note the glandular profiles immediately adjacent to bone. Upper right, Estrogen receptor highlights the nuclei. Lower right, Her-2/neu yields a strong, membrane reaction. The primary tumor would also need to be positive with these markers before they could be differentially used in this setting.
Summary
some poorly differentiated tumors can lose PSA reactivity.336 It can be difficult to differentiate metastatic adenocarcinoma from salivary duct carcinoma (see the section on salivary gland tumors) owing to possibly overlapping PSA, AR, and PAP staining profiles. Clinical and radiographic correlation will be essential. Breast carcinoma can metastasize anywhere in the head and neck. The glandular or single-file infiltration can be quite characteristic, supported by positive ERs, PRs, and/or Her-2/neu (Fig. 9-100).
Summary A plethora of primary tumors can arise in the head and neck region, and this region may also serve as a
321
repository for metastatic tumors. The differential diagnosis by light microscopy, in concert with the immunostaining insight supplied here, should help resolve the majority of diagnostic problems.
Acknowledgements Thank you to Dr. Veronica A. Levy for her incomparable medical text editing combined with the rich sentiment behind it: to make this chapter accessible to all. A special thanks to Ms. Hannah Herrera for her research assistance. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Barbareschi M, Pecciarini L, Cangi MG, et al: p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol. 25:1054–1060, 2001. 2. Wenig BM: Squamous cell carcinoma of the upper aerodigestive tract: precursors and problematic variants. Mod Pathol. 15:229– 254, 2002. 3. van Oijen MG, Leppers VD, Straat FG, et al: The origins of multiple squamous cell carcinomas in the aerodigestive tract. Cancer. 88:884–893, 2000. 4. Sturgis EM, Wei Q: Genetic susceptibility–molecular epidemiology of head and neck cancer. Curr Opin Oncol. 14:310–317, 2002. 5. Crowe DL, Hacia JG, Hsieh CL, et al: Molecular pathology of head and neck cancer. Histol Histopathol. 17:909–914, 2002. 6. van Oijen MG, Slootweg PJ: Oral field cancerization: carcinogen-induced independent events or micrometastatic deposits? Cancer Epidemiol Biomarkers Prev. 9:249–256, 2000. 7. Herrero R, Castellsague X, Pawlita M, et al: Human papillomavirus and oral cancer: the International Agency for Research on Cancer multicenter study. J Natl Cancer Inst. 95:1772–1783, 2003. 8. Hafkamp HC, Speel EJ, Haesevoets A, et al: A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8. Int J Cancer. 107:394– 400, 2003. 9. Ha PK, Pai SI, Westra WH, et al: Real-time quantitative PCR demonstrates low prevalence of human papillomavirus type 16 in premalignant and malignant lesions of the oral cavity. Clin Cancer Res. 8:1203–1209, 2002. 10. Gillison ML, Shah KV: Human papillomavirus-associated head and neck squamous cell carcinoma: mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers. Curr Opin Oncol. 13:183–188, 2001. 11. Hobbs CG, Sterne JA, Bailey M, et al: Human papillomavirus and head and neck cancer: a systematic review and meta-analysis. Clin Otolaryngol. 31:259–266, 2006. 12. Lewis JS Jr: p16 Immunohistochemistry As a Standalone Test for Risk Stratification in Oropharyngeal Squamous Cell Carcinoma. Head Neck Pathol. 6(Suppl 1):75–82, 2012. 13. Isayeva T, Li Y, Maswahu D, et al: Human papillomavirus in non-oropharyngeal head and neck cancers: a systematic literature review. Head Neck Pathol. 6(Suppl 1):104–120, 2012. 14. Mulvany NJ, Allen DG, Wilson SM: Diagnostic utility of p16INK4a: a reappraisal of its use in cervical biopsies. Pathology. 40:335–344, 2008. 15. Benevolo M, Vocaturo A, Mottolese M, et al: Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis. Am J Clin Pathol. 129:606–612, 2008. 16. Bernard JE, Butler MO, Sandweiss L, et al: Anal intraepithelial neoplasia: correlation of grade with p16INK4a immunohistochemistry and HPV in situ hybridization. Appl Immunohistochem Mol Morphol. 16:215–220, 2008. 17. O’Regan EM, Toner ME, Finn SP, et al: p16(INK4A) genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas. Hum Pathol. 39:452–458, 2008. 18. Slebos RJ, Yi Y, Ely K, et al: Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma. Clin Cancer Res. 12:701–709, 2006. 19. Lewis JS Jr, Ukpo OC, Ma XJ, et al: Transcriptionally-active high-risk human papillomavirus is rare in oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas–a tissue microarray study utilizing E6/E7 mRNA in situ hybridization. Histopathology. 60:982–991, 2012. 20. Paradiso A, Ranieri G, Stea B, et al: Altered p16INK4a and Fhit expression in carcinogenesis and progression of human oral cancer. Int J Oncol. 24:249–255, 2004. 21. Robinson M, Schache A, Sloan P, et al: HPV Specific Testing: A Requirement for Oropharyngeal Squamous Cell Carcinoma Patients. Head Neck Pathol. 6(Suppl 1):83–90, 2012. 22. Lopes V, Murray P, Williams H, et al: Squamous cell carcinoma of the oral cavity rarely harbours oncogenic human papillomavirus. Oral Oncol. 47:698–701, 2011.
321.e1
23. Hesselink AT, van den Brule AJ, Brink AA, et al: Comparison of hybrid capture 2 with in situ hybridization for the detection of high-risk human papillomavirus in liquid-based cervical samples. Cancer. 102:11–18, 2004. 24. Cobo F, Talavera P, Concha A: Review article: relationship of human papillomavirus with papillary squamous cell carcinoma of the upper aerodigestive tract: a review. Int J Surg Pathol. 16:127–136, 2008. 25. Pintos J, Black MJ, Sadeghi N, et al: Human papillomavirus infection and oral cancer: a case-control study in Montreal, Canada. Oral Oncol. 44:242–250, 2008. 26. Lewis JS Jr, Thorstad WL, Chernock RD, et al: p16 positive oropharyngeal squamous cell carcinoma:an entity with a favorable prognosis regardless of tumor HPV status. Am J Surg Pathol. 34:1088–1096, 2010. 27. McGregor F, Muntoni A, Fleming J, et al: Molecular changes associated with oral dysplasia progression and acquisition of immortality: potential for its reversal by 5-azacytidine. Cancer Res. 62:4757–4766, 2002. 28. Sudbo J, Lippman SM, Lee JJ, et al: The influence of resection and aneuploidy on mortality in oral leukoplakia. N Engl J Med. 350:1405–1413, 2004. 29. Lydiatt WM, Anderson PE, Bazzana T, et al: Molecular support for field cancerization in the head and neck. Cancer. 82:1376– 1380, 1998. 30. Chernock RD, Lewis JS Jr, Zhang Q, et al: Human papillomavirus-positive basaloid squamous cell carcinomas of the upper aerodigestive tract: a distinct clinicopathologic and molecular subtype of basaloid squamous cell carcinoma. Hum Pathol. 41:1016–1023, 2010. 31. Lewis JS Jr, Khan RA, Masand RP, et al: Recognition of nonkeratinizing morphology in oropharyngeal squamous cell carcinoma—a prospective cohort and interobserver variability study. Histopathology. 60:427–436, 2012. 32. Chernock RD: Morphologic features of conventional squamous cell carcinoma of the oropharynx: ‘keratinizing’ and ‘nonkeratinizing’ histologic types as the basis for a consistent classification system. Head Neck Pathol. 6(Suppl 1):41–47, 2012. 33. Gasco M, Crook T: The p53 network in head and neck cancer. Oral Oncol. 39:222–231, 2003. 34. Blons H, Laurent-Puig P: TP53 and head and neck neoplasms. Hum Mutat. 21:252–257, 2003. 35. Schoelch ML, Regezi JA, Dekker NP, et al: Cell cycle proteins and the development of oral squamous cell carcinoma. Oral Oncol. 35:333–342, 1999. 36. Calli C, Calli A, Pinar E, et al: Prognostic significance of p63, p53 and ki67 expression in laryngeal basaloid squamous cell carcinomas. B-ENT. 7:37–42, 2011. 37. Tandon S, Tudur-Smith C, Riley RD, et al: A systematic review of p53 as a prognostic factor of survival in squamous cell carcinoma of the four main anatomical subsites of the head and neck. Cancer Epidemiol Biomarkers Prev. 19:574–587, 2010. 38. Sandulache VC, Skinner HD, Ow TJ, et al: Individualizing antimetabolic treatment strategies for head and neck squamous cell carcinoma based on TP53 mutational status. Cancer. 118:711– 721, 2012. 39. Masunaga S, Tano K, Nakamura J, et al: Usefulness of hexamethylenetetramine as an adjuvant to radiation and cisplatin in the treatment of solid tumors: its independency of p53 status. J Radiat Res. 51:27–35, 2010. 40. Wayne S, Robinson RA: Upper aerodigestive tract squamous dysplasia: correlation with p16, p53, pRb, and Ki-67 expression. Arch Pathol Lab Med. 130:1309–1314, 2006. 41. Oliver RJ, MacDonald DG, Felix DH: Aspects of cell proliferation in oral epithelial dysplastic lesions. J Oral Pathol Med. 29:49–55, 2000. 42. Nathan CO, Amirghahri N, Rice C, et al: Molecular analysis of surgical margins in head and neck squamous cell carcinoma patients. Laryngoscope. 112:2129–2140, 2002. 43. van HV, Tabor MP, van den Brekel MW, et al: Mutated p53 as a molecular marker for the diagnosis of head and neck cancer. J Pathol. 198:476–486, 2002. 44. Tabor MP, Brakenhoff RH, van HV, et al: Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications. Clin Cancer Res. 7:1523–1532, 2001.
321.e2
Immunohistology of Head and Neck Lesions
45. Namazie A, Alavi S, Olopade OI, et al: Cyclin D1 amplification and p16(MTS1/CDK4I) deletion correlate with poor prognosis in head and neck tumors. Laryngoscope. 112:472–481, 2002. 46. Nadal A, Cardesa A: Molecular biology of laryngeal squamous cell carcinoma. Virchows Arch. 442:1–7, 2003. 47. Vielba R, Bilbao J, Ispizua A, et al: p53 and cyclin D1 as prognostic factors in squamous cell carcinoma of the larynx. Laryngoscope. 113:167–172, 2003. 48. Mehra R, Cohen RB, Burtness BA: The role of cetuximab for the treatment of squamous cell carcinoma of the head and neck. Clin Adv Hematol Oncol. 6:742–750, 2008. 49. Gold KA, Lee HY, Kim ES: Targeted therapies in squamous cell carcinoma of the head and neck. Cancer. 115:922–935, 2009. 50. Prince A, guirre-Ghizo J, Genden E, et al: Head and neck squamous cell carcinoma: new translational therapies. Mt Sinai J Med. 77:684–699, 2010. 51. Ford AC, Grandis JR: Targeting epidermal growth factor receptor in head and neck cancer. Head Neck. 25:67–73, 2003. 52. Brockstein B, Lacouture M, Agulnik M: The role of inhibitors of the epidermal growth factor in management of head and neck cancer. J Natl Compr Canc Netw. 6:696–706, 2008. 53. Loeffler-Ragg J, Schwentner I, Sprinzl GM, et al: EGFR inhibition as a therapy for head and neck squamous cell carcinoma. Expert Opin Investig Drugs. 17:1517–1531, 2008. 54. Egloff AM, Grandis JR: Targeting epidermal growth factor receptor and SRC pathways in head and neck cancer. Semin Oncol. 35:286–297, 2008. 55. Yamatodani T, Ekblad L, Kjellen E, et al: Epidermal growth factor receptor status and persistent activation of Akt and p44/42 MAPK pathways correlate with the effect of cetuximab in head and neck and colon cancer cell lines. J Cancer Res Clin Oncol. 135:395–402, 2009. 56. Serrano MF, El-Mofty SK, Gnepp DR, et al: Utility of high molecular weight cytokeratins, but not p63, in the differential diagnosis of neuroendocrine and basaloid carcinomas of the head and neck. Hum Pathol. 39:591–598, 2008. 57. Morice WG, Ferreiro JA: Distinction of basaloid squamous cell carcinoma from adenoid cystic and small cell undifferentiated carcinoma by immunohistochemistry. Hum Pathol. 29:609–612, 1998. 58. Chapman-Fredricks J, Jorda M, Gomez-Fernandez C: A limited immunohistochemical panel helps differentiate small cell epithelial malignancies of the sinonasal cavity and nasopharynx. Appl Immunohistochem Mol Morphol. 17:207–210, 2009. 59. Begum S, Westra WH: Basaloid squamous cell carcinoma of the head and neck is a mixed variant that can be further resolved by HPV status. Am J Surg Pathol. 32:1044–1050, 2008. 60. Nikitakis NG, Tosios KI, Papanikolaou VS, et al: Immunohistochemical expression of cytokeratins 7 and 20 in malignant salivary gland tumors. Mod Pathol. 17:407–415, 2004. 61. French CA: NUT midline carcinoma. Cancer Genet Cytogenet. 203:16–20, 2010. 62. Stelow EB: A review of NUT midline carcinoma. Head Neck Pathol. 5:31–35, 2011. 63. Poetsch M, Lorenz G, Bankau A, et al: Basaloid in contrast to nonbasaloid head and neck squamous cell carcinomas display aberrations especially in cell cycle control genes. Head Neck. 25:904–910, 2003. 64. Szentirmay Z, Polus K, Tamas L, et al: Human papillomavirus in head and neck cancer: molecular biology and clinicopathological correlations. Cancer Metastasis Rev. 24:19–34, 2005. 65. Thompson LD, Heffner DK: The clinical importance of cystic squamous cell carcinomas in the neck: a study of 136 cases. Cancer. 82:944–956, 1998. 66. Chaturvedi AK: Epidemiology and Clinical Aspects of HPV in Head and Neck Cancers. Head Neck Pathol. 6(Suppl 1):16–24, 2012. 67. Westra WH: The Morphologic Profile of HPV-Related Head and Neck Squamous Carcinoma: Implications for Diagnosis, Prognosis, and Clinical Management. Head Neck Pathol. 6(Suppl 1):48– 54, 2012. 68. Banks ER, Frierson HF Jr, Mills SE, et al: Basaloid squamous cell carcinoma of the head and neck. A clinicopathologic and immunohistochemical study of 40 cases. Am J Surg Pathol. 16:939– 946, 1992.
69. Coletta RD, Cotrim P, Almeida OP, et al: Basaloid squamous carcinoma of oral cavity: a histologic and immunohistochemical study. Oral Oncol. 38:723–729, 2002. 70. Emanuel P, Wang B, Wu M, et al: p63 Immunohistochemistry in the distinction of adenoid cystic carcinoma from basaloid squamous cell carcinoma. Mod Pathol. 18:645–650, 2005. 71. El-Mofty SK: HPV-Related Squamous Cell Carcinoma Variants in the Head and Neck. Head Neck Pathol. 6(Suppl 1):55–62, 2012. 72. Muller S, Barnes L: Basaloid squamous cell carcinoma of the head and neck with a spindle cell component. An unusual histologic variant. Arch Pathol Lab Med. 119:181–182, 1995. 73. Barnes L, Ferlito A, Altavilla G, et al: Basaloid squamous cell carcinoma of the head and neck: clinicopathological features and differential diagnosis. Ann Otol Rhinol Laryngol. 105:75–82, 1996. 74. Ide F, Shimoyama T, Horie N, et al: Basaloid squamous cell carcinoma of the oral mucosa: a new case and review of 45 cases in the literature. Oral Oncol. 38:120–124, 2002. 75. Raslan WF, Barnes L, Krause JR, et al: Basaloid squamous cell carcinoma of the head and neck: a clinicopathologic and flow cytometric study of 10 new cases with review of the English literature. Am J Otolaryngol. 15:204–211, 1994. 76. Wieneke JA, Thompson LD, Wenig BM: Basaloid squamous cell carcinoma of the sinonasal tract. Cancer. 85:841–854, 1999. 77. Ukpo OC, Flanagan JJ, Ma XJ, et al: High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am J Surg Pathol. 35:1343–1350, 2011. 78. Lewis JS Jr, Chernock RD, Ma XJ, et al: Partial p16 staining in oropharyngeal squamous cell carcinoma: extent and pattern correlate with human papillomavirus RNA status. Mod Pathol. 2012. 79. Genden EM: The Role for Surgical Management of HPV-Related Oropharyngeal Carcinoma. Head Neck Pathol. 6(Suppl 1):98– 103, 2012. 80. Lui VW, Grandis JR: Primary Chemotherapy and Radiation as a Treatment Strategy for HPV-Positive Oropharyngeal Cancer. Head Neck Pathol. 6(Suppl 1):91–97, 2012. 81. Zhang MQ, El-Mofty SK, Davila RM: Detection of human papillomavirus-related squamous cell carcinoma cytologically and by in situ hybridization in fine-needle aspiration biopsies of cervical metastasis: a tool for identifying the site of an occult head and neck primary. Cancer. 114:118–123, 2008. 82. El-Mofty SK, Lu DW: Prevalence of high-risk human papillomavirus DNA in nonkeratinizing (cylindrical cell) carcinoma of the sinonasal tract: a distinct clinicopathologic and molecular disease entity. Am J Surg Pathol. 29:1367–1372, 2005. 83. Worden FP, Kumar B, Lee JS, et al: Chemoselection as a strategy for organ preservation in advanced oropharynx cancer: response and survival positively associated with HPV16 copy number. J Clin Oncol. 26:3138–3146, 2008. 84. Soriano E, Faure C, Lantuejoul S, et al: Course and prognosis of basaloid squamous cell carcinoma of the head and neck: a casecontrol study of 62 patients. Eur J Cancer. 44:244–250, 2008. 85. Klijanienko J, El-Naggar A, Ponzio-Prion A, et al: Basaloid squamous carcinoma of the head and neck. Immunohistochemical comparison with adenoid cystic carcinoma and squamous cell carcinoma. Arch Otolaryngol Head Neck Surg. 119:887–890, 1993. 86. Tsubochi H, Suzuki T, Suzuki S, et al: Immunohistochemical study of basaloid squamous cell carcinoma, adenoid cystic and mucoepidermoid carcinoma in the upper aerodigestive tract. Anticancer Res. 20:1205–1211, 2000. 87. Holst VA, Marshall CE, Moskaluk CA, et al: KIT protein expression and analysis of c-kit gene mutation in adenoid cystic carcinoma. Mod Pathol. 12:956–960, 1999. 88. Mino M, Pilch BZ, Faquin WC: Expression of KIT (CD117) in neoplasms of the head and neck: an ancillary marker for adenoid cystic carcinoma. Mod Pathol. 16:1224–1231, 2003. 89. Bilal H, Handra-Luca A, Bertrand JC, et al: P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues. J Histochem Cytochem. 51:133–139, 2003. 90. Kiyoshima T, Shima K, Kobayashi I, et al: Expression of p53 tumor suppressor gene in adenoid cystic and mucoepidermoid
References carcinomas of the salivary glands. Oral Oncol. 37:315–322, 2001. 91. Owonikoko T, Loberg C, Gabbert HE, et al: Comparative analysis of basaloid and typical squamous cell carcinoma of the oesophagus: a molecular biological and immunohistochemical study. J Pathol. 193:155–161, 2001. 92. Seethala RR, Hunt JL, Baloch ZW, et al: Adenoid cystic carcinoma with high-grade transformation: a report of 11 cases and a review of the literature. Am J Surg Pathol. 31:1683–1694, 2007. 93. Nagao T, Gaffey TA, Serizawa H, et al: Dedifferentiated adenoid cystic carcinoma: a clinicopathologic study of 6 cases. Mod Pathol. 16:1265–1272, 2003. 94. Thompson LD, Wieneke JA, Miettinen M, et al: Spindle cell (sarcomatoid) carcinomas of the larynx: a clinicopathologic study of 187 cases. Am J Surg Pathol. 26:153–170, 2002. 95. Lewis JE, Olsen KD, Sebo TJ: Spindle cell carcinoma of the larynx: review of 26 cases including DNA content and immunohistochemistry. Hum Pathol. 28:664–673, 1997. 96. Thompson LD: Diagnostically challenging lesions in head and neck pathology. Eur Arch Otorhinolaryngol. 254:357–366, 1997. 97. Bridge JA, Bowen JM, Smith RB: The small round blue cell tumors of the sinonasal area. Head Neck Pathol. 4:84–93, 2010. 98. Thompson LD, Wieneke JA, Miettinen M: Sinonasal tract and nasopharyngeal melanomas: a clinicopathologic study of 115 cases with a proposed staging system. Am J Surg Pathol. 27:594– 611, 2003. 99. Thompson LD: Olfactory neuroblastoma. Head Neck Pathol. 3:252–259, 2009. 100. Thompson LD, Seethala RR, Muller S: Ectopic Sphenoid Sinus Pituitary Adenoma (ESSPA) with Normal Anterior Pituitary Gland: A Clinicopathologic and Immunophenotypic Study of 32 Cases with a Comprehensive Review of the English Literature. Head Neck Pathol. 6:75–100, 2012. 101. Wooff JC, Weinreb I, Perez-Ordonez B, et al: Calretinin staining facilitates differentiation of olfactory neuroblastoma from other small round blue cell tumors in the sinonasal tract. Am J Surg Pathol. 35:1786–1793, 2011. 102. Wenig BM: Undifferentiated malignant neoplasms of the sinonasal tract. Arch Pathol Lab Med. 133:699–712, 2009. 103. Iezzoni JC, Mills SE: “Undifferentiated” small round cell tumors of the sinonasal tract: differential diagnosis update. Am J Clin Pathol. 124(Suppl):S110–S121, 2005. 104. Devaney K, Wenig BM, Abbondanzo SL: Olfactory neuroblastoma and other round cell lesions of the sinonasal region. Mod Pathol. 9:658–663, 1996. 105. Devoe K, Weidner N: Immunohistochemistry of small roundcell tumors. Semin Diagn Pathol. 17:216–224, 2000. 106. Franchi A, Palomba A, Cardesa A: Current diagnostic strategies for undifferentiated tumours of the nasal cavities and paranasal sinuses. Histopathology. 59:1034–1045, 2011. 107. Cordes B, Williams MD, Tirado Y, et al: Molecular and phenotypic analysis of poorly differentiated sinonasal neoplasms: an integrated approach for early diagnosis and classification. Hum Pathol. 40:283–292, 2009. 108. Broich G, Pagliari A, Ottaviani F: Esthesioneuroblastoma: a general review of the cases published since the discovery of the tumour in 1924. Anticancer Res. 17:2683–2706, 1997. 109. Dulguerov P, Allal AS, Calcaterra TC: Esthesioneuroblastoma: a meta-analysis and review. Lancet Oncol. 2:683–690, 2001. 110. Argani P, Perez-Ordonez B, Xiao H, et al: Olfactory neuroblastoma is not related to the Ewing family of tumors: absence of EWS/FLI1 gene fusion and MIC2 expression. Am J Surg Pathol. 22:391–398, 1998. 111. Bourne TD, Bellizzi AM, Stelow EB, et al: p63 Expression in olfactory neuroblastoma and other small cell tumors of the sinonasal tract. Am J Clin Pathol. 130:213–218, 2008. 112. Slootweg PJ, Lubsen H: Rhabdomyoblasts in olfactory neuroblastoma. Histopathology. 19:182–184, 1991. 113. Miyagami M, Katayama Y, Kinukawa N, et al: An ultrastructural and immunohistochemical study of olfactory neuroepithelioma with rhabdomyoblasts. Med Electron Microsc. 35:160–166, 2002. 114. Ejaz A, Wenig BM: Sinonasal undifferentiated carcinoma: clinical and pathologic features and a discussion on classification, cellular differentiation, and differential diagnosis. Adv Anat Pathol. 12:134–143, 2005.
321.e3
115. Franchi A, Moroni M, Massi D, et al: Sinonasal undifferentiated carcinoma, nasopharyngeal-type undifferentiated carcinoma, and keratinizing and nonkeratinizing squamous cell carcinoma express different cytokeratin patterns. Am J Surg Pathol. 26:1597–1604, 2002. 116. Jeng YM, Sung MT, Fang CL, et al: Sinonasal undifferentiated carcinoma and nasopharyngeal-type undifferentiated carcinoma: two clinically, biologically, and histopathologically distinct entities. Am J Surg Pathol. 26:371–376, 2002. 117. Cerilli LA, Holst VA, Brandwein MS, et al: Sinonasal undifferentiated carcinoma: immunohistochemical profile and lack of EBV association. Am J Surg Pathol. 25:156–163, 2001. 118. Smith SR, Som P, Fahmy A, et al: A clinicopathological study of sinonasal neuroendocrine carcinoma and sinonasal undifferentiated carcinoma. Laryngoscope. 110:1617–1622, 2000. 119. Mills SE, Fechner RE: “Undifferentiated” neoplasms of the sinonasal region: differential diagnosis based on clinical, light microscopic, immunohistochemical, and ultrastructural features. Semin Diagn Pathol. 6:316–328, 1989. 120. Wadsworth B, Bumpous JM, Martin AW, et al: Expression of p16 in sinonasal undifferentiated carcinoma (SNUC) without associated human papillomavirus (HPV). Head Neck Pathol. 5:349– 354, 2011. 121. Chernock RD, Perry A, Pfeifer JD, et al: Receptor tyrosine kinases in sinonasal undifferentiated carcinomas–evaluation for EGFR, c-KIT, and HER2/neu expression. Head Neck. 31:919– 927, 2009. 122. Lopategui JR, Gaffey MJ, Frierson HF Jr, et al: Detection of Epstein-Barr viral RNA in sinonasal undifferentiated carcinoma from Western and Asian patients. Am J Surg Pathol. 18:391–398, 1994. 123. Thompson LD: Update on nasopharyngeal carcinoma. Head Neck Pathol. 1:81–86, 2007. 124. Thompson LD, Miettinen M, Wenig BM: Sinonasal-type hemangiopericytoma: a clinicopathologic and immunophenotypic analysis of 104 cases showing perivascular myoid differentiation. Am J Surg Pathol. 27:737–749, 2003. 125. Prasad ML, Jungbluth AA, Iversen K, et al: Expression of melanocytic differentiation markers in malignant melanomas of the oral and sinonasal mucosa. Am J Surg Pathol. 25:782–787, 2001. 126. Dauer EH, Lewis JE, Rohlinger AL, et al: Sinonasal melanoma: a clinicopathologic review of 61 cases. Otolaryngol Head Neck Surg. 138:347–352, 2008. 127. Rivera RS, Nagatsuka H, Gunduz M, et al: C-kit protein expression correlated with activating mutations in KIT gene in oral mucosal melanoma. Virchows Arch. 452:27–32, 2008. 128. Miettinen M, Fernandez M, Franssila K, et al: Microphthalmia transcription factor in the immunohistochemical diagnosis of metastatic melanoma: comparison with four other melanoma markers. Am J Surg Pathol. 25:205–211, 2001. 129. Kardon DE, Thompson LD: Sinonasal mucosal malignant melanoma: report of an unusual case mimicking schwannoma. Ann Diagn Pathol. 4:303–307, 2000. 130. Torlakovic EE, Slipicevic A, Florenes VA, et al: Fli-1 expression in malignant melanoma. Histol Histopathol. 23:1309–1314, 2008. 131. Feeley C, Theaker J: Epithelial markers in primary sinonasal mucosal melanoma. Histopathology. 45:96–98, 2004. 132. Knott PD, Gannon FH, Thompson LD: Mesenchymal chondrosarcoma of the sinonasal tract: a clinicopathological study of 13 cases with a review of the literature. Laryngoscope. 113:783–790, 2003. 133. Thompson LD, Gyure KA: Extracranial sinonasal tract meningiomas: a clinicopathologic study of 30 cases with a review of the literature. Am J Surg Pathol. 24:640–650, 2000. 134. Gaiger de OM, Thompson LD, Chaves AC, et al: Management of melanotic neuroectodermal tumor of infancy. Ann Diagn Pathol. 8:207–212, 2004. 135. Mills SE: Neuroectodermal neoplasms of the head and neck with emphasis on neuroendocrine carcinomas. Mod Pathol. 15:264–278, 2002. 136. Ordonez NG: Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol. 24:1217–1223, 2000.
321.e4
Immunohistology of Head and Neck Lesions
137. Perez-Ordonez B, Caruana SM, Huvos AG, et al: Small cell neuroendocrine carcinoma of the nasal cavity and paranasal sinuses. Hum Pathol. 29:826–832, 1998. 138. Milroy CM, Ferlito A: Immunohistochemical markers in the diagnosis of neuroendocrine neoplasms of the head and neck. Ann Otol Rhinol Laryngol. 104:413–418, 1995. 139. Cheuk W, Kwan MY, Suster S, et al: Immunostaining for thyroid transcription factor 1 and cytokeratin 20 aids the distinction of small cell carcinoma from Merkel cell carcinoma, but not pulmonary from extrapulmonary small cell carcinomas. Arch Pathol Lab Med. 125:228–231, 2001. 140. Mineta H, Miura K, Takebayashi S, et al: Immunohistochemical analysis of small cell carcinoma of the head and neck: a report of four patients and a review of sixteen patients in the literature with ectopic hormone production. Ann Otol Rhinol Laryngol. 110:76–82, 2001. 141. Hafezi S, Seethala RR, Stelow EB, et al: Ewing’s family of tumors of the sinonasal tract and maxillary bone. Head Neck Pathol. 5:8–16, 2011. 142. Luk IS, Chan JK, Chow SM, et al: Pituitary adenoma presenting as sinonasal tumor: pitfalls in diagnosis. Hum Pathol. 27:605– 609, 1996. 143. Lloyd RV, Chandler WF, Kovacs K, et al: Ectopic pituitary adenomas with normal anterior pituitary glands. Am J Surg Pathol. 10:546–552, 1986. 144. Asa SL, Puy LA, Lew AM, et al: Cell type-specific expression of the pituitary transcription activator pit-1 in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab. 77:1275–1280, 1993. 145. Ahmed AA, Tsokos M: Sinonasal rhabdomyosarcoma in children and young adults. Int J Surg Pathol. 15:160–165, 2007. 146. Montone KT, Barr FG, Zhang PJ, et al: Embryonal and alveolar rhabdomyosarcoma of parameningeal sites in adults: a report of 13 cases. Int J Surg Pathol. 17:22–30, 2009. 147. Callender TA, Weber RS, Janjan N, et al: Rhabdomyosarcoma of the nose and paranasal sinuses in adults and children. Otolaryngol Head Neck Surg. 112:252–257, 1995. 148. Cessna MH, Zhou H, Perkins SL, et al: Are myogenin and myoD1 expression specific for rhabdomyosarcoma? A study of 150 cases, with emphasis on spindle cell mimics. Am J Surg Pathol. 25:1150–1157, 2001. 149. Bahrami A, Gown AM, Baird GS, et al: Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol. 21:795–806, 2008. 150. Davis JL, Matsumura L, Weeks DA, et al: PAX2 expression in Wilms tumors and other childhood neoplasms. Am J Surg Pathol. 35:1186–1194, 2011. 151. Folpe AL, Hill CE, Parham DM, et al: Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on CD99-positive mimics of Ewing’s sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol. 24:1657–1662, 2000. 152. Mhawech-Fauceglia P, Herrmann FR, Bshara W, et al: Friend leukaemia integration-1 expression in malignant and benign tumours: a multiple tumour tissue microarray analysis using polyclonal antibody. J Clin Pathol. 60:694–700, 2007. 153. Mercado GE, Barr FG: Fusions involving PAX and FOX genes in the molecular pathogenesis of alveolar rhabdomyosarcoma: recent advances. Curr Mol Med. 7:47–61, 2007. 154. Heffner DK, Hyams VJ: Teratocarcinosarcoma (malignant teratoma?) of the nasal cavity and paranasal sinuses A clinicopathologic study of 20 cases. Cancer. 53:2140–2154, 1984. 155. Smith SL, Hessel AC, Luna MA, et al: Sinonasal teratocarcinosarcoma of the head and neck: a report of 10 patients treated at a single institution and comparison with reported series. Arch Otolaryngol Head Neck Surg. 134:592–595, 2008. 156. Heffner DK, Gnepp DR: Sinonasal fibrosarcomas, malignant schwannomas, and “Triton” tumors. A clinicopathologic study of 67 cases. Cancer. 70:1089–1101, 1992. 157. Lae ME, Roche PC, Jin L, et al: Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol. 26:823–835, 2002.
158. de AE, Gerald WL: Molecular biology of the Ewing’s sarcoma/ primitive neuroectodermal tumor family. J Clin Oncol. 18:204– 213, 2000. 159. Nikitakis NG, Salama AR, O’Malley BW Jr, et al: Malignant peripheral primitive neuroectodermal tumor-peripheral neuroepithelioma of the head and neck: a clinicopathologic study of five cases and review of the literature. Head Neck. 25:488–498, 2003. 160. Thompson LD: Ewing sarcoma and primitive neuroectodermal tumor. Ear Nose Throat J. 86:79–80, 2007. 161. Mhawech-Fauceglia P, Herrmann F, Penetrante R, et al: Diagnostic utility of FLI-1 monoclonal antibody and dual-colour, breakapart probe fluorescence in situ (FISH) analysis in Ewing’s sarcoma/primitive neuroectodermal tumour (EWS/PNET). A comparative study with CD99 and FLI-1 polyclonal antibodies. Histopathology. 49:569–575, 2006. 162. Gu M, Antonescu CR, Guiter G, et al: Cytokeratin immunoreactivity in Ewing’s sarcoma: prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol. 24:410–416, 2000. 163. Schuetz AN, Rubin BP, Goldblum JR, et al: Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: additional evidence of epithelial differentiation. Mod Pathol. 18:1403–1410, 2005. 164. Franchi A, Santucci M, Wenig BM: Adenocarcinoma. In Barnes EL, Eveson JW, Reichart P, et al, editors: Pathology and Genetics Head and Neck Tumours, Lyon, France, 2005, IARC Press, pp 20–23. 165. Eveson JW: Salivary gland-type carcinoma. In Barnes EL, Eveson JW, Reichart P, et al, editors: Pathology and Genetics Head and Neck Tumours, Lyon, France, 2005, IARC Press, pp 24–25. 166. Jo VY, Mills SE, Cathro HP, et al: Low-grade sinonasal adenocarcinomas: the association with and distinction from respiratory epithelial adenomatoid hamartomas and other glandular lesions. Am J Surg Pathol. 33:401–408, 2009. 167. Barnes L: Intestinal-type adenocarcinoma of the nasal cavity and paranasal sinuses. Am J Surg Pathol. 10:192–202, 1986. 168. Kleinsasser O, Schroeder HG: Adenocarcinomas of the inner nose after exposure to wood dust. Morphological findings and relationships between histopathology and clinical behavior in 79 cases. Arch Otorhinolaryngol. 245:1–15, 1988. 169. Llorente JL, Perez-Escuredo J, varez-Marcos C, et al: Genetic and clinical aspects of wood dust related intestinal-type sinonasal adenocarcinoma: a review. Eur Arch Otorhinolaryngol. 266:1– 7, 2009. 170. Franquemont DW, Fechner RE, Mills SE: Histologic classification of sinonasal intestinal-type adenocarcinoma. Am J Surg Pathol. 15:368–375, 1991. 171. McKinney CD, Mills SE, Franquemont DW: Sinonasal intestinaltype adenocarcinoma: immunohistochemical profile and comparison with colonic adenocarcinoma. Mod Pathol. 8:421–426, 1995. 172. Choi HR, Sturgis EM, Rashid A, et al: Sinonasal adenocarcinoma: evidence for histogenetic divergence of the enteric and nonenteric phenotypes. Hum Pathol. 34:1101–1107, 2003. 173. Bashir AA, Robinson RA, Benda JA, et al: Sinonasal adenocarcinoma: immunohistochemical marking and expression of oncoproteins. Head Neck. 25:763–771, 2003. 174. Cathro HP, Mills SE: Immunophenotypic differences between intestinal-type and low-grade papillary sinonasal adenocarcinomas: an immunohistochemical study of 22 cases utilizing CDX2 and MUC2. Am J Surg Pathol. 28:1026–1032, 2004. 175. Abecasis J, Viana G, Pissarra C, et al: Adenocarcinomas of the nasal cavity and paranasal sinuses: a clinicopathological and immunohistochemical study of 14 cases. Histopathology. 45:254– 259, 2004. 176. Ozolek JA, Barnes EL, Hunt JL: Basal/myoepithelial cells in chronic sinusitis, respiratory epithelial adenomatoid hamartoma, inverted papilloma, and intestinal-type and nonintestinal-type sinonasal adenocarcinoma: an immunohistochemical study. Arch Pathol Lab Med. 131:530–537, 2007. 177. Franchi A, Massi D, Palomba A, et al: CDX-2, cytokeratin 7 and cytokeratin 20 immunohistochemical expression in the differential diagnosis of primary adenocarcinomas of the sinonasal tract. Virchows Arch. 445:63–67, 2004.
References 178. Kennedy MT, Jordan RC, Berean KW, et al: Expression pattern of CK7, CK20, CDX-2, and villin in intestinal-type sinonasal adenocarcinoma. J Clin Pathol. 57:932–937, 2004. 179. Ortiz-Rey JA, Alvarez C, San MP, et al: Expression of CDX2, cytokeratins 7 and 20 in sinonasal intestinal-type adenocarcinoma. Appl Immunohistochem Mol Morphol. 13:142–146, 2005. 180. Resto VA, Krane JF, Faquin WC, et al: Immunohistochemical distinction of intestinal-type sinonasal adenocarcinoma from metastatic adenocarcinoma of intestinal origin. Ann Otol Rhinol Laryngol. 115:59–64, 2006. 181. Castillo C, Buob D, Mortuaire G, et al: Signet-ring cell adenocarcinoma of sinonasal tract: an immunohistochemical study of the mucins profile. Arch Pathol Lab Med. 131:961–964, 2007. 182. Franchi A, Palomba A, Fondi C, et al: Immunohistochemical investigation of tumorigenic pathways in sinonasal intestinaltype adenocarcinoma. A tissue microarray analysis of 62 cases. Histopathology. 59:98–105, 2011. 183. Saber AT, Nielsen LR, Dictor M, et al: K-ras mutations in sinonasal adenocarcinomas in patients occupationally exposed to wood or leather dust. Cancer Lett. 126:59–65, 1998. 184. az-Molina JP, Llorente JL, Vivanco B, et al: Wnt-pathway activation in intestinal-type sinonasal adenocarcinoma. Rhinology. 49:593–599, 2011. 185. Perez-Ordonez B, Huynh NN, Berean KW, et al: Expression of mismatch repair proteins, beta catenin, and E cadherin in intestinal-type sinonasal adenocarcinoma. J Clin Pathol. 57:1080– 1083, 2004. 186. Stelow EB, Jo VY, Mills SE, et al: A histologic and immunohistochemical study describing the diversity of tumors classified as sinonasal high-grade nonintestinal adenocarcinomas. Am J Surg Pathol. 35:971–980, 2011. 187. Heffner DK, Hyams VJ, Hauck KW, et al: Low-grade adenocarcinoma of the nasal cavity and paranasal sinuses. Cancer. 50:312–322, 1982. 188. Ozolek JA, Hunt JL: Tumor suppressor gene alterations in respiratory epithelial adenomatoid hamartoma (REAH): comparison to sinonasal adenocarcinoma and inflamed sinonasal mucosa. Am J Surg Pathol. 30:1576–1580, 2006. 189. Thompson LD: Sinonasal tract glomangiopericytoma (hemangiopericytoma). Ear Nose Throat J. 83:807, 2004. 190. Chu PG, Chang KL, Wu AY, et al: Nasal glomus tumors: report of two cases with emphasis on immunohistochemical features and differential diagnosis. Hum Pathol. 30:1259–1261, 1999. 191. Tse LL, Chan JK: Sinonasal haemangiopericytoma-like tumour: a sinonasal glomus tumour or a haemangiopericytoma? Histopathology. 40:510–517, 2002. 192. Eichhorn JH, Dickersin GR, Bhan AK, et al: Sinonasal hemangiopericytoma. A reassessment with electron microscopy, immunohistochemistry, and long-term follow-up. Am J Surg Pathol. 14:856–866, 1990. 193. Abraham SC, Montgomery EA, Giardiello FM, et al: Frequent beta-catenin mutations in juvenile nasopharyngeal angiofibromas. Am J Pathol. 158:1073–1078, 2001. 194. Beham A, Regauer S, Beham-Schmid C, et al: Expression of CD34-antigen in nasopharyngeal angiofibromas. Int J Pediatr Otorhinolaryngol. 44:245–250, 1998. 195. Stelow EB, Bellizzi AM, Taneja K, et al: NUT rearrangement in undifferentiated carcinomas of the upper aerodigestive tract. Am J Surg Pathol. 32:828–834, 2008. 196. Kalinsky K, Haluska FG: Novel inhibitors in the treatment of metastatic melanoma. Expert Rev Anticancer Ther. 7:715–724, 2007. 197. Wong CW, Fan YS, Chan TL, et al: BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs. J Clin Pathol. 58:640–644, 2005. 198. Curtin JA, Busam K, Pinkel D, et al: Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol. 24:4340–4346, 2006. 199. Lutzky J, Bauer J, Bastian BC: Dose-dependent, complete response to imatinib of a metastatic mucosal melanoma with a K642E KIT mutation. Pigment Cell Melanoma Res. 21:492–493, 2008. 200. Yang XR, Diehl S, Pfeiffer R, et al: Evaluation of risk factors for nasopharyngeal carcinoma in high-risk nasopharyngeal
321.e5
carcinoma families in Taiwan. Cancer Epidemiol Biomarkers Prev. 14:900–905, 2005. 201. Spano JP, Busson P, Atlan D, et al: Nasopharyngeal carcinomas: an update. Eur J Cancer. 39:2121–2135, 2003. 202. Marks JE, Phillips JL, Menck HR: The National Cancer Data Base report on the relationship of race and national origin to the histology of nasopharyngeal carcinoma. Cancer. 83:582–588, 1998. 203. Heng DM, Wee J, Fong KW, et al: Prognostic factors in 677 patients in Singapore with nondisseminated nasopharyngeal carcinoma. Cancer. 86:1912–1920, 1999. 204. Chan JKC, Bray F, McCarron P, et al: Nasopharyngeal carcinoma. In Barnes EL, Eveson JW, Reichart P, et al, editors: Pathology and Genetics Head and Neck Tumours, Lyon, France, 2005, IARC Press, pp 85–97. 205. Wei KR, Xu Y, Liu J, et al: Histopathological classification of nasopharyngeal carcinoma. Asian Pac J Cancer Prev. 12:1141– 1147, 2011. 206. Dubey P, Ha CS, Ang KK, et al: Nonnasopharyngeal lymphoepithelioma of the head and neck. Cancer. 82:1556–1562, 1998. 207. Leung SY, Yuen ST, Chung LP, et al: Epstein-Barr virus is present in a wide histological spectrum of sinonasal carcinomas. Am J Surg Pathol. 19:994–1001, 1995. 208. Tsai ST, Jin YT, Su IJ: Expression of EBER1 in primary and metastatic nasopharyngeal carcinoma tissues using in situ hybridization. A correlation with WHO histologic subtypes. Cancer. 77:231–236, 1996. 209. Sheu LF, Chen A, Tseng HH, et al: Assessment of p53 expression in nasopharyngeal carcinoma. Hum Pathol. 26:380–386, 1995. 210. Bar-Sela G, Kuten A, Ben-Eliezer S, et al: Expression of HER2 and C-KIT in nasopharyngeal carcinoma: implications for a new therapeutic approach. Mod Pathol. 16:1035–1040, 2003. 211. Thompson LDR, Fanburg-Smith JC: Nasopharyngeal angiofibroma. In Barnes EL, Eveson JW, Reichart P, et al, editors: Pathology and Genetics of Head and Neck Tumours, Lyon, France, 2005, IARC Press, pp 102–103. 212. Bremer JW, Neel HB III, DeSanto LW, et al: Angiofibroma: treatment trends in 150 patients during 40 years. Laryngoscope. 96:1321–1329, 1986. 213. Beham A, Kainz J, Stammberger H, et al: Immunohistochemical and electron microscopical characterization of stromal cells in nasopharyngeal angiofibromas. Eur Arch Otorhinolaryngol. 254:196–199, 1997. 214. Hwang HC, Mills SE, Patterson K, et al: Expression of androgen receptors in nasopharyngeal angiofibroma: an immunohistochemical study of 24 cases. Mod Pathol. 11:1122–1126, 1998. 215. Montag AG, Tretiakova M, Richardson M: Steroid hormone receptor expression in nasopharyngeal angiofibromas. Consistent expression of estrogen receptor beta. Am J Clin Pathol. 125:832–837, 2006. 216. Zhang PJ, Weber R, Liang HH, et al: Growth factors and receptors in juvenile nasopharyngeal angiofibroma and nasal polyps: an immunohistochemical study. Arch Pathol Lab Med. 127:1480– 1484, 2003. 217. Carlos R, Thompson LD, Netto AC, et al: Epstein-Barr virus and human herpes virus-8 are not associated with juvenile nasopharyngeal angiofibroma. Head Neck Pathol. 2:145–149, 2008. 218. Wenig BM, Hyams VJ, Heffner DK: Nasopharyngeal papillary adenocarcinoma. A clinicopathologic study of a low-grade carcinoma. Am J Surg Pathol. 12:946–953, 1988. 219. Ohe C, Sakaida N, Tadokoro C, et al: Thyroid-like low-grade nasopharyngeal papillary adenocarcinoma: report of two cases. Pathol Int. 60:107–111, 2010. 220. Nojeg MM, Jalaludin MA, Jayalakshmi P: Papillary adenocarcinoma of the nasopharynx–case report and review of the literature. Med J Malaysia. 53:104–106, 1998. 221. Pineda-Daboin K, Neto A, Ochoa-Perez V, et al: Nasopharyngeal adenocarcinomas: a clinicopathologic study of 44 cases including immunohistochemical features of 18 papillary phenotypes. Ann Diagn Pathol. 10:215–221, 2006. 222. Fu CH, Chang KP, Ueng SH, et al: Primary thyroid-like papillary adenocarcinoma of the nasopharynx. Auris Nasus Larynx. 35:579–582, 2008.
321.e6
Immunohistology of Head and Neck Lesions
223. Carrizo F, Luna MA: Thyroid transcription factor-1 expression in thyroid-like nasopharyngeal papillary adenocarcinoma: report of 2 cases. Ann Diagn Pathol. 9:189–192, 2005. 224. Filie AC, Lage JM, Azumi N: Immunoreactivity of S100 protein, alpha-1-antitrypsin, and CD68 in adult and congenital granular cell tumors. Mod Pathol. 9:888–892, 1996. 225. Kaiserling E, Xiao JC, Ruck P, et al: Aberrant expression of macrophage-associated antigens (CD68 and Ki-M1P) by Schwann cells in reactive and neoplastic neural tissue. Lightand electron-microscopic findings. Mod Pathol. 6:463–468, 1993. 226. Kurtin PJ, Bonin DM: Immunohistochemical demonstration of the lysosome-associated glycoprotein CD68 (KP-1) in granular cell tumors and schwannomas. Hum Pathol. 25:1172–1178, 1994. 227. Mazur MT, Shultz JJ, Myers JL: Granular cell tumor. Immunohistochemical analysis of 21 benign tumors and one malignant tumor. Arch Pathol Lab Med. 114:692–696, 1990. 228. Thompson LD: Laryngeal granular cell tumor. Ear Nose Throat J. 88:824–825, 2009. 229. Le BH, Boyer PJ, Lewis JE, et al: Granular cell tumor: immunohistochemical assessment of inhibin-alpha, protein gene product 9.5, S100 protein, CD68, and Ki-67 proliferative index with clinical correlation. Arch Pathol Lab Med. 128:771–775, 2004. 230. Smith BC, Ellis GL, Meis-Kindblom JM, et al: Ectomesenchymal chondromyxoid tumor of the anterior tongue. Nineteen cases of a new clinicopathologic entity. Am J Surg Pathol. 19:519–530, 1995. 231. Angiero F: Ectomesenchymal chondromyxoid tumour of the tongue. A review of histological and immunohistochemical features. Anticancer Res. 30:4685–4689, 2010. 232. Pires FR, Abrahao AC, Cabral MG, et al: Clinical, histological and immunohistochemical features of ectomesenchymal chondromyxoid tumor. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 108:914–919, 2009. 233. Kaplan I, Anavi Y, Calderon S: Ectomesenchymal chondromyxoid tumour of the anterior tongue. Int J Oral Maxillofac Surg. 33:404–407, 2004. 234. Allen CM: The ectomesenchymal chondromyxoid tumor: a review. Oral Dis. 14:390–395, 2008. 235. Goveas N, Ethunandan M, Cowlishaw D, et al: Ectomesenchymal chondromyxoid tumour of the tongue: Unlikely to originate from myoepithelial cells. Oral Oncol. 42:1026–1028, 2006. 236. Woo VL, Angiero F, Fantasia JE: Myoepithelioma of the tongue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 99:581–589, 2005. 237. O’Hara BJ, Paetau A, Miettinen M: Keratin subsets and monoclonal antibody HBME-1 in chordoma: immunohistochemical differential diagnosis between tumors simulating chordoma. Hum Pathol. 29:119–126, 1998. 238. Barnes EL: Neuroendocrine tumours. In Barnes EL, Eveson JW, Reichart P, et al, editors: Pathology and Genetics Head and Neck Tumours, Lyon, France, 2005, IARC Press, pp 135–139. 239. Barnes L: Paraganglioma of the larynx. A critical review of the literature. ORL J Otorhinolaryngol Relat Spec. 53:220–234, 1991. 240. El-Naggar AK, Batsakis JG: Carcinoid tumor of the larynx. A critical review of the literature. ORL J Otorhinolaryngol Relat Spec. 53:188–193, 1991. 241. Woodruff JM, Senie RT: Atypical carcinoid tumor of the larynx. A critical review of the literature. ORL J Otorhinolaryngol Relat Spec. 53:194–209, 1991. 242. Woodruff JM, Huvos AG, Erlandson RA, et al: Neuroendocrine carcinomas of the larynx. A study of two types, one of which mimics thyroid medullary carcinoma. Am J Surg Pathol. 9:771– 790, 1985. 243. Gnepp DR: Small cell neuroendocrine carcinoma of the larynx. A critical review of the literature. ORL J Otorhinolaryngol Relat Spec. 53:210–219, 1991. 244. Oliveira AM, Tazelaar HD, Myers JL, et al: Thyroid transcription factor-1 distinguishes metastatic pulmonary from welldifferentiated neuroendocrine tumors of other sites. Am J Surg Pathol. 25:815–819, 2001. 245. Salim SA, Milroy C, Rode J, et al: Immunocytochemical characterization of neuroendocrine tumours of the larynx. Histopathology. 23:69–73, 1993.
246. Buchner A, Merrell PW, Carpenter WM: Relative frequency of intra-oral minor salivary gland tumors: a study of 380 cases from northern California and comparison to reports from other parts of the world. J Oral Pathol Med. 36:207–214, 2007. 247. Toida M, Shimokawa K, Makita H, et al: Intraoral minor salivary gland tumors: a clinicopathological study of 82 cases. Int J Oral Maxillofac Surg. 34:528–532, 2005. 248. Gnepp DR, el-Mofty S: Polymorphous low-grade adenocarcinoma: glial fibrillary acidic protein staining in the differential diagnosis with cellular mixed tumors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 83:691–695, 1997. 249. Castle JT, Thompson LD, Frommelt RA, et al: Polymorphous low grade adenocarcinoma: a clinicopathologic study of 164 cases. Cancer. 86:207–219, 1999. 250. Darling MR, Schneider JW, Phillips VM: Polymorphous lowgrade adenocarcinoma and adenoid cystic carcinoma: a review and comparison of immunohistochemical markers. Oral Oncol. 38:641–645, 2002. 251. Vargas V, Sudilovsky D, Kaplan MJ: Mixed tumor, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma of the salivary gland: pathogenic implications and differential diagnosis by Ki-67 (Mib 1), Bcl 2 and S-100 immunohistochemistry. Appl Immunohistochem. 5:8–16, 1997. 252. Curran AE, White DK, Damm DD, et al: Polymorphous lowgrade adenocarcinoma versus pleomorphic adenoma of minor salivary glands: resolution of a diagnostic dilemma by immunohistochemical analysis with glial fibrillary acidic protein. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 91:194–199, 2001. 253. Curran AE, Allen CM, Beck FM, et al: Distinctive pattern of glial fibrillary acidic protein immunoreactivity useful in distinguishing fragmented pleomorphic adenoma, canalicular adenoma and polymorphous low grade adenocarcinoma of minor salivary glands. Head Neck Pathol. 1:27–32, 2007. 254. Neves CO, Soares AB, Costa AF, et al: CD10 (Neutral Endopeptidase) Expression in Myoepithelial Cells of Salivary Neoplasms. Appl Immunohistochem Mol Morphol. 18:172–178, 2010. 255. Shah SS, Chandan VS, Wilbur DC, et al: Glial fibrillary acidic protein and CD57 immunolocalization in cell block preparations is a useful adjunct in the diagnosis of pleomorphic adenoma. Arch Pathol Lab Med. 131:1373–1377, 2007. 256. Andreadis D, Epivatianos A, Poulopoulos A, et al: Detection of C-KIT (CD117) molecule in benign and malignant salivary gland tumours. Oral Oncol. 42:57–65, 2006. 257. Andreadis D, Epivatianos A, Mireas G, et al: Immunohistochemical detection of E-cadherin in certain types of salivary gland tumours. J Laryngol Otol. 120:298–304, 2006. 258. Vargas PA, Cheng Y, Barrett AW, et al: Expression of Mcm-2, Ki-67 and geminin in benign and malignant salivary gland tumours. J Oral Pathol Med. 37:309–318, 2008. 259. Antony J, Gopalan V, Smith RA, et al: Carcinoma ex pleomorphic adenoma: a comprehensive review of clinical, pathological and molecular data. Head Neck Pathol. 6:1–9, 2012. 260. Martins C, Fonseca I, Roque L, et al: PLAG1 gene alterations in salivary gland pleomorphic adenoma and carcinoma ex-pleomorphic adenoma: a combined study using chromosome banding, in situ hybridization and immunocytochemistry. Mod Pathol. 18:1048–1055, 2005. 261. Prasad AR, Savera AT, Gown AM, et al: The myoepithelial immunophenotype in 135 benign and malignant salivary gland tumors other than pleomorphic adenoma. Arch Pathol Lab Med. 123:801–806, 1999. 262. Savera AT, Zarbo RJ: Defining the role of myoepithelium in salivary gland neoplasia. Adv Anat Pathol. 11:69–85, 2004. 263. Barnes L, Appel BN, Perez H, et al: Myoepithelioma of the head and neck: case report and review. J Surg Oncol. 28:21–28, 1985. 264. de AV, Carvalho YR, de Araujo NS: Actin versus vimentin in myoepithelial cells of salivary gland tumors. A comparative study. Oral Surg Oral Med Oral Pathol. 77:387–391, 1994. 265. Rosa JC, Felix A, Fonseca I, et al: Immunoexpression of c-erbB-2 and p53 in benign and malignant salivary neoplasms with myoepithelial differentiation. J Clin Pathol. 50:661–663, 1997. 266. Zarbo RJ, Prasad AR, Regezi JA, et al: Salivary gland basal cell and canalicular adenomas: immunohistochemical demonstration of myoepithelial cell participation and morphogenetic considerations. Arch Pathol Lab Med. 124:401–405, 2000.
References 267. Michal M, Skalova A, Simpson RH, et al: Clear cell malignant myoepithelioma of the salivary glands. Histopathology. 28:309– 315, 1996. 268. Nagao T, Sugano I, Ishida Y, et al: Salivary gland malignant myoepithelioma: a clinicopathologic and immunohistochemical study of ten cases. Cancer. 83:1292–1299, 1998. 269. Savera AT, Sloman A, Huvos AG, et al: Myoepithelial carcinoma of the salivary glands: a clinicopathologic study of 25 patients. Am J Surg Pathol. 24:761–774, 2000. 270. Brandwein MS, Ivanov K, Wallace DI, et al: Mucoepidermoid carcinoma: a clinicopathologic study of 80 patients with special reference to histological grading. Am J Surg Pathol. 25:835–845, 2001. 271. Luna MA: Salivary mucoepidermoid carcinoma: revisited. Adv Anat Pathol. 13:293–307, 2006. 272. Ferlito A, Recher G, Bottin R: Mucoepidermoid carcinoma of the larynx. A clinicopathological study of 11 cases with review of the literature. ORL J Otorhinolaryngol Relat Spec. 43:280–299, 1981. 273. Yang CS, Kuo KT, Chou TY, et al: Mucoepidermoid tumors of the lung: analysis of 11 cases. J Chin Med Assoc. 67:565–570, 2004. 274. Vedrine PO, Coffinet L, Temam S, et al: Mucoepidermoid carcinoma of salivary glands in the pediatric age group: 18 clinical cases, including 11 second malignant neoplasms. Head Neck. 28:827–833, 2006. 275. Rahbar R, Grimmer JF, Vargas SO, et al: Mucoepidermoid carcinoma of the parotid gland in children: A 10-year experience. Arch Otolaryngol Head Neck Surg. 132:375–380, 2006. 276. Shapiro NL, Bhattacharyya N: Clinical characteristics and survival for major salivary gland malignancies in children. Otolaryngol Head Neck Surg. 134:631–634, 2006. 277. Martinez-Madrigal F, Pineda-Daboin K, Casiraghi O, et al: Salivary gland tumors of the mandible. Ann Diagn Pathol. 4:347– 353, 2000. 278. Jahan-Parwar B, Huberman RM, Donovan DT, et al: Oncocytic mucoepidermoid carcinoma of the salivary glands. Am J Surg Pathol. 23:523–529, 1999. 279. Weinreb I, Seethala RR, Perez-Ordonez B, et al: Oncocytic mucoepidermoid carcinoma: clinicopathologic description in a series of 12 cases. Am J Surg Pathol. 33:409–416, 2009. 280. Auclair PL, Goode RK, Ellis GL: Mucoepidermoid carcinoma of intraoral salivary glands. Evaluation and application of grading criteria in 143 cases. Cancer. 69:2021–2030, 1992. 281. Goode RK, Auclair PL, Ellis GL: Mucoepidermoid carcinoma of the major salivary glands: clinical and histopathologic analysis of 234 cases with evaluation of grading criteria. Cancer. 82:1217– 1224, 1998. 282. Guzzo M, Andreola S, Sirizzotti G, et al: Mucoepidermoid carcinoma of the salivary glands: clinicopathologic review of 108 patients treated at the National Cancer Institute of Milan. Ann Surg Oncol. 9:688–695, 2002. 283. Haddad R, Colevas AD, Krane JF, et al: Herceptin in patients with advanced or metastatic salivary gland carcinomas. A phase II study. Oral Oncol. 39:724–727, 2003. 284. Nguyen LH, Black MJ, Hier M, et al: HER2/neu and Ki-67 as prognostic indicators in mucoepidermoid carcinoma of salivary glands. J Otolaryngol. 32:328–331, 2003. 285. Penner CR, Thompson LD: Necrotizing sialometaplasia. Ear Nose Throat J. 82:493–494, 2003. 286. Wolfish EB, Nelson BL, Thompson LD: Sinonasal Tract Mucoepidermoid Carcinoma: A Clinicopathologic and Immunophenotypic Study of 19 Cases Combined with a Comprehensive Review of the Literature. Head Neck Pathol. 2011. 287. Ellis GL: Clear cell neoplasms in salivary glands: clearly a diagnostic challenge. Ann Diagn Pathol. 2:61–78, 1998. 288. Freedman PD, Lumerman H: Lobular carcinoma of intraoral minor salivary gland origin. Report of twelve cases. Oral Surg Oral Med Oral Pathol. 56:157–166, 1983. 289. Batsakis JG, Pinkston GR, Luna MA, et al: Adenocarcinomas of the oral cavity: a clinicopathologic study of terminal duct carcinomas. J Laryngol Otol. 97:825–835, 1983. 290. Anderson C, Krutchkoff D, Pedersen C, et al: Polymorphous low grade adenocarcinoma of minor salivary gland: a clinicopathologic and comparative immunohistochemical study. Mod Pathol. 3:76–82, 1990.
321.e7
291. Ghazy SE, Helmy IM, Baghdadi HM: Maspin and MCM2 immunoprofiling in salivary gland carcinomas. Diagn Pathol. 6:89, 2011. 292. Zarbo RJ, Regezi JA, Batsakis JG: S-100 protein in salivary gland tumors: an immunohistochemical study of 129 cases. Head Neck Surg. 8:268–275, 1986. 293. Gnepp DR, Chen JC, Warren C: Polymorphous low-grade adenocarcinoma of minor salivary gland. An immunohistochemical and clinicopathologic study. Am J Surg Pathol. 12:461–468, 1988. 294. Beltran D, Faquin WC, Gallagher G, et al: Selective immunohistochemical comparison of polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma. J Oral Maxillofac Surg. 64:415–423, 2006. 295. Perez-Ordonez B, Linkov I, Huvos AG: Polymorphous low-grade adenocarcinoma of minor salivary glands: a study of 17 cases with emphasis on cell differentiation. Histopathology. 32:521– 529, 1998. 296. Martinez-Rodriguez N, Leco-Berrocal I, Rubio-Alonso L, et al: Epidemiology and treatment of adenoid cystic carcinoma of the minor salivary glands: a meta-analytic study. Med Oral Patol Oral Cir Bucal. 16:e884–e889, 2011. 297. Papaspyrou G, Hoch S, Rinaldo A, et al: Chemotherapy and targeted therapy in adenoid cystic carcinoma of the head and neck: a review. Head Neck. 33:905–911, 2011. 298. Seethala RR: An update on grading of salivary gland carcinomas. Head Neck Pathol. 3:69–77, 2009. 299. Dodd RL, Slevin NJ: Salivary gland adenoid cystic carcinoma: a review of chemotherapy and molecular therapies. Oral Oncol. 42:759–769, 2006. 300. de AV, de Sousa SO, Carvalho YR, et al: Application of immunohistochemistry to the diagnosis of salivary gland tumors. Appl Immunohistochem Mol Morphol. 8:195–202, 2000. 301. Edwards PC, Bhuiya T, Kelsch RD: Assessment of p63 expression in the salivary gland neoplasms adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, and basal cell and canalicular adenomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 97:613–619, 2004. 302. Penner CR, Folpe AL, Budnick SD: C-kit expression distinguishes salivary gland adenoid cystic carcinoma from polymorphous low-grade adenocarcinoma. Mod Pathol. 15:687–691, 2002. 303. Mitani Y, Rao PH, Futreal PA, et al: Novel chromosomal rearrangements and break points at the t(6;9) in salivary adenoid cystic carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome. Clin Cancer Res. 17:7003–7014, 2011. 304. Bell D, Roberts D, Karpowicz M, et al: Clinical significance of Myb protein and downstream target genes in salivary adenoid cystic carcinoma. Cancer Biol Ther. 12:569–573, 2011. 305. Persson F, Fehr A, Sundelin K, et al: Studies of genomic imbalances and the MYB-NFIB gene fusion in polymorphous lowgrade adenocarcinoma of the head and neck. Int J Oncol. 40:80–84, 2012. 306. Brill LB, Kanner WA, Fehr A, et al: Analysis of MYB expression and MYB-NFIB gene fusions in adenoid cystic carcinoma and other salivary neoplasms. Mod Pathol. 24:1169–1176, 2011. 307. West RB, Kong C, Clarke N, et al: MYB expression and translocation in adenoid cystic carcinomas and other salivary gland tumors with clinicopathologic correlation. Am J Surg Pathol. 35:92–99, 2011. 308. Donath K, Seifert G, Schmitz R: [Diagnosis and ultrastructure of the tubular carcinoma of salivary gland ducts. Epithelialmyoepithelial carcinoma of the intercalated ducts]. Virchows Arch A Pathol Pathol Anat. 356:16–31, 1972. 309. Seethala RR, Barnes EL, Hunt JL: Epithelial-myoepithelial carcinoma: a review of the clinicopathologic spectrum and immunophenotypic characteristics in 61 tumors of the salivary glands and upper aerodigestive tract. Am J Surg Pathol. 31:44–57, 2007. 310. Cho KJ, El-Naggar AK, Ordonez NG, et al: Epithelial-myoepithelial carcinoma of salivary glands. A clinicopathologic, DNA flow cytometric, and immunohistochemical study of Ki-67 and HER-2/neu oncogene. Am J Clin Pathol. 103:432–437, 1995. 311. Antic T, Venkataraman G, Oshima K: Oncocytic epithelial-myoepithelial carcinoma: an evolving new variant with comparative immunohistochemistry. Pathology. 40:415–418, 2008.
321.e8
Immunohistology of Head and Neck Lesions
312. Shinozaki A, Nagao T, Endo H, et al: Sebaceous epithelialmyoepithelial carcinoma of the salivary gland: clinicopathologic and immunohistochemical analysis of 6 cases of a new histologic variant. Am J Surg Pathol. 32:913–923, 2008. 313. Kusafuka K, Takizawa Y, Ueno T, et al: Dedifferentiated epithelial-myoepithelial carcinoma of the parotid gland: a rare case report of immunohistochemical analysis and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 106:85–91, 2008. 314. Fonseca I, Soares J: Proliferating cell nuclear antigen immunohistochemistry in epithelial-myoepithelial carcinoma of the salivary glands. Arch Pathol Lab Med. 117:993–995, 1993. 315. Furuse C, Cury PR, Altemani A, et al: Beta-catenin and Ecadherin expression in salivary gland tumors. Int J Surg Pathol. 14:212–217, 2006. 316. Wang B, Brandwein M, Gordon R, et al: Primary salivary clear cell tumors–a diagnostic approach: a clinicopathologic and immunohistochemical study of 20 patients with clear cell carcinoma, clear cell myoepithelial carcinoma, and epithelialmyoepithelial carcinoma. Arch Pathol Lab Med. 126:676–685, 2002. 317. Seifert G: Classification and differential diagnosis of clear and basal cell tumors of the salivary glands. Semin Diagn Pathol. 13:95–103, 1996. 318. O’Sullivan-Mejia ED, Massey HD, Faquin WC, et al: Hyalinizing clear cell carcinoma: report of eight cases and a review of literature. Head Neck Pathol. 3:179–185, 2009. 319. Antonescu CR, Katabi N, Zhang L, et al: EWSR1-ATF1 fusion is a novel and consistent finding in hyalinizing clear-cell carcinoma of salivary gland. Genes Chromosomes Cancer. 50:559– 570, 2011. 320. Lai G, Nemolato S, Lecca S, et al: The role of immunohistochemistry in the diagnosis of hyalinizing clear cell carcinoma of the minor salivary gland: a case report. Eur J Histochem. 52:251– 254, 2008. 321. Solar AA, Schmidt BL, Jordan RC: Hyalinizing clear cell carcinoma: case series and comprehensive review of the literature. Cancer. 115:75–83, 2009. 322. Rezende RB, Drachenberg CB, Kumar D, et al: Differential diagnosis between monomorphic clear cell adenocarcinoma of salivary glands and renal (clear) cell carcinoma. Am J Surg Pathol. 23:1532–1538, 1999. 323. Barnes L, Rao U, Krause J, et al: Salivary duct carcinoma. Part I. A clinicopathologic evaluation and DNA image analysis of 13 cases with review of the literature. Oral Surg Oral Med Oral Pathol. 78:64–73, 1994. 324. Brandwein MS, Jagirdar J, Patil J, et al: Salivary duct carcinoma (cribriform salivary carcinoma of excretory ducts). A clinicopathologic and immunohistochemical study of 12 cases. Cancer. 65:2307–2314, 1990. 325. Delgado R, Vuitch F, bores-Saavedra J: Salivary duct carcinoma. Cancer. 72:1503–1512, 1993. 326. Guzzo M, Di PS, Grandi C, et al: Salivary duct carcinoma: clinical characteristics and treatment strategies. Head Neck. 19:126– 133, 1997. 327. Lewis JE, McKinney BC, Weiland LH, et al: Salivary duct carcinoma. Clinicopathologic and immunohistochemical review of 26 cases. Cancer. 77:223–230, 1996. 328. Wick MR, Ockner DM, Mills SE, et al: Homologous carcinomas of the breasts, skin, and salivary glands. A histologic and immunohistochemical comparison of ductal mammary carcinoma, ductal sweat gland carcinoma, and salivary duct carcinoma. Am J Clin Pathol. 109:75–84, 1998. 329. Nagao T, Gaffey TA, Serizawa H, et al: Sarcomatoid variant of salivary duct carcinoma: clinicopathologic and immunohistochemical study of eight cases with review of the literature. Am J Clin Pathol. 122:222–231, 2004. 330. Skalova A, Starek I, Vanecek T, et al: Expression of HER-2/neu gene and protein in salivary duct carcinomas of parotid gland as revealed by fluorescence in-situ hybridization and immunohistochemistry. Histopathology. 42:348–356, 2003. 331. Fan CY, Wang J, Barnes EL: Expression of androgen receptor and prostatic specific markers in salivary duct carcinoma: an immunohistochemical analysis of 13 cases and review of the literature. Am J Surg Pathol. 24:579–586, 2000.
332. Felix A, El-Naggar AK, Press MF, et al: Prognostic significance of biomarkers (c-erbB-2, p53, proliferating cell nuclear antigen, and DNA content) in salivary duct carcinoma. Hum Pathol. 27:561–566, 1996. 333. Kapadia SB, Barnes L: Expression of androgen receptor, gross cystic disease fluid protein, and CD44 in salivary duct carcinoma. Mod Pathol. 11:1033–1038, 1998. 334. Hunt JL, Tomaszewski JE, Montone KT: Prostatic adenocarcinoma metastatic to the head and neck and the workup of an unknown epithelioid neoplasm. Head Neck. 26:171–178, 2004. 335. Delgado R, Klimstra D, bores-Saavedra J: Low grade salivary duct carcinoma. A distinctive variant with a low grade histology and a predominant intraductal growth pattern. Cancer. 78:958– 967, 1996. 336. Brandwein-Gensler M, Hille J, Wang BY, et al: Low-grade salivary duct carcinoma: description of 16 cases. Am J Surg Pathol. 28:1040–1044, 2004. 337. Weinreb I, Tabanda-Lichauco R, Van der KT, et al: Low-grade intraductal carcinoma of salivary gland: report of 3 cases with marked apocrine differentiation. Am J Surg Pathol. 30:1014– 1021, 2006. 338. Skalova A, Vanecek T, Sima R, et al: Mammary analogue secretory carcinoma of salivary glands, containing the ETV6-NTRK3 fusion gene: a hitherto undescribed salivary gland tumor entity. Am J Surg Pathol. 34:599–608, 2010. 339. Lei Y, Chiosea SI: Re-evaluating historic cohort of salivary acinic cell carcinoma with new diagnostic tools. Head Neck Pathol. 6(2):166–170, 2012. 340. Griffith C, Seethala R, Chiosea SI: Mammary analogue secretory carcinoma: a new twist to the diagnostic dilemma of zymogen granule poor acinic cell carcinoma. Virchows Arch. 459:117–118, 2011. 341. Chiosea SI, Griffith C, Assaad A, et al: Clinicopathological characterization of mammary analogue secretory carcinoma of salivary glands. Histopathology. 61(3):387–394, 2012. 342. Petersson F, Lian D, Chau YP, et al: Mammary analogue secretory carcinoma: the first submandibular case reported including findings on fine needle aspiration cytology. Head Neck Pathol. 6:135– 139, 2012. 343. Hotte SJ, Winquist EW, Lamont E, et al: Imatinib mesylate in patients with adenoid cystic cancers of the salivary glands expressing c-kit: a Princess Margaret Hospital phase II consortium study. J Clin Oncol. 23:585–590, 2005. 344. Alcedo JC, Fabrega JM, Arosemena JR, et al: Imatinib mesylate as treatment for adenoid cystic carcinoma of the salivary glands: report of two successfully treated cases. Head Neck. 26:829–831, 2004. 345. Tonon G, Modi S, Wu L, et al: t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway. Nat Genet. 33:208–213, 2003. 346. Okabe M, Miyabe S, Nagatsuka H, et al: MECT1-MAML2 fusion transcript defines a favorable subset of mucoepidermoid carcinoma. Clin Cancer Res. 12:3902–3907, 2006. 347. Behboudi A, Enlund F, Winnes M, et al: Molecular classification of mucoepidermoid carcinomas-prognostic significance of the MECT1-MAML2 fusion oncogene. Genes Chromosomes Cancer. 45:470–481, 2006. 348. Martins C, Cavaco B, Tonon G, et al: A study of MECT1MAML2 in mucoepidermoid carcinoma and Warthin’s tumor of salivary glands. J Mol Diagn. 6:205–210, 2004. 349. Alkhalidi H, de Tilly LN, Fenton R, et al: Spontaneous middleear encephalocele: report of two cases and brief review. Clin Neuropathol. 27:357–360, 2008. 350. Gyure KA, Thompson LD, Morrison AL: A clinicopathological study of 15 patients with neuroglial heterotopias and encephaloceles of the middle ear and mastoid region. Laryngoscope. 110:1731–1735, 2000. 351. Penner CR, Thompson LD: Nasal glial heterotopia: a clinicopathologic and immunophenotypic analysis of 10 cases with a review of the literature. Ann Diagn Pathol. 7:354–349, 2003. 352. Wind JJ, Caputy AJ, Roberti F: Spontaneous encephaloceles of the temporal lobe. Neurosurg Focus. 25:E11, 2008.
References 353. Thompson LD, Nelson BL, Barnes EL: Ceruminous adenomas: a clinicopathologic study of 41 cases with a review of the literature. Am J Surg Pathol. 28:308–318, 2004. 354. Hicks GW: Tumors arising from the glandular structures of the external auditory canal. Laryngoscope. 93:326–340, 1983. 355. Kacerovska D, Cermak J, Kreuzberg B, et al: Neuroendocrine adenoma of the middle ear with extension into the external auditory canal. Cesk Patol. 48:36–38, 2012. 356. Fayad JN, Keles B, Brackmann DE: Jugular foramen tumors: clinical characteristics and treatment outcomes. Otol Neurotol. 31:299–305, 2010. 357. Erickson D, Kudva YC, Ebersold MJ, et al: Benign paragangliomas: clinical presentation and treatment outcomes in 236 patients. J Clin Endocrinol Metab. 86:5210–5216, 2001. 358. Sennaroglu L, Sungur A: Histopathology of paragangliomas. Otol Neurotol. 23:104–105, 2002. 359. Rao AB, Koeller KK, Adair CF: From the archives of the AFIP. Paragangliomas of the head and neck: radiologic-pathologic correlation. Armed Forces Institute of Pathology. Radiographics. 19:1605–1632, 1999. 360. Gulya AJ: The glomus tumor and its biology. Laryngoscope. 103:7–15, 1993. 361. Pellitteri PK, Rinaldo A, Myssiorek D, et al: Paragangliomas of the head and neck. Oral Oncol. 40:563–575, 2004. 362. Maroldi R, Farina D, Palvarini L, et al: Computed tomography and magnetic resonance imaging of pathologic conditions of the middle ear. Eur J Radiol. 40:78–93, 2001. 363. Myssiorek D: Head and neck paragangliomas: an overview. Otolaryngol Clin North Am. 34:829–836, v, 2001. 364. Lam KY, Lo CY, Wat NM, et al: The clinicopathological features and importance of p53, Rb, and mdm2 expression in phaeochromocytomas and paragangliomas. J Clin Pathol. 54:443–448, 2001. 365. Barnes L, Taylor SR: Carotid body paragangliomas. A clinicopathologic and DNA analysis of 13 tumors. Arch Otolaryngol Head Neck Surg. 116:447–453, 1990. 366. Martinez-Madrigal F, Bosq J, Micheau C, et al: Paragangliomas of the head and neck. Immunohistochemical analysis of 16 cases in comparison with neuro-endocrine carcinomas. Pathol Res Pract. 187:814–823, 1991. 367. Johnson TL, Zarbo RJ, Lloyd RV, et al: Paragangliomas of the head and neck: immunohistochemical neuroendocrine and intermediate filament typing. Mod Pathol. 1:216–223, 1988. 368. Min KW: Diagnostic usefulness of sustentacular cells in paragangliomas: immunocytochemical and ultrastructural investigation. Ultrastruct Pathol. 22:369–376, 1998. 369. Achilles E, Padberg BC, Holl K, et al: Immunocytochemistry of paragangliomas–value of staining for S-100 protein and glial fibrillary acid protein in diagnosis and prognosis. Histopathology. 18:453–458, 1991. 370. Thompson LD: Pheochromocytoma of the Adrenal gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 cases. Am J Surg Pathol. 26:551–566, 2002. 371. Offergeld C, Brase C, Yaremchuk S, et al: Head and neck paragangliomas: clinical and molecular genetic classification. Clinics (Sao Paulo). 67(Suppl 1):19–28, 2012. 372. Buffet A, Venisse A, Nau V, et al: A decade (2001-2010) of genetic testing for pheochromocytoma and paraganglioma. Horm Metab Res. 44:359–366, 2012. 373. Torske KR, Thompson LD: Adenoma versus carcinoid tumor of the middle ear: a study of 48 cases and review of the literature. Mod Pathol. 15:543–555, 2002. 374. Thompson LD, Bouffard JP, Sandberg GD, et al: Primary ear and temporal bone meningiomas: a clinicopathologic study of 36 cases with a review of the literature. Mod Pathol. 16:236–245, 2003. 375. Ferlito A, Devaney KO, Rinaldo A: Primary extracranial meningioma in the vicinity of the temporal bone: a benign lesion which is rarely recognized clinically. Acta Otolaryngol. 124:5–7, 2004. 376. Wu ZB, Yu CJ, Guan SS: Posterior petrous meningiomas: 82 cases. J Neurosurg. 102:284–289, 2005. 377. Rushing EJ, Bouffard JP, McCall S, et al: Primary extracranial meningiomas: an analysis of 146 cases. Head Neck Pathol. 3:116– 130, 2009.
321.e9
378. Bierry G, Riehm S, Marcellin L, et al: Middle ear adenomatous tumor: a not so rare glomus tympanicum-mimicking lesion. J Neuroradiol. 37:116–121, 2010. 379. Devaney KO, Ferlito A, Rinaldo A: Epithelial tumors of the middle ear–are middle ear carcinoids really distinct from middle ear adenomas? Acta Otolaryngol. 123:678–682, 2003. 380. Thompson LD: Neuroendocrine adenoma of the middle ear. Ear Nose Throat J. 84:560–561, 2005. 381. Berns S, Pearl G: Middle ear adenoma. Arch Pathol Lab Med. 130:1067–1069, 2006. 382. Bold EL, Wanamaker JR, Hughes GB, et al: Adenomatous lesions of the temporal bone immunohistochemical analysis and theories of histogenesis. Am J Otol. 16:146–152, 1995. 383. Heffner DK: Low-grade adenocarcinoma of probable endolymphatic sac origin A clinicopathologic study of 20 cases. Cancer. 64:2292–2302, 1989. 384. Michaels L: Origin of endolymphatic sac tumor. Head Neck Pathol. 1:104–111, 2007. 385. Bisceglia M, D’Angelo VA, Wenig BM: Endolymphatic sac papillary tumor (Heffner tumor). Adv Anat Pathol. 13:131–138, 2006. 386. Gaffey MJ, Mills SE, Boyd JC: Aggressive papillary tumor of middle ear/temporal bone and adnexal papillary cystadenoma. Manifestations of von Hippel-Lindau disease. Am J Surg Pathol. 18:1254–1260, 1994. 387. Kim HJ, Butman JA, Brewer C, et al: Tumors of the endolymphatic sac in patients with von Hippel-Lindau disease: implications for their natural history, diagnosis, and treatment. J Neurosurg. 102:503–512, 2005. 388. El-Naggar AK, Pflatz M, Ordonez NG, et al: Tumors of the middle ear and endolymphatic sac. Pathol Annu. 29 (Pt 2):199– 231, 1994. 389. Choo D, Shotland L, Mastroianni M, et al: Endolymphatic sac tumors in von Hippel-Lindau disease. J Neurosurg. 100:480– 487, 2004. 390. Devaney KO, Ferlito A, Rinaldo A: Endolymphatic sac tumor (low-grade papillary adenocarcinoma) of the temporal bone. Acta Otolaryngol. 123:1022–1026, 2003. 391. Jensen RL, Gillespie D, House P, et al: Endolymphatic sac tumors in patients with and without von Hippel-Lindau disease: the role of genetic mutation, von Hippel-Lindau protein, and hypoxia inducible factor-1alpha expression. J Neurosurg. 100:488–497, 2004. 392. Lonser RR, Kim HJ, Butman JA, et al: Tumors of the endolymphatic sac in von Hippel-Lindau disease. N Engl J Med. 350:2481–2486, 2004. 393. Megerian CA, McKenna MJ, Nuss RC, et al: Endolymphatic sac tumors: histopathologic confirmation, clinical characterization, and implication in von Hippel-Lindau disease. Laryngoscope. 105:801–808, 1995. 394. Mukherji SK, Albernaz VS, Lo WW, et al: Papillary endolymphatic sac tumors: CT, MR imaging, and angiographic findings in 20 patients. Radiology. 202:801–808, 1997. 395. Horiguchi H, Sano T, Toi H, et al: Endolymphatic sac tumor associated with a von Hippel-Lindau disease patient: an immunohistochemical study. Mod Pathol. 14:727–732, 2001. 396. Megerian CA, Pilch BZ, Bhan AK, et al: Differential expression of transthyretin in papillary tumors of the endolymphatic sac and choroid plexus. Laryngoscope. 107:216–221, 1997. 397. Taguchi D, Takeda T, Kakigi A, et al: Expressions of aquaporin-2, vasopressin type 2 receptor, transient receptor potential channel vanilloid (TRPV)1, and TRPV4 in the human endolymphatic sac. Laryngoscope. 117:695–698, 2007. 398. Gyure KA, Morrison AL: Cytokeratin 7 and 20 expression in choroid plexus tumors: utility in differentiating these neoplasms from metastatic carcinomas. Mod Pathol. 13:638–643, 2000. 399. Bishop JA, Sharma R, Illei PB: Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol. 41:20–25, 2010. 400. Ozolek JA, Bastacky SI, Myers EN, et al: Immunophenotypic comparison of salivary gland oncocytoma and metastatic renal cell carcinoma. Laryngoscope. 115:1097–1100, 2005.
321.e10
Immunohistology of Head and Neck Lesions
401. Shen SS, Truong LD, Scarpelli M, et al: Role of immunohistochemistry in diagnosing renal neoplasms: when is it really useful? Arch Pathol Lab Med. 136:410–417, 2012. 402. Truong LD, Shen SS: Immunohistochemical diagnosis of renal neoplasms. Arch Pathol Lab Med. 135:92–109, 2011. 403. Austin JR, Kershiznek MM, McGill D, et al: Breast carcinoma metastatic to paranasal sinuses. Head Neck. 17:161–165, 1995. 404. Batsakis JG, McBurney TA: Metastatic neoplasms to the head and neck. Surg Gynecol Obstet. 133:673–677, 1971.
405. Ogunyemi O, Rojas A, Hematpour K, et al: Metastasis of genitourinary tumors to the head and neck region. Eur Arch Otorhinolaryngol. 267:273–279, 2010. 406. Manjunatha BS, Kumar GS, Raghunath V: Immunohistochemical expression of Bcl-2 in benign and malignant salivary gland tumors. Med Oral Patol Oral Cir Bucal. 16:e503–e507, 2011. 407. Ferreiro JA: Immunohistochemical analysis of salivary gland canalicular adenoma. Oral Surg Oral Med Oral Pathol. 78:761– 765, 1994.
C H A P T E R 1 0
IMMUNOHISTOLOGY OF ENDOCRINE TUMORS SANDRA J. SHIN, DIANA O. TREABA, RONALD A. DELELLIS
Overview 322 Biology of Antigens and Antibodies 322 Tumors of Specific Sites 326 Endocrine Tumors in Other Sites 358 Summary 362
Overview Immunohistochemical (IHC) methods have had a profound impact on the understanding of the endocrine system and its changes in a wide variety of disease states.1 In particular, these methods have led to the development of a series of functional classifications of endocrine tumors (ETs) that have supplemented, and in some cases replaced, traditional morphologic classifications. The use of IHC in endocrine pathology has been critical for the recognition of new tumor entities, identification of sites of origin of metastatic tumors, and prognostic assessments based on patterns of hormone expression and the presence of a variety of other markers. Moreover, these methods have played a key role both in the identification of precursors of ETs and in elucidating the steps in the hyperplasia-neoplasia sequence. The goals of this chapter are to review the major classes of IHC markers used in the assessment of ETs, the diagnostic approaches of these methods for ETs of specific sites, selected theranostic approaches based on these studies, and to highlight advances in the molecular diagnosis of these tumors.
Biology of Antigens and Antibodies Hormones An important approach to the diagnosis and classification of ETs relies on the demonstration of their hormonal content.1,2 This goal can be accomplished by the use of antibodies directed against the mature hormones 322
and hormone precursors. An additional approach involves the use of in situ hybridization (ISH, hybridization histochemistry) for the demonstration of specific hormonal messenger RNAs (mRNAs). The latter approach is discussed in detail in several reviews.3,4 Virtually all classes of hormones—small peptides, large polypeptide hormones, steroids, amines—and hormone receptors can be visualized in IHC formats.2,5 With the advent of microwave-based antigen retrieval methods, the vast majority of these products can be demonstrated in formalin-fixed, paraffin-embedded (FFPE) samples. However, hormonal products by themselves cannot be used as lineage-specific markers.6 For example, somatostatin is present in the D cells of the pancreatic islets, gastrointestinal (GI) and bronchopulmonary endocrine cells, thymic endocrine cells, and thyroid C cells and also in their corresponding tumors; therefore the presence of immunoreactive somatostatin by itself does not provide evidence of the site of origin of a metastatic lesion. The discussion of individual hormones is addressed in sections on specific endocrine cell types and their corresponding tumors.
Enzymes Enzymes that are active in the biosynthesis and processing of hormones are important markers of endocrine cells.6 Immunoreactivity for aromatic L-amino acid decarboxylase, for example, is widely distributed in neuroendocrine (NE) cells.7 In contrast, tyrosine hydroxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyl transferase have a more limited tissue distribution and are confined to known sites of catecholamine biosynthesis.8 Immunolocalization of these enzymes permits catecholamine synthesizing abilities to be deduced from paraffin sections. The presence of an immunoreactive enzyme, however, does not necessarily imply that the enzyme is present in a functional form.6 A variety of endopeptidases and carboxypeptidases required for the formation of biologically active peptides from precursor molecules are present in the transGolgi region and in secretory granules of NE cells. They include the prohormone convertases PC1/PC3 and PC2 and carboxypeptidases H and E.9,10 The proconvertases are widely distributed in NE cells and their
Biology of Antigens and Antibodies
corresponding tumors, whereas other types of endocrine cells—thyroid follicular cells, parathyroid chief cells, adrenal cortical cells, and testis—are negative.10 NE cells with a neural phenotype (e.g., adrenal medullary cells) contain a predominance of PC2, whereas epithelial NE cells contain a predominance of PC1/PC3. With the exception of parathyroid cells, the presence of PC2 and PC3 correlates with the presence of chromogranins and secretogranins. PC2 and PC1/PC3 are present in normal pituitaries and in pituitary adenomas, adrenocorticotropic hormone (ACTH)–producing adenomas contain a predominance of PC1/PC3, and other adenomas express a predominance of PC2.9 Both peptidylglycine α-amidating monooxygenase and peptidylamidoglycolate lyase are present in NE secretory granules.11 These enzymes are responsible for the alpha amidation of the C-terminal regions of peptide hormones. This function is critical for biologic activity of the peptides. Neuron-specific enolase (NSE) is an additional enzyme that has been studied extensively in NE cells.12 The staining of NE tumors is unrelated to the cellular content of secretory granules, and even degranulated cells are NSE positive. This enzyme is the most acidic isoenzyme of the glycolytic enzyme enolases and is present both in neurons and in NE cells.12 The enolases are products of three genetic loci that have been designated alpha, beta, and gamma. Nonneuronal enolase (α-α) is present in fetal tissues of different types, in glial cells, and in many non-NE tissues in the adult. Muscle enolase is of the β-β type, whereas the neuronal form of enolase has been designated γ-γ. Hybrid enolases are present in megakaryocytes and in a variety of other cell types. NSE (γ-γ) replaces nonneuronal enolase during the migration and differentiation of neurons, and the appearance of this isoenzyme heralds the formation of synapses and electrical excitability. Although many earlier studies used NSE as a marker of NE cells, more recent studies have indicated that the specificity of this marker is limited.13,14 The protein gene product 9.5 (PGP9.5) is a ubiquitin carboxyterminal hydrolase that plays a role in the catalytic degradation of abnormal denatured proteins.15-17 PGP9.5 is present in neurons and nerve fibers and in a variety of NE cells, with the possible exception of those in the normal GI tract. In contrast, carcinoid tumors and a variety of other NE tumors contain PGP9.5. The patterns of staining for NSE and PGP9.5 are generally similar in that positive cells show diffuse cytoplasmic reactivity unrelated to the type of hormone produced or the degree of cellular differentiation.18 Comparative studies, however, have demonstrated that some NE tumors may be positive for PGP9.5 and negative for NSE, whereas others may be positive for NSE and negative for PGP9.5. Antibodies to PGP9.5 are particularly useful for the demonstration of neurons and cells with neuronal differentiation. It should be recognized that some non-NE tumors, such as those of the exocrine pancreas, may also be positive for PGP9.5.19 Histaminase (diamine oxidase) has been used as a marker for some NE cells and their tumors. This enzyme is present in high concentrations in medullary thyroid carcinomas and has been reported in small cell
323
carcinomas of the lung and other NE neoplasms.20 High serum levels of histaminase also occur in pregnancy, and IHC studies have revealed the presence of this enzyme in decidual cells. Thyroid peroxidase is responsible for the oxidation of thyroidal iodide, and antibodies to the enzyme have been used in IHC formats for the identification of normal and neoplastic thyroid tissue.21 Enzymes of the biosynthetic pathway of steroid hormones can also be demonstrated effectively by IHC. Among the enzymes that have been localized are P450scc (cholesterol side-chain cleavage), 3-β-hydroxysteroid dehydrogenase (3β-HSD), 21hydroxylase, 17-α-hydroxylase, 11-β-hydroxylase, and 17-β-hydroxysteroid dehydrogenase type 12 (17βHSD12).22-25 To date, relatively few studies have evaluated antibodies to these enzymes as diagnostic reagents. Immunolocalization of 17β-HSD12 invasive ductal carcinoma was relatively recently associated with poor prognosis and tumor progression.25 Deficiency of a DNA repair enzyme O6methylguanine DNA methyltransferase (MGMT) is more common in pancreatic NE tumors than in carcinoid tumors and is associated with sensitivity to temozolomide. Absence of MGMT identified by IHC may explain the sensitivity of some pancreatic NE tumors to treatment.26
Chromogranins, Secretogranins, and Other Granule Proteins The chromogranins and secretogranins represent the major constituents of NE secretory granules.27-32 Three major chromogranin proteins have been identified and categorized and have been designated chromogranin A, chromogranin B, and secretogranin II, also known as chromogranin C. Additional granins that have been characterized include secretogranin (1B1075), secretogranin IV (HISL-19), and secretogranin V (7B2).33 The chromogranin and secretogranin proteins contain multiple dibasic residues that are sites for endogenous proteolytic processing to smaller peptides.33 For example, chromogranin A contains 439 amino acids with 10 pairs of amino acids that represent potential cleavage sites by proteases such as the prohormone convertases. Resultant peptides include chromostatin, pancreastatin, parastatin, and vasostatin. Functional roles for these smaller peptides include intracellular hormone-binding functions, inhibitory effects on the secretion of other hormones, and antibacterial and antifungal effects. Many NE cells contain all the major granins, whereas others show distinctive patterns of chromogranin distribution. The monoclonal antibody LK2H10, developed by Lloyd and Wilson, is directed against chromogranin A and is currently the most commonly used chromogranin antibody.27 Chromogranins are present within the matrices of secretory granules of NE cells. As a result, tumors with abundant secretory granules demonstrate intense chromogranin immunoreactivity, whereas those with fewer granules are less intensely stained. Numerous studies have demonstrated that chromogranin A represents the single most specific marker of NE differentiation in general use. Antibodies to
324
Immunohistology of Endocrine Tumors
chromogranin B and secretogranin II are available but are not in general use. Tissue-specific patterns and ratios of the chromogranin proteins are typically maintained in NE tumors. For example, chromogranin A is the major granin expressed by gastric carcinoids and serotonin-producing carcinoids of the appendix and ileum. In contrast, strong immunoreactivity for chromogranin B and secretogranin II is typical of rectal NE tumors (carcinoids and small cell carcinomas) and of prolactinomas, which lack chromogranin A.34,35 NE secretory protein-55 (NESP-55) is a 241–amino acid polypeptide that is a member of the chromogranin family.36 It is expressed exclusively in endocrine and neuronal tissue but has a less wide distribution than chromogranin A in human tissues. The reactivity of NESP-55 appears to be restricted to ETs of the pancreas and adrenal medulla, and several studies have indicated that it may be useful in the identification of sites of origin of metastatic ETs.37,38
Synaptophysin and Other Synaptic Vesicle Proteins Synaptophysin is a calcium-binding glycoprotein (38,000 kD), which is the most abundant integral membrane protein constituent of synaptic vesicles of neurons.39 It is also present in a wide spectrum of NE cells and in many of their corresponding tumors. Typically, synaptophysin reactivity is present in a punctate pattern in synaptic regions of neurons and is present diffusely throughout the cytoplasm of NE cells. Ultrastructurally, synaptophysin is present in microvesicles, whereas chromogranin is present in secretory granules.39 These differences indicate that chromogranins and synaptophysin are complementary generic NE markers. Synaptophysin immunoreactivity, however, is not specific to NE cells, because it is also present in adrenal cortical cells and their tumors.40 Synaptic vesicle protein 2 (SVP-2) is present in the central and peripheral nervous systems and in a wide variety of NE cell types. Comparative studies of the distribution of SVP-2, synaptophysin, and chromogranin A in NE tumors have shown excellent agreement, with the exception of hindgut ETs, which showed weak synaptophysin immunoreactivity, no staining for chromogranin A, but strong staining for SVP-2.41 Gastrointestinal stromal tumors also express SVP-2, suggesting that these tumors may have an NE phenotype.42 Vesicular monoamine transporters (VMATs) mediate the transport of amines into vesicles of neurons and endocrine cells. VMAT1 and VMAT2 are differentially expressed by GI ETs with patterns specific for each tumor type.43 For example, serotonin-producing ETs expressed VMAT1 predominantly, whereas histamineproducing ETs (gastric ETs) expressed VMAT2 almost exclusively. On the other hand, peptide hormone–producing GI tumors (rectal carcinoids) and pancreatic ETs (PETs) contained few VMAT1- or VMAT2-positive cells.43 Synaptotagmins (p65), which form a large calciumbinding family, are implicated in neurotransmitter
release, although synaptotagmin I is the only isoform demonstrated to have a role in vesicle fusion. In the pancreatic islets, synaptotagmins have been colocalized with insulin, but the roles of this family of proteins have not been fully explored as markers of NE tumors.44,45 The vesicle-associated membrane proteins (VAMPs, or synaptobrevins) occur in three isoforms and are proteins that are anchored to the cytoplasmic portion of synaptic membrane vesicles and secretory granules. VAMP2 and VAMP3 are present in pancreatic β cells, but the roles of this family of proteins have not been widely studied as markers of NE tumors.46 In contrast to synaptophysin and other synaptic vesicle proteins, synaptosomal-associated protein, 25 kD (SNAP-25), and syntaxin are present in the plasma membranes. At present, only a few studies have reported on the application of these markers in diagnostic pathology.47
CD57 The CD57 antigen is present on subsets of T cells and natural killer (NK) cells.48-50 Antibodies to CD57 also react with Schwann, oligodendroglial, and a variety of NE cells of both neural and epithelial types. Additionally, CD57 positivity is present in prostatic, renal, and cortical thymic epithelial cells. Antibodies to CD57 react with varying proportions of neural tumors, including schwannomas, neurofibromas, neuromas, and granular cell tumors. Among ETs, CD57 has been used most commonly as a marker for NE tumors. For example, CD57 is present in 100% of pheochromocytomas, 85% of extraadrenal paragangliomas and ETs of diverse origins, and 50% of small cell bronchogenic carcinomas. However, CD57 is not restricted in its distribution to NE tumors, because reactivity is present in more than 95% of papillary thyroid carcinomas and approximately 70% of follicular carcinomas.51 Nonendocrine tumors that are frequently CD57 positive include prostatic carcinomas, thymomas, and a variety of small round blue cell tumors. These results indicate that the use of CD57 antibodies alone is unreliable for the specific identification of NE tumors.
Neural Cell Adhesion Molecule (CD56) The neural cell adhesion molecules (NCAMs) comprise a family of glycoproteins that play critical roles in cell binding, migration, and differentiation.52 The NCAM family includes three principal moieties that are generated from alternative splicing of RNA from a gene that is a member of the immunoglobulin supergene family. The molecules are modified posttranslationally by phosphorylation, glycosylation, and sulfation. The homophilic binding properties of NCAMs are modulated by the differential expression of polysialic acid. Although initial studies indicated that NCAM was restricted in its distribution to the nervous system, more recent studies indicate a considerably wider distribution, including the adrenal medulla and cortex (zona glomerulosa), cardiac muscle, thyroid follicular/epithelium, proximal renal tubular epithelium, nephrogenic rests, metanephric mesenchyme, hepatocytes, gastric parietal cells, and
Biology of Antigens and Antibodies
islets of Langerhans. Among tumors, both follicular and papillary thyroid carcinomas—as well as renal cell carcinomas, Wilms tumors, and hepatocellular carcinomas—are NCAM positive.53,54 The Leu-7 antigen, recognized by the human natural killer antibody 1 (HNK-1) monoclonal antibody, has now been identified as a carbohydrate epitope present on NCAM and a number of other adhesion molecules. Most NE cells and tumors with neurosecretory granules contain both NCAM mRNA and NCAM protein.55 Antibodies to a long-chain form of polysialic acid (polySia) found on NCAM have been used in studies of normal and neoplastic C cells and NE tumors of the lung.56,57
Intermediate Filaments Cytokeratins (CKs) are the major intermediate filaments of endocrine cells with the exception of steroidproducing cells. These proteins are members of the intermediate filament (10 nm) superfamily of cytoskeletal proteins.58 They differ from other cytoskeletal filaments on the basis of size and other physical and chemical properties. Microfilaments (5 to 15 nm) contain actin, whereas the 25-nm microtubules contain tubulin. Other types of intermediate filaments present in endocrine cells and their supporting elements include vimentin, glial fibrillary acidic protein (GFAP), and the neurofilament proteins (NFPs). The cytokeratins are the largest and most complex group of intermediate filaments and include a family of at least 30 proteins with molecular weights that range from 40 to 68 kD. The type II keratins are basic and include eight epithelial proteins, CK1 through CK8. The type I keratins are more acidic and include 11 epithelial keratins, CK9 through CK20. Pairs of basic and acidic keratins are expressed differentially in epithelial cells at different stages of development and differentiation. They can be identified immunohistochemically by using pancytokeratin antibodies that react with epitopes on many different molecular-weight CK species or with chainspecific monoclonal antibodies that recognize one specific CK type. The cytokeratins are distributed in tissue-specific patterns, and primary tumors tend to recapitulate the CK profiles of the cells from which they are derived.59,60 In some cases, CK expression patterns tend to be simple, whereas in other cases, complex patterns of expression are apparent. Vimentin (57 kD) is also expressed together with cytokeratins in many normal and neoplastic endocrine cell types. In steroidproducing cells, vimentin is the major intermediate filament protein, and ablation of vimentin is suggested to result in defective steroidogenesis.61 The neurofilaments are composed of heteropolymers of three different subunits with molecular weights of 70, 170, and 195 kD, which correspond to low (L), medium (M), and high (H) molecular weight subunits.62 All three neurofilament subunits are phosphorylated in proportion to the molecular weight of each subunit. The neurofilaments represent the major intermediate filaments of mature and developing neurons, paraganglionic cells, and certain normal NE cells. These intermediate filaments are expressed in tumors with evidence
325
of neuronal differentiation and are also present to varying degrees in NE tumors of epithelial type, which also express cytokeratins. Normal epithelial NE-type cells (pancreatic islets, Merkel cells) most commonly lack neurofilament immunoreactivity, whereas their corresponding neoplasms are commonly positive for this marker. Moreover, the pattern of staining in a dotlike area that corresponds to the Golgi region is typical of NE neoplasms. The studies of Perez and coworkers63 have suggested that the differential expression of neurofilament subtypes is related to tumor site. GFAP (50 kD) is the major intermediate filament type of fibrous and protoplasmic astrocytes. GFAP is also present in nonmyelinated Schwann cells, supporting cells of the anterior pituitary and paraganglia, and in a variety of carcinomas. Immunoreactive GFAP is also present in mixed tumors of the skin and salivary glands and in nerve sheath tumors and chordomas.
Transcription Factors Transcription factors are proteins that bind to regulatory elements in the promoter and enhancer regions of DNA and either stimulate or inhibit gene expression and protein synthesis.1,64 They play critical roles in embryogenesis and development. Transcription factors may be tissue specific, or they may be present in a variety of different tissue types. Many of the so-called tissuespecific transcription factors, however, are not restricted to a single tissue type. For example, thyroid transcription factor 1 (TTF-1) is present both in thyroid follicular cells and in lung, whereas the adrenal 4 binding protein/ steroidogenic factor 1 (Ad4BP/SF-1) is present in steroid-producing cells and in certain anterior pituitary cell types. Pituitary transcription factor Pit-1 is present in certain cells of the adenohypophysis and is also present in the placenta. Additional transcription factors that have been used by IHC include mammalian achaete-scute complex–like protein 1 (MASH-1), TTF, Pax-2, Pax-8, PDX-2, neurogenin 3, Isl1, and the guanine adenine thymine adenine (GATA) family of transcription factors.64-66 Applications of transcription factor localization are discussed in subsequent sections.
Somatostatin Receptors Somatostatin acts via specific receptors that belong to the seven transmembrane G-protein–coupled superfamilies. Somatostatin receptors sst-1 through sst-5 represent the five major subtypes. The inhibitory action of somatostatin on hormone secretion is mediated by sst-2, whereas suppression of cell growth is mediated by sst-1, sst-2, and sst-5; the effects of somatostatin on apoptosis are mediated by sst-2 and sst-3. IHC analysis of somatostatin receptors has been used to gauge the responsiveness of NE tumors to somatostatin analogs.67
Cell-Cycle Markers Antibodies to cell-cycle markers have been used in endocrine pathology primarily as an adjunct for the distinction of benign and malignant tumors.68-70 In
326
Immunohistology of Endocrine Tumors
A
B
Figure 10-1 Normal human thyroid gland stained for thyroid transcription factor 1. A, Streptavidin biotin peroxidase technique with incomplete blocking of endogenous biotin. Both the nuclei and cytoplasm are stained positively. B, Polymer-based (EnVision FLEX+; Dako, Glostrup, Denmark) technique. Positive staining is restricted to nuclei.
general, malignant tumors have a higher labeling index than benign tumors, as assessed with antibodies to Ki-67 (MIB-1). However, there may be considerable overlap in proliferative indices between benign and malignant ETs. The cyclin-dependent kinase inhibitor p27 is decreased in many malignant ETs, compared with their benign counterparts. In some instances, the combined use of Ki-67 and p27 is more effective than the use of either antibody alone.68
Pitfalls of Immunohistochemistry of Endocrine Tumors Many endocrine cells, including thyroid follicular and adrenal cortical cells, contain high levels of biotin-like activity, which is enhanced following heat-induced epitope retrieval (Fig. 10-1). Because endogenous biotin is often incompletely blocked following standard blocking procedures, the use of polymer-based detection systems is recommended for all studies of endocrine cells and tumors.71 The use of such a system essentially circumvents the nonspecific background staining observed with avidin- or streptividin-based system.
Tumors of Specific Sites Adenohypophysis The cell types of the adenohypophysis were categorized originally on the basis of their reactivities with hematoxylin and eosin (H&E) as acidophils, basophils, and
chromophobes. With more sophisticated histochemical staining sequences, the three cell types were subdivided further. For example, acidophils were further differentiated into the orange, G-positive, prolactin-positive cells and the erythrosin-positive growth hormone–producing cells, whereas basophils could be demonstrated by their periodic acid–Schiff (PAS) positivity. The subsequent development of IHC methods allowed for the distinction of cell types based on their content of specific hormones.72 The major cell types and their corresponding products include somatotrophs (growth hormone), lactotrophs (prolactin), mammosomatotrophs (growth hormone, prolactin), thyrotrophs (thyroid-stimulating hormone [TSH]), corticotrophs (adrenocorticotropin hormone [ACTH], β-endorphin, melanocyte-stimulating hormone), and gonadotrophs (follicle-stimulating hormone [FSH], luteinizing hormone [LH]). The somatotrophs are present predominantly in the lateral wings and account for approximately 50% of the cells of the adenohypophysis. Lactotrophs predominate at the posterolateral edges of the gland and account for 15% to 25% of the cells. The corticotrophs are present primarily in the central mucoid wedge and account for 15% to 20% of the cells. Thyrotrophs account for 5% of the cells and are located in the anteromedial regions of the gland. The gonadotrophs compose approximately 5% of the cell populations and are scattered throughout the anterior lobe. In addition to the hormone-producing cells, a second cell population is also present (folliculostellate cells) in the normal gland.73 The latter cells have a dendritic
Tumors of Specific Sites
327
TABLE 10-1 Classification and Immunohistochemistry of Pituitary Adenomas Type Sparsely granulated prolactin
Frequency
Immunohistochemistry PRL (paranuclear), α-su (rare), Pit-1, ER-α
27%
Densely granulated prolactin cell adenoma
0.4%
PRL (diffuse), Pit-1, ER-α
Sparsely granulated GH cell adenoma
7.6%
GH (weak), α-su (weak), Pit-1
Densely granulated GH cell adenoma
7.1%
GH (strong), α-su (~50%), Pit-1
Mixed GH cell and prolactin cell adenoma
3.5%
GH and PRL in different cells, GATA-2
Mammosomatotroph cell adenoma
1.2%
GH and PRL in same cells, Pit-1, ER-α, α-su
Corticotroph cell adenoma
9.6%
ACTH, β-end, β-LPH, neuro-D1, Pit-1, Tbx19
Thyrotroph cell adenoma
1.1%
β-TSH, α-su (variable), GATA-2
Gonadotroph cell adenoma
9.8%
β-FSH, β-LH, α-su, SF-1, ER-α, GATA-2
Silent corticotroph cell adenoma (subtype 1)
2.0%
ACTH, β-end (β-end > ACTH)
Silent corticotroph cell adenoma (subtype 2)
1.5%
ACTH (focal), β-end (β-end > ACTH)
Silent adenoma (subtype 3)
1.4%
GH, PRL, α-su
Null cell adenoma
12.4%
Usually negative for hormones, but some cases are positive for β-FSH and α-su; SF-1
Oncocytoma
13.4%
Similar to null cell
Unclassified
8.8%
ACTH, Adrenocorticotropin hormone; α-su, alpha subunit; end, endorphin; FSH, follicle stimulating hormone; LH, luteinizing hormone; LPH, lipotropin; TSH, thyroid stimulating hormone; ER, estrogen receptor; GATA, GATA-binding protein; GH, growth hormone; PRL, prolactin; SF, steroid factor; Tbx19, T-box 19 transcription factor.
shape and typically encircle the hormone-positive cells. The folliculostellate cells are positive for S-100 protein and are variably positive for GFAP. Pituitary adenomas are currently classified according to their content of specific hormones as summarized in Table 10-1 and Figures 10-2 and 10-3. The tumors also have distinctive patterns of reactivity with antibodies to transcription factors and cytokeratins.74 More than 90% of adenomas contain CK8, and in sparsely granulated growth hormone (GH) cell adenomas, the staining is globular and corresponds to the presence of fibrous bodies. Perinuclear staining is typical of densely granulated GH cell and mammosomatotroph adenomas,
A
whereas corticotroph cell adenomas exhibit more diffuse cytoplasmic staining for CK8. Approximately 50% of adenomas exhibit keratin immunoreactivity with the AE1/AE3 antibody cocktail, but only 7% and 10% are reactive with CK19 and CK7, respectively (Fig. 10-4).59 Pituitary adenomas are typically positive for NE markers that include chromogranin (100%), synaptophysin (92%), and NSE (80%).1,36,75 The hormonal content of these tumors can be demonstrated with monoclonal antibodies to specific anterior pituitary hormones and hormone precursor fragments (see Table 10-1). In contrast to their presence in the normal
B
Figure 10-2 A, Pituitary adenoma (hematoxylin and eosin). B, Immunoperoxidase stain for prolactin. The cells show weak granular cytoplasmic positivity (sparsely granulated prolactinoma).
328
Immunohistology of Endocrine Tumors
A
B
Figure 10-3 A, Pituitary adenoma (hematoxylin and eosin). B, Immunoperoxidase stain for growth hormone. All the cells contain immunoreactive growth hormone.
anterior pituitary, S-100–positive folliculostellate cells are generally absent from pituitary adenomas.76 Pituitary carcinomas are rare, and their diagnosis rests on the demonstration of metastases. These tumors are typically positive for generic NE markers and for one or more anterior pituitary hormones.77 The most frequently synthesized hormones are prolactin and ACTH, whereas the production of growth hormone, TSH, and FSH/LH is rare.77 The distinction of adenomas from carcinomas in the absence of metastases is difficult, if not impossible. Significant differences are found in MIB-1 labeling indices among adenomas, invasive adenomas, and carcinomas, but overlaps in these indices exist, and in some carcinomas, the labeling index is in the range of adenomas.78 Thappar and coworkers79 have
Pituitary adenoma 110 100 90 80 Percent
70 60 50 40 30 20 10
P S10 0
9
FA G
K7
K1 C
C
N SE E1 /A E3 ) H BM E1 (A
R
2
N
5.
SY
AM
)C
AP
KE
(K
ER
C H
R -A
0
Figure 10-4 Distribution of markers in pituitary adenomas. CHR-A, Chromogranin A; KER (CAM5.2), keratins detected with monoclonal antibody CAM5.2; SYNAP, synaptophysin; NSE, neuron-specific enolase; KER (AE1/AE3), keratins detected with antibodies AE1 and AE3; HBME-1, Hector Battifora mesothelial cell 1; CK7, cytokeratin 7; CK19, cytokeratin 19; GFAP, glial fibrillary acid protein.
reported p53 in 0%, 15%, and 100% of adenomas, atypical adenomas, and carcinomas, respectively, but exceptions to these generalizations exist.
Molecular Approaches Relatively few studies have examined the diagnostic molecular aspects of pituitary tumors.80 The distinction between benign and malignant pituitary tumors is difficult, if not impossible, by standard histopathologic criteria. In a study of prolactin-producing tumors that used molecular and IHC approaches, Wierinckx and colleagues81 identified nine genes implicated in invasion, proliferation, and differentiation that were differentially expressed in noninvasive, invasive, and invasive/ aggressive tumors. By routine histology and IHC, the presence of four markers of differentiation—mitoses, Ki-67, pituitary tumor transforming gene (PTTG), and p53—and a marker of invasion, polysialic acid of NCAM, demonstrated that no single marker could distinguish invasive from noninvasive tumors. However, mitoses and Ki-67 labeling were statistically different in the invasive tumors, whereas p53 and PTTG nuclear labeling were common in the invasive group. PTTG expression was restricted to the cytoplasm in noninvasive and invasive tumors, but it was present in both the nucleus and cytoplasm of the invasive/aggressive tumors.81 Galectin-3 and high mobility group A-1 (HMGA-1) may also play a role in pituitary tumor progression.82-84 As noted by Asa,85 the best predictive marker for pituitary tumors is the classification based on hormone content and cell structure. For example, the response to long-acting somatostatin analogs in acromegalic patients who do not respond to surgical resection is best determined by the classification of tumors into sparsely and densely granulated types. Responders to octreotide are more likely to have densely granulated adenomas, which typically have a weak perinuclear pattern of CK immunoreactivity with CAM5.2. Sparsely granulated adenomas, on the other hand, have a characteristic globular, juxtanuclear fibrous body. Additionally, silent corticotrophic adenomas will recur more often and more aggressively than will silent gonadotrophic adenomas.
Tumors of Specific Sites
Pineal Gland Tumors of the pineal gland include parenchymal neoplasms (pineocytoma, pineoblastoma, and pineal parenchymal tumor of intermediate differentiation), germ-cell neoplasms, gliomas, meningeal tumors, and a variety of other tumor types that include lymphomas and lipomatous tumors. Parenchymal tumors comprise a spectrum of lesions that ranges from the most immature lesion (pineoblastoma) to the well-differentiated pineocytoma. Tumors with intermediate features are referred to as pineal parenchymal tumors of intermediate differentiation (PPT-ID).86 Most primary tumors of the pineal gland originate from pineocytes, which represent modified neurons similar to retinal photoreceptor cells.87 Pineocytomas are typically positive for NSE, synaptophysin, neurofilament proteins, tau protein, and microtubule-associated protein 2 (MAP-2; Fig. 10-5).86-88 GFAP and S-100 protein are present in 75% and 83% of cases, respectively. S-antigen, a protein localized in photoreceptor cells, has been demonstrated in 28% of pineocytomas and 50% of pineoblastomas.89,90 Most pineoblastomas are negative for NFPs but are typically positive for synaptophysin. In general, neurofilament positivity indicates a better prognosis in pineal parenchymal tumors, whereas the MIB-1 labeling index is 1.58, 16.1, and 23.5 in pineocytomas, PPT-IDs, and pineoblastomas, respectively.91 In contrast to germ-cell tumors, pineocytomas are negative for placental alkaline phosphatase (PAP), human chorionic gonadotropin (hCG), and α-fetoprotein (AFP).86 More recently, CRX, a homeobox transcription factor, has been found to demonstrate strong nuclear expression in more than 95% of retinal and pineal tumors, although not in the majority of tumors considered in the differential diagnosis of lesions of the pineal region, with the exception of medulloblastomas.92,93 Intensity of CRX staining did not correlate with the subclassification of pineal tumors studied (four pineocytomas, four PPT-IDs, and five pineoblastomas); and more common than not, distribution of staining was heterogeneous.92
A
329
Follicular Cells and Their Neoplasms THYROGLOBULIN, T3 AND T4, THYROID PEROXIDASE, AND THYROID TRANSCRIPTION FACTORS 1 AND 2
Thyroglobulin (TGB) is a 660-kD glycoprotein with a sedimentation constant of 19S. Iodoproteins of higher and lower sedimentation constants have also been identified immunohistochemically.94 Considerable variation is found in TGB staining intensity in normal thyroid gland. The cuboidal to columnar cells of the normal gland consistently exhibit greater degrees of TGB immunoreactivity than the flattened (atrophic) cells of follicles distended with colloid. Hyperplastic cells are typically strongly stained for TGB, whereas cells that line involuted follicles are weakly reactive or negative. Follicular cells both in Graves disease and in the hyperplastic phase of Hashimoto disease are moderately to strongly reactive for TGB. Variation in the staining of colloid is also apparent. Follicular adenomas are positive for TGB but also show considerable variability in staining intensity based on the functional status of their component cells.94 As would be expected, hyperfunctional adenomas exhibit strong positivity for TGB, whereas inactive follicular cells, such as those present in dilated follicles, have considerably less reactivity and may be negative. Normofollicular adenomas generally demonstrate moderate immunoreactivity for TGB, whereas adenomas of solid and oncocytic types contain smaller amounts of TGB. Hyalinizing trabecular tumors are typically positive for TGB and may occasionally exhibit positivity for some NE markers, including chromogranin A and hormonal peptides (neurotensin, endorphins).95 These tumors have plasma membrane patterns of staining with the monoclonal antibody MIB-196; however, this pattern of reactivity occurs only when staining is performed at room temperature rather than at 37° C (Fig. 10-6).97 The most likely explanation for the plasma membrane pattern of reactivity is that the antibody cross-reacts with an epitope present in the plasma membrane under these conditions.97
B
Figure 10-5 A, Pineocytoma (hematoxylin and eosin). B, Immunoperoxidase stain for synaptophysin. The cell processes show strong reactivity.
330
Immunohistology of Endocrine Tumors
A
B
Figure 10-6 Hyalinizing trabecular tumor of thyroid. A, Hematoxylin and eosin. B, Immunoperoxidase stain for MIB-1 performed at room temperature. Staining of the plasma membranes of tumor cells is prominent.
The frequency of TGB positivity in thyroid carcinomas is dependent on the degree of differentiation and the histologic subtype. Generally, poorly differentiated carcinomas contain less TGB than better differentiated tumors. The levels of TGB mRNA are also correspondingly lower in poorly differentiated than in well-differentiated thyroid carcinomas.98 TGB immunoreactivity is present in more than 95% of papillary carcinomas (Figs. 10-7 and 10-8) and follicular tumors. Because TGB is also expressed in metastatic lesions, stains for this marker are particularly valuable in establishing the origins of metastatic tumors. Immunoreactivity for TGB in differentiated follicular and papillary tumors is generally present in a patchy distribution. Although some cells exhibit diffuse and uniform staining, others have focal apical or basal positivity. Some tumor cells may be completely unreactive, and for this
W ) CK 7 VI CD M 5 TT 7 FG 1 AL -3 TG CK B 19 EM CA A 19 S- -9 HB 10 M 0 E1 PR P ER CK P CH 20 RA
KE
R
(H
M
Percent
Papillary thyroid carcinoma 100 90 80 70 60 50 40 30 20 10 0
reason, the absence of TGB in a small biopsy sample does not completely exclude the possibility of a thyroid origin in a metastatic site. Rarely, TGB immunoreactivity, as demonstrated both with monoclonal antibodies and polyclonal antisera, has been reported in nonthyroid malignancies.99 Poorly differentiated thyroid carcinomas, including those of the insular type, are most often TGB positive, although the extent of cellular staining is generally weak and focal.100 Undifferentiated (anaplastic) thyroid carcinomas are most commonly negative for TGB. In the series reported by Ordonez and coworkers,101 approximately 15% of cases of anaplastic carcinoma exhibited TGB immunoreactivity in a small number of cells by using both monoclonal antibodies and polyclonal antisera. Examination of serial sections in these cases failed to reveal evidence of entrapped normal follicular cells or foci of differentiated thyroid carcinoma. Other authors, however, have failed to demonstrate any TGB immunoreactivity in anaplastic carcinomas except in foci of residual differentiated tumors.102 Antibodies to T3 and T4 have been used less extensively than TGB in studies of thyroid carcinoma. Kawaoi
Figure 10-7 Distribution of markers in papillary thyroid carcinoma. KER (HMW), high molecular weight cytokeratins; CK7, cytokeratin 7; VIM, vimentin; TTF-1, thyroid transcription factor 1; GAL-3, galectin-3; TGB, thyroglobulin; CK19, cytokeratin 19; EMA, epithelial membrane antigen; HBME-1, Hector Battifora mesothelial cell 1; PRP, progesterone receptor protein; ERP, estrogen receptor protein; CK20, cytokeratin 20; CHR-A, chromogranin A.
Figure 10-8 Papillary thyroid carcinoma. The cells in this welldifferentiated tumor reveal uniform reactivity for thyroglobulin (immunoperoxidase stain for thyroglobulin).
Tumors of Specific Sites
Figure 10-9 Well-differentiated follicular thyroid carcinoma. Immunoperoxidase stain for thyroid transcription factor 1 shows the typical nuclear positivity.
and coworkers103 reported T4 positivity in 95% of papillary carcinomas and 54% of follicular carcinomas but in no cases of anaplastic thyroid carcinoma. T3 was present in 66% of papillary carcinomas, 81% of follicular carcinomas, and 45% of anaplastic carcinomas. The significance of T3 staining in the absence of T4 immunoreactivity, however, is unknown. IHC studies have demonstrated that thyroid carcinomas are associated with changes in the quantity and antigenic properties of thyroid peroxidase (TPO).104 However, TPO immunostaining is not sufficiently discriminatory for the differential diagnosis of thyroid malignancies versus benign thyroid lesions.21 TTF-1 is a homeodomain-containing transcription factor expressed in the thyroid, diencephalon, and lung. TTF-1 regulates the expression of TPO and Tg (thyroglobulin) genes in the thyroid,105 and in the lung, it plays a key role in the specific expression of surfactant proteins A, B, and C and Clara cell secretory protein.106,107 TTF-1 immunoreactivity has been reported in 96% of papillary, 100% of follicular, 20% of Hürthle cell, 100% of insular, and 90% of medullary carcinomas (Fig. 10-9).107 In general, the intensity of TTF-1 staining in C-cell tumors is less than that observed in follicular cell tumors. Undifferentiated (anaplastic) carcinomas, on the other hand, are generally negative. In the lung, this marker has been reported in 72.5% of adenocarcinomas, 10% of squamous carcinomas, 26% of large cell carcinomas, 75% of large cell neuroendocrine carcinomas (NECs), more than 90% of small cell carcinomas, and 100% of alveolar adenomas. In contrast, only a small fraction of adenocarcinomas of nonpulmonary and nonthyroid types are positive for this marker. In their study of thyroid and pulmonary carcinomas, Kaufmann and Dietel demonstrated reactivity for surfactant protein A in 3 of 7 thyroid carcinomas in a focal pattern.108 Byrd-Gloster and coworkers109 reported that TTF-1 is useful in the distinction of pulmonary small cell carcinomas from Merkel cell carcinomas; in their study, 97% of small cell bronchogenic carcinomas were TTF-1 positive, whereas none of 21 Merkel cell tumors exhibited positivity. However, TTF-1 has been reported
331
in some nonpulmonary small cell carcinomas, including those that arise in the prostate, urinary bladder, and uterine cervix.110 Thyroid transcription factor 2 (TTF-2) and Pax-8, which are essential for thyroid organogenesis and differentiation, have also been studied in thyroid tumors.111 TTF-1 and -2 and Pax-8 were expressed in differentiated and poorly differentiated thyroid carcinomas, whereas TTF-1 and -2 were expressed in 18% and 7% of anaplastic carcinomas, respectively. Pax-8, on the other hand, was present in 79% of anaplastic carcinomas.111 TTF-2 was negative in all other neoplastic and nonneoplastic tissues, including those of pulmonary origin. Although Pax-8 was present in a variety of normal and neoplastic tissues, it was not expressed in pulmonary tumors or normal pulmonary tissue. These findings suggest that Pax-8 may be a useful marker for the diagnosis of anaplastic thyroid carcinoma, particularly when the differential diagnosis includes pulmonary carcinoma.111 INTERMEDIATE FILAMENTS
An extensive body of literature describes the distribution of intermediate filaments in normal and neoplastic follicular cells (Table 10-2).112-124 Broad-spectrum keratin antibodies react with normal and hyperplastic follicular cells, follicular cells in chronic thyroiditis, and virtually all thyroid epithelial malignancies. In contrast, antibodies to high-molecular-weight (HMW) keratins have been reported to react with some follicular cells in 8% of normal thyroid, 44% of hyperplastic glands, and all cases of thyroiditis. HMW keratins were present in 100% of papillary carcinomas, 6% of follicular carcinomas, and 20% of anaplastic carcinomas in one study.113 Studies reported by Schelfhout and coworkers115 have demonstrated uniform reactivity for CK19 in 100% of
TABLE 10-2 Cytokeratin Distribution in Papillary Carcinomas and Follicular Tumors Cytokeratin Type
Papillary (%)
Follicular (%)
8
100
100
18
100
100
7
100
100
19
98*
84*
1, 5, 10, 11/14
97
22
5, 6
68
8
17
40
15
13
30
0
20
26
12
14
11
10
4
2.4
0
*Although CK19 is present in papillary carcinoma and in follicular tumors, the extent of staining is consistently higher in papillary carcinoma.
332
Immunohistology of Endocrine Tumors
Figure 10-10 Papillary thyroid carcinoma. Immunoperoxidase stain for cytokeratin 19 demonstrates intense cytoplasmic reactivity.
papillary carcinomas (Fig. 10-10). Focal reactivity for CK19 (in <5% of the tumor cells) was present in 80% of follicular carcinomas and 90% of follicular adenomas, whereas 90% of colloid (adenomatous) nodules demonstrated more diffuse positivity in less than 50% of the cells. These data were largely confirmed by Raphael and associates.116 Generally, the extent of CK19 staining in follicular is considerably less than that present in papillary carcinomas, except in areas of degenerative changes that may be strongly positive.124 More recent studies have demonstrated that normal thyroid strongly expresses the simple epithelial cytokeratins 7 and 18, and to a lesser extent 8 and 19, but not stratified epithelial cytokeratins.117 The same patterns of staining were present in lymphocytic thyroiditis, but reactivity for CK19 was more intense. Immunoreactivity for CK7, CK8, CK18, and CK19 was present in both papillary and follicular carcinomas, although the extent and intensity of CK19 staining were greater in papillary carcinomas; CK19 was present in all cases of follicular carcinoma, at least focally.117 The stratified epithelial keratins, CK5/6 and CK13, were present in 66% (27/41) and 34% (14/41) of papillary carcinomas, respectively, but these keratins were absent from other tumor types. Miettinen and associates118 observed CK19 in all papillary carcinomas and in approximately 50% of follicular carcinomas, whereas CK5/6 was present focally in papillary carcinomas. Kragsterman and coworkers119 concluded that CK19 is of limited value as a marker for routine histopathologic diagnosis, but that the presence of this marker should raise the suspicion of papillary carcinoma. Baloch and coworkers120 examined a large series of papillary carcinomas of both usual types and follicular variants for a spectrum of cytokeratins that included CK5/6/18, CK18, CK10/13, CK20, CK17, and CK19. In this series, all cases of papillary carcinoma, including the follicular variant, were positive for CK19 (Fig. 10-11). The follicular variants showed strong immunoreactivity in areas with nuclear features of papillary carcinoma, whereas the remaining areas had moderate to strong staining. Normal thyroid parenchyma immediately adjacent to the follicular variants was also
positive, but normal thyroid tissue adjacent to the conventional papillary carcinomas was negative. The significance of the latter observations is unknown. Follicular adenomas, follicular carcinomas, and hyperplastic nodules were negative for CK19. The reasons for the discrepancies in CK19 immunoreactivity in follicular tumors between this and other series are unknown. Considerable controversy surrounds the presence of CK19 in hyalinizing trabecular tumors of the thyroid. Fonseca and coworkers121 reported CK19 in all cases of hyalinizing trabecular tumors and suggested that this tumor represents a peculiar encapsulated variant of papillary carcinoma. In contrast, Hirokawa and colleagues122 found minimal to no CK19 in their series of cases. Liberman and Weidner123 studied the distribution of HMW cytokeratins as demonstrated with the monoclonal antibody 34βE12 (CK1, CK5, CK10, and CK14) and antibodies to involucrin, a structural protein of the stratum corneum, in a series of papillary and follicular carcinomas. Antibodies to HMW cytokeratins reacted with 91% of papillary carcinomas, including the follicular variant, and 20% of follicular neoplasms (adenomas and carcinomas). In general, the staining pattern in papillary carcinomas was strong and patchy, whereas follicular neoplasms stained weakly. Involucrin was positive in 72.5% of papillary carcinomas and 29% of follicular tumors. It has been suggested that the pattern of staining with 34βE12 might be best explained by the presence of an epitope on CK1 or by an epitope that is not recognized by other monoclonal antibodies to CK5, CK10, and CK14.123 Cytokeratins are demonstrable in 70% to 75% of anaplastic thyroid carcinomas by using antibodies AE1/ AE3, 34βH11, and CAM5.2, and approximately 30% exhibit reactivity with 34βE12 (Fig. 10-12).101,102 Poorly differentiated carcinomas exhibit positivity in 100% of cases with broad spectrum cytokeratin antibodies.1 Vimentin is coexpressed with cytokeratins in the vast majority of normal and neoplastic thyroids. In the series of Miettinen and colleagues,113 follicular and papillary tumors expressed vimentin in more than 50% of the
Figure 10-11 Papillary thyroid carcinoma, follicular variant. Immunoperoxidase stain for cytokeratin 19 demonstrates intense cytoplasmic reactivity.
Tumors of Specific Sites
A
333
B
Figure 10-12 Anaplastic (undifferentiated) thyroid carcinoma. A, Hematoxylin and eosin. B, Immunoperoxidase stain for broad-spectrum cytokeratins (AE1/AE3) shows cytoplasmic staining.
tumor cells. Immunoreactivity for vimentin was generally present in the basal portions of the cells in contrast to the more diffuse cytoplasmic reactivity for cytokeratins, and vimentin immunoreactivity has been reported in 94% of anaplastic thyroid carcinomas. ONCOGENES AND TUMOR SUPPRESSOR GENES
The distribution of p53 has been examined in cases of thyroid carcinoma. In the series reported by Soares and coworkers,125 stains for p53 were negative in adenomas and papillary thyroid carcinomas. However, p53 was present in 20% of follicular carcinomas, predominantly those of the widely invasive type, and in 16% of poorly differentiated carcinomas and 67% of undifferentiated carcinomas. In the series reported by Holm and Nesland,126 19% of papillary carcinomas, 17% of follicular carcinomas, and 75% of undifferentiated carcinomas were p53 positive. In contrast, the RB protein was present in all thyroid carcinomas, suggesting that it does not play a major role in thyroid carcinogenesis. The use of the RET oncogene protein has been explored by IHC as a marker for papillary carcinomas and a subset of hyalinizing trabecular tumors.127-130 However, correlation is low between expression of the protein by using commercially available antibodies to the C-terminal domain of the protein and the presence of RET/PTC rearrangements as determined by molecular studies.131 The t(2;3)(q13; p25) translocation found in a subset of thyroid follicular carcinomas results in fusion of the DNA binding domains of the thyroid transcription factor Pax-8 to domains A through F of the peroxisome proliferation–activated receptor (PPAR) γ-1 (Fig. 10-13).132 The translocation results in dramatic overexpression of the PPARG protein. Generally, only strong nuclear staining correlates with the presence of rearrangements.133 Studies using polymerase chain reaction (PCR) and IHC demonstrated that most follicular carcinomas positive for PPARG were widely invasive, whereas tumors that lacked the rearrangement were minimally invasive.134 Papillary carcinomas and Hürthle
cell tumors were negative, but approximately 10% of follicular adenomas are positive for PPARG.135,136 It has been suggested that positivity for this protein in tumors classified as adenomas should prompt the search for invasion in additional levels and sections.133 Strong staining for PPARG has also been reported in follicular cells adjacent to foci of chronic inflammation.137 The use of antibodies to the mutated BRAF V600 protein has also been explored for the diagnosis of papillary thyroid carcinoma.138,139 In one study, no difference was found in the expression of the protein between microcarcinomas and macrocarcinomas.138 The expression of the protein was significantly more common in tumors with tall cell and oncocytic features than in tumors of conventional type, whereas follicular, diffuse sclerosing, and solid variants were negative.138 Bullock and coworkers139 have concluded that IHC for the mutated protein is more sensitive and specific than Sanger sequencing in the routine diagnostic setting.
Figure 10-13 Well-differentiated follicular carcinoma stained for PAX8/PPARG. Positive staining is confined to the nuclei of the tumor cells. (Courtesy Dr. Yuri Nikiforov.)
334
Immunohistology of Endocrine Tumors
HBME-1
The monoclonal antibody Hector Battifora mesothelial cell 1 (HBME-1) recognizes an uncharacterized antigen in the microvilli of mesothelial cells, tracheal epithelium, and adenocarcinomas of the pancreas, lung, and breast. It has also been assessed for its efficacy in differentiating benign and malignant thyroid lesions, both in aspirates (direct smears and cell blocks) and in tissue sections.140 Miettinen and Kerkkainen141 demonstrated positivity for HBME-1 in 100% of papillary (145/145) and follicular (27/27) carcinomas, whereas benign lesions were either negative or showed focal positivity in approximately 30% of the cases (Table 10-3). In a more recent series, Mase and colleagues142 demonstrated positivity in 13% of adenomatous goiters, 27% of adenomas, 84% of follicular carcinomas, and 97% of papillary carcinomas (Fig. 10-14). Among follicular neoplasms, the sensitivity for the detection of carcinomas was 84.6%, whereas specificity, positive predictive value, and overall accuracy were 72.6%, 66%, and 77.2%, respectively.142 Sack and colleagues140 concluded that a positive result for HBME-1 on fine needle aspiration (FNA) is supportive evidence that the lesion is a carcinoma but that a negative result does not exclude the diagnosis of malignancy. This marker has been considered a useful discriminant for the distinction of papillary hyperplasia and papillary carcinoma; however, a positive finding does not guarantee a diagnosis of malignancy.143 Generally, oncocytic neoplasms of both papillary and follicular types are less commonly positive for HBME-1 than their nononcocytic counterparts.144-146 As many as 90% of poorly differentiated thyroid carcinomas and approximately 20% of undifferentiated thyroid carcinomas are positive for HBME-1.145 GALECTIN-3
Galectin-3 is a β-galactoside–binding lectin expressed in a large number of normal and neoplastic tissues.147 In 1995, Xu and coworkers147 examined the expression of galectin-1 and galectin-3 in a small series of thyroid tumors and found expression of these lectins in
Figure 10-14 Papillary thyroid carcinoma. Immunoperoxidase stain for Hector Battifora mesothelial cell 1 demonstrates positive staining of plasma membranes of tumor cells.
papillary and follicular carcinomas but not in adenomas, nodular goiter, or normal thyroid tissue. On the basis of these studies, they concluded that the galectins could be useful in the distinction of benign and malignant thyroid tumors. Generally similar results were reported in a number of other studies (Fig. 10-15).148-150 Bartolazzi and colleagues151 performed a large retrospective and prospective study of galectin-3 expression in more than 1000 benign and malignant thyroid lesions. The sensitivity, specificity, positive predictive value, and diagnostic accuracy for the discrimination of benign and malignant tumors were 99%, 98%, 92%, and 99% respectively. These findings suggest that galectin-3 is a useful marker for the diagnosis of low-grade thyroid carcinomas. It should be noted, however, that in a more recent study that compared galectin-3 expression in a series of cases of papillary carcinoma and papillary hyperplasia, a considerable overlap was noted in the frequency of expression of this marker.143 Both galectin-3 and HBME-1 have also been analyzed.152 The sensitivity of galectin-3 for oncocytic carcinomas and oncocytic variants of papillary carcinoma was 95%, whereas that
TABLE 10-3 Marker Expression in Benign and Malignant Thyroid Lesions Diagnosis
CK19 (%)
HBME-1 (%)
GAL (%)
CITED (%)
Nodular hyperplasia
20
0-10
20-55
20-25
5-10
40
Follicular adenoma
10
5-25
10
10-15
5-10
30-40
80-90
60-100
60-100
85-90
80-90
70-90
Follicular carcinoma
35
35-100
45-95
10-50
50
80-90
Poorly differentiated thyroid carcinoma
50
65-90
60-70
–
–
50
Undifferentiated (anaplastic carcinoma)
60-70
10-50
90-100
–
100
40
Papillary thyroid carcinoma
FN1 (%)
CD44v6 (%)
The expression of most of the markers is generally highest in papillary carcinoma followed by follicular carcinomas. The extent of staining in cases of nodular (adenomatous) hyperplasia and follicular adenomas is considerably less than that observed in papillary and follicular carcinomas. Follicular tumors of uncertain malignant potential often have intermediate patterns of staining. The percentage in this table represents averages from multiple reported series. CD44v6, Heparan sulfate proteoglycan; CK19, cytokeratin 19; HBME-1, Hector Battifora mesothelial cell 1; GAL, galectin-3; CITED, Cbp/ p300-interacting transactivator 1; FN1, fibronectin 1.
Tumors of Specific Sites
Figure 10-15 Papillary thyroid carcinoma. Immunoperoxidase stain demonstrates cytoplasmic and nuclear positivity for galectin-3.
for HBME-1 was 53%. The combination of the two markers increased the sensitivity to 99%; however, the specificity was 88% for both markers.152 Galectin-3 is present in approximately 60% of poorly differentiated thyroid carcinomas and in as many as 90% of undifferentiated carcinomas.151
335
malignancy. A group of follicular lesions with questionable features of follicular tumors of uncertain malignant potential most often had intermediate patterns of staining. The cadherins are an important class of adhesion molecules the expression of which has also been studied in thyroid tumors. Expression of E-cadherin mRNA is high in normal thyroid cells, becomes variably reduced in differentiated thyroid carcinomas, and is lost in undifferentiated carcinomas.159 These changes are reflected at the protein level, suggesting that the loss of E-cadherin is associated with the process of thyroid tumor dedifferentiation. In addition, β-catenin plays a role in cell adhesion and Wnt signaling. Typically, membrane β-catenin is reduced in follicular cell adenomas and carcinomas (Fig. 10-16) with further loss of membrane expression correlating with loss of differentiation.160 CTNNB1 exon 3 mutations and nuclear β-catenin mutations were restricted to poorly differentiated and undifferentiated thyroid carcinomas. These studies indicate that low membrane β-catenin expression, as well as its nuclear localization, and CTNNB1 mutations are significantly associated with poor prognosis, independent of conventional prognostic indicators. Decreased expression of E-cadherin and β-catenin have also been observed by Wiseman and coworkers.161 These studies also demonstrated that transformation was characterized by decreased expression of thyroglobulin (TGB),
OTHER MARKERS AND MARKER PANELS
Numerous other proteins have been explored as potential markers for different types of thyroid malignancies (see Table 10-3).153-175 Cbp/p300-interacting transactivator 1 (CITED1) is a nuclear protein involved in the coregulation of transcription factors.155,156 This protein is present in a variety of cell types and is overexpressed in papillary thyroid carcinomas (87% to 93%).156 However, CITED1 immunoreactivity is also expressed in as many as 50% of follicular carcinomas, 16% of follicular adenomas, and 24% of nodular goiters. Fibronectin 1 (FN1) is upregulated in thyroid carcinomas compared with normal thyroid tissue.156 In the study of Prasad and colleagues,157 FN was present in 91% of papillary carcinomas, 50% of follicular carcinomas, 100% of anaplastic carcinomas, 5% of follicular adenomas, and 7% of nodular goiters. Studies of multiple markers that included galectin-3, FN1, CITED1, CK19, and HBME-1 have demonstrated a significant association of expression with malignancy.157 Expression of galectin-3, FN1, and/or HBME-1 was seen in 100% of carcinomas and 24% of adenomas. Coexpression of multiple proteins was present in 95% of carcinomas and in only 5% of adenomas (P < .0001). Among nonneoplastic thyroid tissues, adenomatous hyperplasia frequently expressed galectin-3, CK19, and CITED1, but their expression was frequently focal. Sconamiglio and colleagues158 found that galectin-3, CK19, HBME-1, and CITED1 were more highly expressed in papillary carcinomas than in follicular adenomas. In this study, HBME-1 was the most specific and CK19 the most sensitive marker of malignancy. The expression of CK19 and HBME-1 was 100% specific for
Figure 10-16 β-Catenin stain of papillary thyroid carcinoma. The tumor cells exhibit prominent staining of their plasma membranes and moderate cytoplasmic positivity. Intranuclear pseudoinclusions are positively stained (arrows).
336
Immunohistology of Endocrine Tumors
Bcl-2, and VEGF with increased expression of p53, MIB-1, and topoisomerase II-α.161 Carcinoembryonic antigen (CEA) is generally absent from nonmedullary thyroid carcinomas. Using six different monoclonal CEA (mCEA) antibodies, DasovicKnezevic and colleagues162 found that 10 papillary, 10 follicular, and 8 anaplastic carcinomas were negative for CEA. In contrast, Ordonez and coworkers101 reported CEA immunoreactivity in 9% of anaplastic thyroid carcinomas using a monoclonal antibody. CD15 is present in approximately 30% of papillary carcinomas, and its expression is more likely to occur in tumors at advanced stages.163 Thus CD15 has been considered a prognostic marker for these tumors. Ghali and coworkers51 reported strong positive staining for CD57 in 100% of papillary and follicular carcinomas, whereas focal positive staining was present in 25% of colloid goiters and 21% of follicular adenomas. Other authors, however, have questioned the specificity of CD57 as a marker of malignancy in thyroid tumors.164,165 CA 15-3 and CA 19-9 are variably expressed in thyroid follicular tumors. Both follicular and papillary carcinomas reveal CA 15-3 positivity in 100% of cases, whereas CA 19-9 is present in 70% of papillary tumors but is not found in any follicular carcinomas.166 CA 125 immunoreactivity has been reported in approximately 40% of papillary carcinomas.99 The distribution of mucin-related antigens (MUCs) has also been analyzed in thyroid tumors.167 However, the expression of MUC1 is not restricted to thyroid malignancies, because it also has been reported in thyroid adenomas and hyperplasias.176 Although some studies have indicated that MUC1 expression is a marker of high-risk papillary carcinomas,177 other studies have failed to support this observation.176 Cyclooxygenases 1 and 2 (COX-1 and -2) have been investigated in thyroid tumors by using both IHC and in situ hybridization (ISH).168 These studies have demonstrated that the expression of COX-1 mRNA and its corresponding protein is similar and weak in normal and neoplastic thyroid tissue. On the other hand, COX-2 mRNA and its corresponding protein are strongly expressed in well-differentiated thyroid carcinomas compared with normal thyroid tissue and follicular adenomas. COX-2 appears to be upregulated particularly in papillary microcarcinomas, whereas larger and more advanced tumors had reduced expression.169,170 CD44v6 (heparan sulfate proteoglycan) mediates cell-to-cell and cell-to-matrix interactions. Typically, papillary and follicular carcinomas strongly express CD44v6151; however, a significant proportion of benign thyroid lesions are also positive. Steroid hormone receptors for estrogen, progesterone, and androgen are variably expressed in thyroid tumors. Bur and associates171 reported estrogen receptor (ER) positivity in approximately 20% of papillary carcinomas, whereas follicular carcinomas of conventional and oncocytic types were negative. Progesterone receptor (PR) positivity was present in 30% of papillary carcinomas; 40% of follicular tumors, including adenomas and carcinomas; and approximately 50% of oncocytic tumors. No significant correlation was apparent among
gender, age, or pathologic findings associated with aggressive behavior and the ER/PR status of the tumors. S-100 protein has also been studied in neoplastic lesions of the thyroid gland. McLaren and Cossar172 reported positivity in 100% of papillary carcinomas, 75% of follicular carcinomas, 37.5% of follicular adenomas, and 28.5% of papillary hyperplasias. More recently, overexpression of S-100A10, S-100A6, and thioredoxin has been proposed as a biomarker of papillary thyroid carcinoma with metastatic potential.178 Antibodies to mitochondrial antigens are of value in the identification of thyroid tumors with oncocytic (Hürthle cell) features.173 Positive cells typically exhibit intense cytoplasmic positivity that corresponds to the distribution of mitochondria (Fig. 10-17). p63, a homolog of p53, is expressed in a variety of cells that include basal cells of squamous epithelium, myoepithelial cells of breast and salivary glands, and basal cells of the prostate. In the thyroid, it is expressed selectively in solid cell nests that represent remnants of the ultimobranchial bodies (Fig. 10-18).174 In thyroid tumors, p63 is expressed in sclerosing mucoepidermoid carcinomas with eosinophilia, which are thought to be derived from remnants of the ultimobranchial bodies.175 Papillary and anaplastic carcinomas are uncommonly positive for p63.
A
B Figure 10-17 A, Papillary thyroid carcinoma, oncocytic type (hematoxylin and eosin). B, Immunoperoxidase stain for mitochondria by using the antibody MITO-113 demonstrates intense granular cytoplasmic staining.
Tumors of Specific Sites
Figure 10-18 Normal thyroid stained for p63. Positive nuclear staining is confined to the cells of the solid cell nest.
KEY DIAGNOSTIC POINTS Thyroid Carcinomas • TGB, TTF-1 and TTF-2, and thyroid peroxidase are present in benign thyroid lesions and in more than 95% of differentiated thyroid carcinomas. • HBME-2 and galectin-3 are useful markers, although not completely specific, for differentiated thyroid carcinomas. • CK19 is usually expressed in papillary carcinomas but may also be present in other thyroid malignancies, adenomas, and some benign conditions (e.g., thyroiditis). • Marker panels that include CK19, HBME-1, and galectin-3 are often positive in the follicular variant of papillary carcinomas. • Follicular tumors with questionable features of papillary carcinoma (follicular tumors of uncertain malignant potential) often demonstrate intermediate and/or irregular patterns of staining for CK19, HBME-1, and galectin-3. • Undifferentiated thyroid carcinomas are usually negative for TGB, TTF-1, and TTF-2 but commonly express cytokeratins and Pax-8.
337
Figure 10-19 Normal thyroid C cells. Immunoperoxidase stain for calcitonin demonstrates the intrafollicular topography of the normal C cells.
are characterized by invasion of C cells through the follicular basement membrane. In addition to its occurrence in patients with MEN 2, C-cell hyperplasia may occur in patients with hypercalcemia or hypergastrinemia, around follicular or papillary neoplasms, and it may occur in some normal individuals. This type of C-cell hyperplasia has been termed secondary or physiologic in contrast to the hyperplasia seen in patients with MEN 2 (primary or “neoplastic” hyperplasia).181 Medullary thyroid carcinomas are positive for calcitonin in more than 95% of cases (Figs. 10-21 and 1022).180,182,183 In rare cases negative for calcitonin peptide, calcitonin mRNA may be demonstrated by ISH.184 Several studies have suggested that the patterns of calcitonin staining in these tumors may have prognostic significance and that tumors with low levels of calcitonin may behave more aggressively. Franc and
C Cells and Medullary Thyroid Carcinoma C cells, the second major endocrine cell population of the thyroid gland, are the primary sites of synthesis and storage of calcitonin. These cells also synthesize a variety of other regulatory products, including somatostatin, gastrin-releasing peptide, and amines.179,180 C cells can be distinguished from the follicular cells on the basis of their content of calcitonin and the presence of generic NE markers, including chromogranin A and synaptophysin. C cells in normal glands have an exclusive intrafollicular topography and are concentrated at the junctions of the upper and middle thirds of the lobes (Fig. 10-19).179 In patients with multiple endocrine neoplasia type II (MEN 2), C-cell hyperplasia has been recognized as the precursor of medullary thyroid carcinoma (Fig. 10-20). The earliest phases of medullary carcinoma
Figure 10-20 C-cell hyperplasia from a patient with multiple endocrine neoplasia 2A. This immunoperoxidase stain for calcitonin is strongly positive in the C cells, whereas the follicular cells are negative.
338
Immunohistology of Endocrine Tumors
Medullary thyroid carcinoma 100 90 80
Percent
70 60 50 40 30 20 10
KE R
(P AN CK ) CH 7 R C -B SN GR AP P SY -25 NA CA P TT LC CH F-1 R m -A CE A NF P V CDIM G 57 AL CD -3 1 CK 0 20 ER P PR P
0
Figure 10-21 Distribution of markers in medullary thyroid carcinoma. KER(PAN), Pancytokeratin; CK7, cytokeratin 7; CHR-B, chromogranin B; CGRP, calcitonin gene–related protein; SNAP-25, synaptosomal associated protein 25; SYNAP, synaptophysin; CALC, calcitonin; TTF-1, thyroid transcription factor 1; CHR-A, chromogranin A; mCEA, monoclonal carcinoembryonic antigen; NFP, neurofilament protein; VIM, vimentin; GAL-3, galectin-3; CK20, cytokeratin 20; ERP, estrogen receptor protein; PRP, progesterone receptor protein.
A
coworkers185 demonstrated in univariate analyses that patients with tumors that contained fewer than 50% calcitonin-immunoreactive cells had less favorable survival patterns than patients whose tumors contained more than 50% immunoreactive cells. Rarely, small cell carcinomas that resemble oat cell carcinomas may occur within the thyroid and have been reported to be negative for calcitonin peptide and the corresponding mRNA.186 In addition to calcitonin, normal and neoplastic C cells contain the calcitonin gene–related peptide (CGRP).187 CGRP results from alternate splicing of the primary transcript of the calcitonin gene. The normal thyroid expresses calcitonin predominantly, whereas CGRP is found primarily in the central and peripheral nervous systems. In medullary carcinomas, calcitonin and CGRP are produced in a concordant manner.187 A variety of other peptides have been demonstrated by IHC, and their presence has been confirmed by correlative radioimmunoassays of tumor extracts. Somatostatin and gastrin-releasing peptide are found commonly in medullary thyroid carcinomas.188,189 Scopsi and coworkers1 used antisera raised against four different regions of the prosomatostatin molecule and demonstrated positive staining in all cases. Most, but not all, of the somatostatin-positive cells were also positive for calcitonin. Somatostatin immunoreactive cells are
B
Figure 10-22 Medullary thyroid carcinoma. A, Hematoxylin and eosin. B, Immunoperoxidase stain for calcitonin. The tumor cells are strongly positive for calcitonin.
Tumors of Specific Sites
generally present singly or in small groups and represent less than 5% of the entire tumor cell population. The somatostatin-positive cells have a dendritic shape with branching cell processes that extend between adjacent tumor cells. Gastrin-releasing peptide is present in approximately 30% of medullary carcinomas.189 Other peptide products that are present in these tumors include ACTH and other proopiomelanocortin (POMC) peptides, neurotensin, substance P, and vasoactive intestinal peptide (VIP).182 The alpha chain of hCG has been demonstrated in 46% (17/37) of cases.190 Both catecholamines and serotonin are present in medullary thyroid carcinomas. Uribe and coworkers183 demonstrated serotonin immunoreactivity in 70% (14/20) of cases. Serotonin immunoreactivity in these tumors is generally present in cells with a dendritic morphology, similar to that of the somatostatinpositive cells. Medullary thyroid carcinomas are typically positive for TTF-1, although the staining intensity is often less than that seen in follicular cells.107 TGB may occur in these tumors as entrapped follicles, single follicular cells, or extracellular deposits (Fig. 10-23). This phenomenon is most likely to occur at the junction of the tumor and the adjacent thyroid parenchyma or along fibrovascular septa. In one series, TGB immunoreactivity was present in approximately 60% of primary thyroid tumors but in no case of metastatic medullary thyroid carcinoma.191 True mixed tumors with C-cell and follicular features have also been reported; these tumors are composed of cells that contain calcitonin or other peptides and TGB (Fig. 10-24).192,193 The existence of such tumors may explain the rare cases of medullary thyroid carcinoma that have the capacity for radioactive iodine uptake. The proposed origin of tumors with mixed medullary and follicular features has been controversial. Volante
Figure 10-23 Medullary thyroid carcinoma. Immunoperoxidase stain for thyroglobulin. Positivity is confined to entrapped follicles.
and coworkers194 proposed an origin from two different progenitors. According to their hypothesis, neoplastic transformation of C cells leads to the development of medullary thyroid carcinoma with entrapped normal follicles. Stimulation of the entrapped follicular cells results in hyperplasia and ultimately follicular (or papillary) neoplasia (hostage hypothesis). Neoplastic C cells and follicular cells would have the capacity to metastasize and could explain the presence of both components in distant sites. Medullary carcinomas are typically positive for the entire battery of generic NE markers, including NSE, the chromogranin proteins, synaptophysin, and histaminase (see Fig. 10-21).179,182 Because NSE is also expressed in a variety of non–C-cell neoplasms, it should never be used as the sole marker to distinguish medullary carcinomas from other thyroid tumor types. In addition to chromogranin A, medullary carcinomas also consistently
Metastatic neuroendocrine lung tumor
Parathyroid tumor
TTF-1 + TGB – Chromo + Calcitonin + – PTH – Thyroid transcription factor 1(TTF-1) Thyroglobulin (TGB) Chromogranin Calcitonin Parathyroid hormone (PTH) TTF-1 + TGB + Chromo – Calcitonin – PTH –
Figure 10-24 Algorithm of head and neck endocrine tumors.
Thyroid follicular cell tumor
339
Paraganglioma
TTF-1 – TGB – Chromo + Calcitonin – PTH +
CK +
Cytokeratin
TTF-1 + TGB – Chromo + Calcitonin + PTH – Medullary carcinoma
Chromo + Synapto + S-100 +
CK –
Chromogranin Synaptophysin S-100
TTF-1 + TGB + Chromo + Calcitonin + PTH – Mixed follicular and C-cell tumor
340
Immunohistology of Endocrine Tumors
express chromogranin B and secretogranin II.195 The calcium-binding protein calbindin-D28K, which is also regarded as a general NE marker, has been found in 95% (18/19) of medullary carcinomas.196 Polysialic acid (polySia) of NCAM is consistently expressed in medullary carcinomas. Komminoth and coworkers56 demonstrated that all cases of medullary carcinoma were positive, whereas other thyroid tumor types were consistently negative. Strong polySia immunoreactivity occurs in all cases of primary C-cell hyperplasia, whereas normal C cells and C cells in cases of secondary C-cell hyperplasia were negative in most cases. Bcl-2 immunoreactivity is present in 79% (26/33) of cases of medullary carcinoma.197 In the study reported by Viale and associates,197 lack of Bcl-2 immunoreactivity correlated significantly with shorter survival (P = .0001). In multivariate analyses, lack of Bcl-2 was an independent predictor of poor prognosis. Viale and colleagues197 also demonstrated that p53 immunoreactivity was present in 12% (4/33) of medullary carcinomas. Holm and Nesland126 reported p53 immunoreactivity in 13% (6/46) of medullary carcinomas. As detected by both monoclonal antibodies and polyclonal antisera, CEA is present in the vast majority of medullary carcinomas.198 Monoclonal antibodies that are specific to CEA react with approximately 75% of cases of medullary carcinoma but not with other tumor types.199 Antibodies that react with epitopes present on CEA and the nonspecific cross-reacting antigens react with almost 90% of medullary carcinomas but also give positive reactions with other thyroid tumor types. Several groups have demonstrated that some medullary thyroid carcinomas may lose their ability to synthesize and secrete calcitonin, while maintaining their capacity for CEA production, and that such tumors may have an aggressive course.200 Franc and coworkers186 demonstrated that patients with medullary carcinomas that contained more than 50% CEA-positive cells and less than 50% calcitonin-positive cells had a poorer prognosis compared with other groups. Medullary carcinomas are typically positive for low-molecular-weight (LMW) cytokeratins. Vimentin immunoreactivity is present in approximately 60% of cases, whereas NFPs have been reported in 83% (10/12) cases.201 Normal C cells have been reported to lack
NFPs but are typically positive for LMW cytokeratins and are variably positive for vimentin. KEY DIAGNOSTIC POINTS Medullary Thyroid Carcinoma • Normal C cells have an intrafollicular topography and can be identified by their positivity for calcitonin, chromogranin, synaptophysin, and other neuroendocrine markers. • Ninety-five percent of medullary carcinomas are positive for calcitonin, and a variety of other peptides may also be present. • Medullary carcinomas are variably positive for TTF-1 and TTF-2 but are negative for TGB. • CEA is present in most medullary carcinomas. • Familial forms of medullary carcinoma are preceded in their development by C-cell hyperplasia. • True mixed C-cell and follicular tumors are rare.
MOLECULAR APPROACHES Papillary Carcinoma
A variety of different genetic alterations, including rearrangements (RET and NTRK1) and point mutations (BRAF and RAS) have been implicated in the development of papillary thyroid carcinoma (PTC; Table 10-4).131,202-206 Although radiation exposure has been linked to the development of rearrangements, the origin of point mutations remains unknown. The various mutations and rearrangements result in the activation of the mitogen-activated protein kinase (MAPK) pathway, which is involved in signaling from a variety of growth factors and cell surface receptors. Together, these genetic alterations are present in approximately 70% of PTCs, and they rarely overlap in the same tumor. The RET protooncogene encodes a tyrosine kinase (TK) receptor that consists of an extracellular domain with a ligand binding site, a transmembrane domain, and an intracellular TK domain.131 Ligand binding results in receptor dimerization, which leads to the autophosphorylation of tyrosine residues and initiation of the signaling cascade. RET/PTC results from rearrangement of the 3′ area of RET with the 5′
TABLE 10-4 Genetic Alterations in Thyroid Cancer Gene
Papillary (%)
BRAF
39
RAS RET/PTC
Follicular (%)
Poorly Differentiated (%)
Anaplastic (%)
0
13
14
15
35
35
53
28
0
9
0
PAX8-PPARG
1
34
0
0
p53
5
7
24
59
β-catenin CTNNB1
0
0
16
66
Modified from Nikiforov Y: Genetic alterations involved in the transition from well to poorly differentiated and anaplastic carcinoma. Endocr Pathol. 2004;15:319-328.
Tumors of Specific Sites
area of several genes that are expressed in normal follicular cells.207 Considerable variations are seen in the frequencies of RET rearrangements in different published series. These differences may be related in part to variations in age groups and history of radiation exposure. In addition, there may be heterogeneous distributions of rearrangements in individual tumors as well as variations in the sensitivities of different detection techniques.131 Clonal rearrangements of RET, as defined by involvement of the majority of tumor cells, occur in approximately 20% of cases.131 RET/PTC1 and RET/PTC3 are the most common rearrangements and result from paracentric inversion of the long arm of chromosome 10; RET/PTC2 results from a 10;17 reciprocal translocation that involves R1α on 17q23. Additional rearrangements have been described in radiation-induced tumors, but their frequencies are low. RET/PTC1 and RET/PTC3 account for 60% to 70% and 20% to 30% of the cases, respectively, whereas RET/PTC2 is responsible for approximately 10%. RET rearrangements are common in pediatric patients and in individuals exposed to accidental or therapeutic irradiation. RET/PTC1 is more common in classic PTCs, micro PTCs, and in the diffuse sclerosing variant than in other types, whereas RET/ PTC3 has been associated with the solid variant.208 RET rearrangements are less common in the follicular variants than in PTCs of conventional type. Interestingly, RET rearrangements are associated with PTCs that lack evidence of progression to poorly differentiated or undifferentiated thyroid carcinomas. The neurotrophic receptor tyrosine kinase (NTRK1) on chromosome 1q22 encodes the receptor for nerve growth factor. Rearrangements of NTRK1 are considerably less common than RET rearrangements in PTCs.209 BRAF is a serine/threonine protein kinase, which is a potent activator of the MAPK pathway. Mutations in this gene have been found in approximately two thirds of malignant melanomas and in a similar proportion of ovarian and colonic adenocarcinomas. The most common mutation results in a thymidine-to-adenine transversion at nucleotide position 1799 and a valineto-glutamate substitution at residue 600 (V600E). An identical substitution has been identified in 40% to 70% of papillary carcinomas.210,211 There is no evidence of similar mutations in other thyroid tumor types. BRAF mutations have been associated with conventional PTCs, tall cell and oncocytic variants, and microcarcinomas.131 In contrast, BRAF mutations are uncommon in the follicular variants.212 Approximately 15% of poorly differentiated thyroid carcinomas and a significantly higher proportion of undifferentiated/anaplastic carcinomas are positive for BRAF mutations.212 Moreover, the mutations occur more commonly in those poorly differentiated and undifferentiated carcinomas with a papillary component. These findings suggest that the high-grade tumors progress from BRAF-positive papillary carcinomas. RAS mutations are uncommon in papillary carcinomas of conventional type, in contrast to the follicular variants, in which they are considerably more common.213
341
Follicular Adenoma and Carcinoma
RAS mutations are present in approximately 50% of follicular carcinomas and 20% to 40% of follicular adenomas (see Table 10-4).214 Among RAS-positive carcinomas, NRAS codon 61 mutations are present in approximately 80% of cases, and HRAS codon 61 mutations are present in almost 20%. Identical mutations are present at lower frequencies in follicular adenomas. RAS mutations also occur in approximately 25% of oncocytic carcinomas and in as many as 5% of oncocytic adenomas. PAX8/PPARG rearrangements in follicular tumors occur as a result of a recurrent translocation t(2;3) (q13;p25) that leads to fusion of the PAX8 and the PPARG genes.132,134 This rearrangement occurs in approximately 35% of conventional follicular carcinomas and in as many as 10% of follicular adenomas. Similar rearrangements have also been noted in a small proportion of follicular carcinomas of the oncocytic type. As noted in a previous section, the rearrangement results in overexpression of the PPARG protein, which can be detected by IHC. It should be remembered, however, that only strong nuclear staining correlates with the presence of the rearrangement and that rearrangements may occur in benign thyroid tumors.135,136 Follicular carcinomas that harbor the PAX8/PPARG rearrangement typically occur in patients who are younger than those who have carcinomas without the rearrangement. These carcinomas tend to be smaller in size and are more likely to be angioinvasive. Deletions and point mutations that involve mitochondrial DNA are common in follicular neoplasms of the oncocytic type, but the role of these mutations in the genesis of tumors is unknown.215 Mutations of the NDUFA13 gene (formerly GRIM-19), which is involved in the mitochondrial respiratory chain and in apoptosis, have been found in 15% of oncocytic carcinomas but do not occur in other thyroid tumor types.216 Poorly Differentiated and Undifferentiated Thyroid Carcinoma
The frequencies of the various mutations in poorly differentiated thyroid carcinomas and other thyroid tumor subtypes are summarized in Table 10-4.217 The PTEN and P1K3CA genes are mutated in 15% to 20% of the cases, respectively.217 In addition, p53 mutations are present in 15% to 30% of poorly differentiated thyroid carcinomas and in 60% to 80% of undifferentiated carcinomas.125 In contrast, relatively few (<2%) differentiated thyroid carcinomas harbor mutations of p53. These findings suggest that progressive loss of differentiation in thyroid tumors occurs as a result of mutations of this gene. The CTNNB1 gene encodes β-catenin, an important intermediary in the Wnt signaling pathway.160,161 Point mutations of CTNNB1 occur in 25% and 66% of poorly differentiated and undifferentiated thyroid carcinomas, respectively. As discussed in a previous section, most tumors with mutations have a nuclear pattern of localization, in contrast to the usual plasma membrane staining pattern.
342
Immunohistology of Endocrine Tumors
Medullary Thyroid Carcinoma
Germline mutations of the RET gene are present in virtually all patients with familial forms of medullary thyroid carcinoma, which includes MEN 2A and 2B and FMTC.218 Codon 634 mutations occur in a high proportion of patients with MEN 2A and FMTC, whereas codon 918 mutations predominate in patients with MEN 2B. Somatic mutations of RET codon 918 occur in 20% to 80% of cases of sporadic MTC, and their distribution, both within the primary tumors and in metastases, is heterogeneous. TESTING FOR PANELS OF MUTATIONS
Testing panels for mutations have been used for analysis of both tissue and cytologic samples. The panel typically includes the most common mutations that occur in approximately 70% of thyroid cancers: BRAF V600E, RET/PTC1, RET/PTC3, NRAS codon 61, HRAS codon 61, KRAS codons 12/13, and PPARG. The results of several studies suggest that the presence of BRAF, RET/ PTC, or PPARG mutations is virtually diagnostic of malignancy. RAS mutations, on the other hand, have a 74% to 100% positive predictive value for the diagnosis of malignancies.219,220
have suggested a more conservative approach for thyroid nodules that are cytologically indeterminate on FNA biopsy and have benign findings based on geneexpression classifier results.224 THERANOSTICS
Molecular targeted therapies have considerable potential for treatment of poorly differentiated, undifferentiated, and medullary carcinomas, each of which has a diminished or absent ability to incorporate radioactive iodine.202,203 An agent that shows considerable clinical promise is ZD6474 (Zactima; AstraZeneca, London), a tyrosine kinase inhibitor that is a potent inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and which effectively blocks RET tyrosine kinase.225 Multikinase inhibitors with potent activity against RAF, VEGFR-2, VEGFR-3, PDGFRB, FLT3, and c-Kit are also being evaluated for therapeutic effects in patients with advanced thyroid tumors. The challenge for investigators is the assessment of the extent to which the various tyrosine kinases are being inhibited and the correlation of these findings with a variety of biomarkers indicative of disease status and outcome data. KEY DIAGNOSTIC POINTS
MICROARRAY GENE PROFILING
Alterations in gene expression of papillary thyroid carcinomas have been studied by using complementary DNA (cDNA) microarrays. The findings have shown that the gene-expression profiles of these tumors are different from those of follicular carcinomas and other thyroid tumor types.156,221 However, distinct sets of variably expressed genes have been found in classic papillary carcinoma, the follicular variant, and possibly in other variants (such as the tall cell variant), which supports the histopathologic and biologic differences between these tumor variants.221,222 The results of gene-expression array studies confirm the overexpression of a number of genes previously known to be upregulated in papillary carcinoma, such as MET, LGALS3 (galectin-3), and KRT19 (cytokeratin 19). Several additional overexpressed genes, such as CITED1, were discovered by using this approach and are now being explored as possible IHC diagnostic markers, as discussed previously.156 In addition, variations in gene-expression profiles between papillary carcinomas carrying BRAF, RAS, RET/PTC, and NTRK1 mutations have been detected, providing a molecular basis for distinct phenotypic and biologic features associated with each mutation type.222,223 Expression microarrays have also been used in FNA biopsy samples of thyroid with indeterminate cytologic results. In one study, 85 of 265 indeterminate cases proved to be malignant.224 The gene-expression classifier correctly identified 78 of the 85 nodules as suspicious (92% sensitivity, 52% specificity). The negative predictive values for “atypia of undetermined significance,” “follicular neoplasm or lesion suspicious for follicular neoplasm,” or “suspicious cytologic findings” were 95%, 94%, and 85% respectively. These findings
Molecular Aspects of Thyroid Carcinoma • Genetic rearrangements (RET and NTRK1) and mutations (BRAF and RAS) are responsible for the development of the majority (70%) of papillary carcinomas. • BRAF mutations are present in 40% to 70% of papillary thyroid carcinomas and also occur in a subset of poorly differentiated and undifferentiated thyroid carcinomas. • Clonal rearrangements of RET (present in the majority of tumor cells) occur in approximately 20% of cases of papillary thyroid carcinoma. • RAS mutations are considerably more common in the follicular variant of papillary carcinoma than in conventional type papillary carcinomas. • RAS mutations are present in approximately 50% of follicular carcinomas and 20% to 40% of follicular adenomas. • The various rearrangements and mutations result in the activation of the mitogen-activated protein kinase pathway and rarely overlap in the same tumor. • PAX8/PPARG rearrangements occur in approximately 35% of follicular carcinomas and in approximately 10% of follicular adenomas. • Deletions and point mutations that involve mitochondrial DNA are common in follicular tumors of the oncocytic type, whereas mutations of the GRIM-19(NDUFA13) gene occur in 15% of these tumors. • Sites of mutation in poorly differentiated and undifferentiated thyroid carcinomas include RAS, PTEN, PIK3CA, p53, BRAF, and CTNNB1 genes. • Germline mutations of RET are present in familial forms of medullary thyroid carcinoma, whereas somatic mutations of RET are common in sporadic forms of medullary carcinoma. • Gene-expression profiles of thyroid tumors differ according to the major histopathologic classifications of these tumors.
Tumors of Specific Sites
Parathyroid adenoma 100 90 80 70 Percent
60 50 40 30 20 10
B
F1
TG
IN CL
TT
D1
A PS CY
L2 BC
H
RA CH
PT
KE
R
(P A
N)
0
Figure 10-25 Distribution of markers in parathyroid adenomas. KER(PAN), Pancytokeratin; PTH, parathyroid hormone; CHR-A, chromogranin A; PSA, prostate-specific antigen; TTF-1, thyroid transcription factor 1; TGB, thyroglobulin.
Parathyroid Glands Both normal and adenomatous glands are positive for CK8, CK18, and CK19, whereas vimentin is restricted to stromal cells (Fig. 10-25).226 The chief cells of normal glands are negative for neurofilaments, but 33% of adenomas contain some neurofilament-positive cells that are also positive for cytokeratins. In contrast to thyroid follicular cells, which are positive for thyroglobulin and TTF-1, normal and neoplastic chief cells are negative for these markers. In the past, the IHC analysis of parathyroid hormone (PTH) was difficult because of both the lack of suitable antibodies and the low level of hormone storage within the chief cells.227 Antigen retrieval methods, however, have greatly facilitated the localization of PTH in FFPE sections (Fig. 10-26).228 PTH and chromogranin A are demonstrable in the vast majority of normal,
A
343
hyperplastic, and neoplastic parathyroid glands. In normal glands, chief cells stain more intensely for PTH and chromogranin A than do oncocytes, whereas hyperplastic glands generally stain less intensely than do normal glands. The intensity of staining for PTH and chromogranin A is less intense in adenomas than in normal and hyperplastic glands. Generally, however, staining of the rim of adjacent normal parathyroid tissue is more intense than that of the adenomas.228 A similar pattern of reactivity has been observed in ISH formats with probes for parathyroid mRNA.229 In a single case of parathyroid carcinoma, Tomita228 reported positive staining for PTH but no significant reaction for chromogranin A. Patterns of PTH reactivity generally correspond to the levels of extractable PTH: the highest levels are present in normal glands, and the lowest levels are shown in adenomatous glands.230 In addition to PTH, PTH-related protein has also been demonstrated by IHC.231 Schmid and coworkers232 reported that 14% (12/86) of hyperplastic parathyroid glands demonstrated focal reactivity for chromogranin B; in 10 of 12 of these cases (83%), calcitonin was colocalized with chromogranin B. CGRP was found in a small proportion of the calcitonin cells in 40% (4/10) of the cases. These observations were confirmed by the demonstration of mRNAs for calcitonin and CGRP. The results of this study indicate that calcitonin and CGRP may be synthesized and stored in hyperplastic parathyroid chief cells. These results have also been confirmed in other studies.233 CD4 immunoreactivity is present both in normal and abnormal parathyroid glands.234 Positive staining is restricted to chief cells, whereas oncocytic cells are nonreactive. Normal, hyperplastic, and neoplastic cells demonstrate positive staining primarily on plasma membranes. In contrast, parathyroid carcinomas demonstrate primarily cytoplasmic staining. Although the functional significance of CD4-like immunoreactivity in the parathyroid is unknown, this moiety may play a role in calcium-regulated PTH release. The renal cell carcinoma antigen (RCC) is commonly expressed in parathyroid tumors 235,236 and some may also be positive for Pax-2.237
B
Figure 10-26 A, Intrathyroidal parathyroid adenoma. B, Immunoperoxidase stain for parathyroid hormone shows strong cytoplasmic positivity in chief cells.
344
Immunohistology of Endocrine Tumors
The distribution of cyclin D1 has also been examined in normal and neoplastic parathyroid tissue.238 In normal glands, cyclin D1 was present in 6% of cases. In contrast, cyclin D1 was present in 91% (10/11) of parathyroid carcinomas, 29% (11/38) of parathyroid adenomas, and 61% (11/18) of hyperplastic parathyroid glands. These studies confirm the high frequency of cyclin D1 expression in adenomas and carcinomas, but they also indicate that high levels of expression may also occur in cases of hyperplasia.238 The distinction between parathyroid adenomas and carcinomas is, on occasion, extremely difficult. Several studies have used MIB-1 to aid in the differential diagnosis. Abbona and coworkers239 reported a significant difference between carcinomas (aggressive and nonaggressive) and adenomas with respect to MIB-1 scores, although no significant differences were noted between nonaggressive carcinomas and adenomas. However, mitotic rates and MIB-1 scores of clinically aggressive carcinomas were significantly higher than in adenomas. Vargas and coworkers240 reported that an MIB-1 fraction in excess of 40 positive signals per 1000 cells correlated strongly with malignancy. In addition, p27 (Kip 1) has been examined in parathyroid hyperplasia, adenoma, and carcinoma.68 The p27 labeling index was 56.8 ± 3.4 for adenomas and 13.9 ± 2.6 for carcinomas, whereas the MIB-1 labeling index was significantly higher in carcinomas than in adenomas. These findings suggest that both p27 and MIB-1 may be helpful when used together for the distinction of parathyroid adenomas and carcinomas. Another approach to the distinction of parathyroid adenomas and carcinomas involves the use of antibodies to the retinoblastoma (RB) protein. Cryns and coworkers241 reported the absence of RB protein in a small series of carcinomas, whereas this protein was present in adenomas. However, Vargas and coworkers240 demonstrated positive staining for RB in 100% of adenomas and 80% of carcinomas, and Farnebo and coworkers242 also demonstrated the lack of utility of RB immunoreactivity for the distinction of adenomas and carcinomas. Presence of p53 has also been examined in normal, hyperplastic, and neoplastic parathyroid tissues. In the study reported by Kayath and colleagues,243 p53 was present in 36% (10/28) of adenomas, 42% (5/12) of cases of primary hyperplasia, 72% (13/18) of cases of diffuse hyperplasia, 44% (17/39) of cases of nodular hyperplasia, and 40% (2/5) of carcinomas. These results indicate that the analysis of p53 by IHC is not useful in the distinction of the various proliferative states of the parathyroid. MOLECULAR APPROACHES
Several groups have demonstrated loss of heterozygosity (LOH) on chromosome 13q, a region that includes RB1 and BRCA2, in parathyroid carcinomas. In the series reported by Cryns and colleagues,241 11 of 11 specimens from patients with parathyroid carcinoma and 1 of 19 adenomas lacked an RB1 allele. BRCA2 has also been suggested as a potential suppressor gene in these tumors.244 However, the contribution of both RB1 and
BRCA2 to the development of carcinomas has been controversial. In a recent study by Cetani and colleagues, LOH for the least one marker of the RB1 locus was found in 6 of 6 carcinomas, whereas LOH for BRCA2 was found in 3 of 5 cases.245 In the same series, LOH for RB1 and BRCA2 was demonstrated in 28.8% and 17.4% of adenomas, respectively. Shattuck and associates246 performed direct sequencing of parathyroid carcinomas that demonstrated lesions of RB1 or BRCA2 and were unable to find microdeletions, insertions, or point mutations of either gene. They concluded that neither RB1 nor BRCA2 were likely to act as tumor suppressor genes in carcinomas. However, these results do not exclude the possibility that the decreased RB1 function in carcinomas, whether secondary or because of epigenetic effects, may play a role in tumor development. It is also possible that other genes on chromosome 13q may be implicated in the development of parathyroid carcinomas. Studies of heritable tumor syndromes have provided considerable insight into the molecular basis of the corresponding sporadic tumors. Mutations of the HRPT2 gene are responsible for the development of the hyperparathyroidism–jaw tumor (HPT-JT) syndrome, which is inherited as an autosomal dominant trait.247 The commonest manifestations of this syndrome include primary hyperparathyroidism, fibroosseous lesions of the mandible and maxilla, and a variety of renal lesions.248 In this syndrome, hyperparathyroidism occurs as a result of neoplasms of one or more parathyroid glands, which frequently show cystic change. Importantly, parathyroid carcinomas occur in 10% to 15% of patients with this syndrome. The role of the HRPT2 gene in the pathogenesis of sporadic parathyroid carcinomas was first demonstrated by Howell and colleagues249 in 2003. Subsequent studies by Shattuck and coworkers250 demonstrated that parathyroid carcinomas from 10 of 15 patients had HRPT2 mutations predicted to inactivate the encoded parafibromin protein. Importantly, the HRPT2 mutations in three of the parathyroid carcinomas of these patients were identified as germline mutations. The latter finding suggests that a subset of patients with apparent sporadic parathyroid carcinomas carry germline mutations in the HRPT2 gene and may, in fact, have the HPT-JT syndrome or a variant of that syndrome. These findings suggest that all patients with parathyroid carcinoma should have jaw and renal imaging studies, and patients with parathyroid carcinoma should be tested for germline HRPT2 mutations. Loss of parafibromin was first reported as a molecular marker for parathyroid carcinoma by Tan and colleagues (Fig. 10-27),251 who noted that loss of parafibromin nuclear staining had a 96% sensitivity and 99% specificity for the definitive diagnosis of parathyroid carcinoma. In addition to parafibromin loss in carcinomas, this protein was also absent from HPT-JT–associated adenomas. Generally, similar results have been reported by other groups, although the reported studies used different scoring systems.252-255 Juhlin and colleagues256 demonstrated that 68% of unequivocal carcinomas exhibited reduced expression
Tumors of Specific Sites
A
345
B
Figure 10-27 Parafibromin stain of parathyroid adenoma (A) and parathyroid carcinoma (B). A, Intense nuclear staining is apparent in the adenoma. B, The tumor cell nuclei are negative, whereas endothelial nuclei (internal control) are positive. (Courtesy Anthony Gill, MD.)
of parafibromin, whereas 100% of adenomas were positive; moreover, 3 of 6 carcinomas with known HRPT2 mutations showed reduced expression of parafibromin. They concluded that 1) parafibromin IHC could be used as an additional marker for parathyroid tumor classification, 2) parafibromin-positive cases have a low risk of malignancy, and 3) cases with reduced protein expression represent either carcinomas or adenomas with HRPT2 mutations. Of particular interest is the observation that 80% (4/5) of metastatic parathyroid carcinomas observed in patients with chronic renal failure were positive for parafibromin. This finding suggests that genetic events other than HRPT2 mutations may be of significance in the genesis of different subsets of parathyroid carcinoma.257 In our own experience,258,259 loss of parafibromin staining was noted in a subset of adenomas unassociated with the HPT-JT syndrome, although some carcinomas have shown positive staining. These observations emphasize the need for the use of well-characterized parafibromin antibodies in addition to standardized fixation and retrieval conditions, staining protocols, and scoring systems before this approach becomes the standard of practice. Although parafibromin IHC represents an important step in the ability to diagnose parathyroid carcinoma, additional studies will be required to test the validity of this approach and to determine the roles of other genes in the development of these tumors. Erovic and coworkers255 studied the expression of a large number of biomarkers for the distinction of parathyroid adenomas and carcinomas. COX-1 and -2; CD9;
MMP-1; FOXO1, VEGFR-2; PDGFRA and PDGFRB; GSTP1; Gli1, Gli2, and Gli3; and Patched (PTCH1) were expressed in the majority of benign and malignant tumors. On the other hand, Bcl-2a, parafibromin, Rb, and p27 were significantly decreased to a variable extent in all parathyroid carcinomas, suggesting their potential value for the distinction of benign and malignant parathyroid tumors.255 KEY DIAGNOSTIC POINTS Parathyroid Tumors • Normal parathyroid chief cells and parathyroid adenomas are positive for parathyroid hormone and a variety of neuroendocrine markers, including synaptophysin and chromogranin A. • Low levels of expression of MIB-1 and high levels of p27 are characteristic of parathyroid adenomas, whereas carcinomas have high levels of MIB-1 and low levels of p27. • Parafibromin is uniformly expressed in parathyroid adenomas, but its expression is often reduced and/or heterogeneous in carcinomas.
ADRENAL GLAND Cortex
Markers that have been used for the identification of adrenal cortical cells include steroidogenic enzymes, monoclonal antibody D11, adrenal 4 binding protein (Ad4BP), A103 (Melan-A), and inhibin A (Fig. 10-28).
346
Immunohistology of Endocrine Tumors
Adrenocortical carcinoma 100 90 80
Percent
70 60 50 40 30 20 10
AD CA 4BP L M RE EL T AN SY -A NA P IN HI B CD 56 D1 1 VI M K BC KE ER L-2 R (PA CA N M ) 5. 2) EM A CD 1 CH 0 RA
0
Figure 10-28 Distribution of markers in adrenocortical tumors. AD4BP, Adrenal 4 binding protein; CALRET, calretinin; MELAN-A, Melan-A protein (A103); SYNAP, synaptophysin; INHIB, inhibin; CD56; monoclonal antibody VIM, vimentin; KER(PAN), pancytokeratin; EMA, epithelial membrane antigen; CHR-A, chromogranin-A.
Studies reported by Sasano and coworkers demonstrated strong staining for P45017a in the fasciculata and reticularis zones of patients with Cushing disease.23,24,27 Cortical adenomas associated with cortisol overproduction demonstrated strong staining for this enzyme, whereas the adjacent zona reticularis showed weak staining consistent with suppression of the normal gland. The monoclonal antibody D11 recognizes several 59-kD proteins capable of binding apolipoprotein E.260,261 Approximately 80% of adrenal cortical tumors show positive nuclear staining for D11; however, 100% of hepatocellular carcinomas, 60% of lung carcinomas, and occasional renal carcinomas have been reported to show cytoplasmic staining for this marker.262 Ad4BP, also known as steroid factor 1 (SF-1), is a transcription factor that regulates steroidogenic cytochrome P450 gene expression. Ad4BP has been reported in 100% of adrenal cortical carcinomas, but no cases of renal cell carcinoma (RCC), hepatocellular carcinoma, or other tumor types, including pheochromocytoma, exhibited positivity.263 The monoclonal antibody A103 (Melan-A) has been used primarily for the identification of malignant melanoma (Fig. 10-29). This antibody cross-reacts with an epitope that is present in steroid-producing cells,
A
B
C
D
Figure 10-29 A, Adrenal cortical carcinoma (hematoxylin and eosin). B, Immunoperoxidase stain for Melan-A (A103) shows granular positivity. C, Immunoperoxidase stain for synaptophysin shows prominent cytoplasmic reactivity. D, Immunoperoxidase stain for cytokeratins (CAM5.2) shows focal cytoplasmic reactivity.
Tumors of Specific Sites
The distinction of benign and malignant adrenocortical tumors is often problematic by standard histologic criteria. Vargas and colleagues277 demonstrated that the mean proliferative fraction, as assessed by counting the proportion of MIB-1 positive cells, was 1.49% in adenomas, 20.8% in carcinomas, and 16.6% in recurrent or metastatic tumors. None of 20 benign lesions had a score that exceeded 8%, whereas only one of 20 carcinomas had a score below 8%. Moreover, 45% of the carcinomas were positive for p53, and none of the adenomas was p53 positive. Schmitt and coworkers278 confirmed the value of the MIB-1 labeling index for the distinction of adenomas and further demonstrated that carcinomas commonly overexpress insulin growth factor 2 (IGF-2) and cyclin-dependent kinase 4 (CDK4). Significant difficulties may lie also in the differentiation of adrenal cortical tumors from adrenal medullary tumors—namely, pheochromocytomas—and in the differentiation of adrenal tumors from extraadrenal tumors, such as metastatic carcinomas or primary carcinomas of neighboring structures, primarily of the kidney or liver (Fig. 10-30). Adrenal cortical carcinomas may show evidence of NE differentiation, as manifested by immunoreactivity for synaptophysin, NFPs, and NSE (see Fig. 10-29, C).40,279 In contrast to pheochromocytomas, however, adrenal cortical carcinomas are typically negative for the chromogranin proteins. In one series, 60% of cortical carcinomas were positive for the LMW neurofilament protein, 80% were positive for synaptophysin, and 60% were positive for NSE.279 In addition, 2 of 4 adrenocortical carcinosarcomas had synaptophysin positivity in their carcinomatous component, and 1 of 4 had synaptophysin positivity in the sarcomatous component.280 The significance of these IHC findings with respect to their histogenesis is unknown. In addition to
Pheochromocytoma 100 90 80 70 Percent
60 50 40 30 20 10
R CH -B R SY -A NA BC P LS- 2 10 G 0 F CD AP 44 S VI M KE CA R LC ( M PA EL N) A CA N-A LR E IN T HI B CK 7 CK 20
0 CH
including those of the adrenal cortex.264 Busam and coworkers264 reported A103 immunoreactivity in 100% of adrenal cortical carcinomas but in no cases of RCC, hepatocellular carcinoma, pheochromocytoma, or other types of epithelial tumors. In a smaller series, Renshaw and Granter265 reported positive staining in two of four adrenal cortical carcinomas. Loy and coworkers266 reported a more extensive study of A103 immunoreactivity in a wide array of tumors that would be considered in the differential diagnosis of adrenocortical tumors. They found that although all 21 adrenal cortical tumors were positive, none of 16 metastatic carcinomas from the lung, kidney, breast, liver, and esophagus and none of the 10 pheochromocytomas showed immunoreactivity. Additionally, these authors also studied 269 extraadrenal carcinomas from various sites that included lung, breast, kidney, pancreas, liver, esophagus, stomach, ovary, colon, biliary tract, bladder, larynx, and gallbladder; all but one case of ovarian serous carcinoma was A103 negative.266 Another study by Ghorab and colleagues267 found that 31 of 32 adrenocortical neoplasms (21 adenomas, 11 carcinomas) were A103 positive. With the exception of one clear cell RCC, all 86 RCCs (67 clear cell, 10 papillary, 4 chromophobe, 4 sarcomatoid, 1 collecting duct) and 57 hepatocellular carcinomas (25 well differentiated, 25 moderately differentiated, 7 poorly differentiated) were negative for A103.267 These studies underscore the utility of A103 in identifying primary adrenal cortical neoplasms and distinguishing them from tumors of other sites, including the adrenal medulla.268 The inhibin A antibody is also useful for the identification of steroid-producing cells.269 Fetsch and coworkers270 used this antibody for the identification of adrenal cortical tumors in cytologic preparations. They reported positive staining in 100% of adrenal cortical tumors and no staining in cases of RCC. Renshaw and Granter265 have demonstrated that inhibin A and A103 are useful in the IHC identification of adrenal cortical neoplasms and that A103 is marginally more specific, and inhibin A is slightly more sensitive, for the identification of cortical tumors. Subsequent studies have found similar results. Overall, the range of positivity for inhibin A in adrenal cortical neoplasms has been 71% to 100% and 75% to 100% in adenomas and carcinomas, respectively, whereas immunoreactivity in metastatic carcinomas—such as renal cell carcinomas (0% to 20%), hepatocellular carcinomas (0% to 4%), and pheochromocytomas (0% to 14%) is considerably less frequent.271-276 Samples of retroperitoneal or right upper quadrant tumors in FNA or needle core biopsies are often small and scant. Also, during sampling, other structures (i.e., the liver) may be inadvertently transversed, which leads to a mixed or nontumorous population of cells that cause diagnostic confusion. For example, a liver FNA sample may contain atypical, reactive hepatocytes that could mimic metastatic adenocarcinoma, RCC, or adrenal cortical carcinoma. These situations can cause considerable diagnostic difficulty and, as a result, clinicians depend increasingly on IHC stains to arrive at the correct histopathologic diagnosis.
347
Figure 10-30 Distribution of markers in pheochromocytoma. CHR, Chromogranin; SYNAP, synaptophysin; GFAP, glial fibrillary acidic protein; VIM, vimentin; CALC, calcitonin; KER(PAN), pancytokeratin; MELAN-A, Melan-A protein (A103); CALRET, calretinin; INHIB, inhibin; CK, cytokeratin.
348
Immunohistology of Endocrine Tumors
chromogranin as a distinguishing characteristic, pheochromocytomas are known to be negative for keratins, although some studies have reported positivity in up to 29% of cases.201,281 Antibodies to Bcl-2 and calretinin have been considered by several authors to assist in this distinction. Bcl-2 is typically present in all cell layers of the normal adrenal cortex but is consistently absent from the medulla. Fogt and coworkers282 demonstrated Bcl-2 immunoreactivity in 23 of 23 cortical adenomas and carcinomas but in only 1 of 11 pheochromocytomas, suggesting that Bcl-2 may be helpful in the differential diagnosis of adrenal cortical and medullary tumors. However, in a later study, Zhang and colleagues283 did not demonstrate similar staining trends using Bcl-2. In their study, only a minority of adrenal cortical neoplasms 13% (2/15) of adenomas and 30% (3 of 10) of carcinomas showed positive staining, and surprisingly, the majority of pheochromocytomas (12/14, or 86%) were Bcl-2 positive. The authors concluded that these seemingly conflicting results may be related to different antigen retrieval methodologies. Calretinin, a calcium-binding protein, is typically used as a marker for neural, mesothelial, or ovarian sexcord stromal tumors but was also found to be expressed in 73% of adrenal cortical tumors.284 These findings were confirmed by Zhang and colleagues,283 who found that 100% (16/16) and 92% (11/12) of adrenal cortical adenomas and carcinomas, respectively, were calretinin positive, but none of 20 pheochromocytomas showed staining. The differentiation of adrenal cortical carcinomas from metastatic carcinomas to the adrenal gland may be difficult. This distinction may be facilitated by studies of the distribution of intermediate filaments, particularly cytokeratins and vimentin. Normal and neoplastic adrenal cortical cells are typically vimentin positive but exhibit considerable differences in patterns of cytokeratin immunoreactivity, depending on factors such as tissue preparation (fixed versus frozen) and the reactivities of the cytokeratin antibodies.285,286 With freshfrozen tissues and FFPE tissues subjected to microwave antigen retrieval, cytokeratin immunoreactivity may be present focally in as many as 60% of adrenal cortical neoplasms, particularly with CAM5.2 (Fig. 10-29, D). The typical intermediate filament profile for cortical carcinomas is, therefore, vimentin positive with variable and generally weak cytokeratin immunoreactivity.287,288 Metastases to the adrenal gland, in contrast, are more likely to exhibit intense cytokeratin staining and are also usually positive for CEA, CD15, and epithelial membrane antigen (EMA), whereas adrenal cortical carcinomas are negative for these markers. It should be remembered that some markers, such as those for RCC, may also be expressed in adrenocortical tumors.236 Furthermore, S-100 protein may be part of a panel when malignant melanoma is being considered; however, sustentacular cells in the adrenal medulla are also typically positive. Additionally, HMB-45, which is known to be positive in malignant melanomas, is occasionally positive in pheochromocytomas.289,290 Both adrenal cortical tumors and pheochromocytomas are positive for
synaptophysin. Inhibin A can also be positive in other steroid-producing tumors, such as those that originate in the ovary. Beyond Immunohistochemistry: Molecular Diagnostic and Theranostic Applications. Over the past decade, considerable advances have been made in understanding both sporadic and heritable adrenocortical tumors at the molecular level. Genetic alterations present in familial tumors include mutations of the MEN1 gene, p53 (Li-Fraumeni syndrome), PRKAR1A (Carney complex), and CDKN1C (formerly p57kip2), KCNQ10T1, H19, and IGF-2 overexpression in Beckwith-Weidemann syndrome.291 Gene-expression profiling studies have demonstrated that the most significantly upregulated genes in cortical carcinomas include ubiquitin-specific protease 4 (USP4) and ubiquitin degradation 1–like (UFD1L).292,293 Additional upregulated genes include members of the insulin-like growth factor (IGF) family such as IGF2, IGF2R, IGFBP3, and IGFBP6. Giordano and coworkers293 also demonstrated increased expression of IGF2 in adrenal cortical carcinomas. Downregulated genes in carcinomas include the chemokine (C-X-C motif) ligand 10 (CXCL10), the retinoic acid receptor responder 2, the aldehyde dehydrogenase family member A1 (ALDH4A1), cytochrome b reductase 1, and glutathione S-transferase A4. Similar patterns of gene expression occur in pediatric adrenal cortical tumors with a consistent marked decrease in the expression of all histocompatibility class II genes in carcinomas as compared with adenomas.294 These results parallel the observations by Marx and colleagues295 that prenatal and postnatal adrenals do not express major histocompatibility complex (MHC) class II antigens, in contrast to adult adrenals, which express these antigens. Adrenocortical carcinomas are generally resistant to chemotherapy, because the tumor cells express high levels of multidrug resistance protein 1 (MDR1) or P-glycoprotein. Clinical trials with the MDR1 efflux pump inhibitor (tariquidar), epidermal growth factor inhibitor (gefitinib), antivascular endothelial growth factor (bevacizumab), and tyrosine kinase inhibitor (sunitinib) are currently in progress.296 Treatment of patients whose tumors were positive for c-Kit and platelet-derived growth factor receptor (PDGFR) did not appear to benefit from treatment with imatinib mesylate (Gleevec; Novartis, Basel, Switzerland). Adrenal Medulla and Extraadrenal Paraganglia
The major cell types of the adrenal medulla and extraadrenal paraganglia include the catecholaminesynthesizing cells and the supporting or sustentacular cells.297,298 Both catecholamines and catecholaminesynthesizing enzymes have been demonstrated in catecholamine-synthesizing cells with immunofluorescent techniques in frozen sections and immunoperoxidase techniques in paraffin-embedded material.8,297-299 The catecholamine-synthesizing cells typically exhibit positivity for a variety of generic NE markers and are variably positive for certain peptide hormones.
Tumors of Specific Sites
Sustentacular cells, in contrast, are positive for S-100 protein.297 Adrenal medullary and extraadrenal paraganglionic cells and their tumors typically exhibit a neurofilamentand vimentin-positive phenotype (see Fig. 10-30).61,285,300 The presence of cytokeratin immunoreactivity in these tumors has been controversial. Kimura and coworkers201 reported CK immunoreactivity in 29% (13/45) of pheochromocytomas using a broad-spectrum CK antibody. CK immunoreactivity was generally sparse, but positive cells were sometimes present in small groups or clusters. CK positivity was also reported in two cases of oncocytic pheochromocytomas.301,302 In contrast to the cytokeratin positivity in pheochromocytomas, extraadrenal paragangliomas in Kimura and coworkers201 study were CK-negative. Other authors, however, have failed to demonstrate cytokeratins in pheochromocytomas and extraadrenal paragangliomas. Chetty and colleagues281 examined 18 extraadrenal paragangliomas and 7 pheochromocytomas for cytokeratins using the antibodies AE1/AE3, CAM5.2, and 34βE12 after microwave antigen retrieval. Reactivity with AE1/AE3 and CAM5.2 was present in three extraadrenal paragangliomas (cauda equina, intravagal, and orbital). None of the pheochromocytomas was positive. Other epithelial markers, such as EMA, are typically negative in pheochromocytomas and paragangliomas. NSE is present in virtually all pheochromocytomas and paragangliomas.297,303 Synaptophysin is present in 100% of cases, whereas chromogranin A is expressed in more than 95%.297,303,304 Generally, chromogranin immunoreactivity is more intense in normal than in neoplastic cells of paraganglionic tissue (Fig. 10-31, A). Chromogranin immunoreactivity appears in a distinctive granular pattern, whereas NSE immunoreactivity appears diffusely within the cytoplasm. Synaptophysin immunoreactivity is present in 100% of cases. Another generic NE marker that has been analyzed in these tumors is PGP9.5, which is present in approximately 80% of reported cases.305 S-100 protein is present only in the sustentacular cells (see Fig. 10-31, B). In the normal adrenal gland, CD56 is present in the medulla and zona glomerulosa.54 Pheochromocytomas are typically strongly positive. Komminoth and
A
349
associates40 used a monoclonal antibody that binds specifically to a long-chain form of polysialic acid (polySia) found on NCAM and demonstrated staining restricted to the medulla of normal human glands. All pheochromocytomas were diffusely polySia positive, whereas 29% (8/28) of cortical carcinomas exhibited focal positivity. In addition to catecholamines, serotonin immunoreactivity has been demonstrated in approximately 80% of pheochromocytomas.303 Both pheochromocytomas and extraadrenal paragangliomas may also contain peptide hormones, including neuropeptide Y (64%), substance P (36%), calcitonin (21%), and leu- and metenkephalin (70%).303,305 Several studies have indicated that determination of circulating levels of neuropeptide Y may be useful in the diagnosis and monitoring of patients with these tumors. Helman and coworkers306 demonstrated that neuropeptide Y mRNA is present in all benign pheochromocytomas but is found in only 30% of malignant pheochromocytomas. Although inhibin A is considered to be a specific marker for cortical neoplasms, Pelkey and coworkers269 reported immunoreactivity in 2 of 19 pheochromocytomas. Clarke and coworkers307 used a variety of markers to aid in the distinction of benign and malignant pheochromocytomas.307 In their study, an MIB-1 labeling index of greater than 3% yielded a specificity of 100% and a sensitivity of 50% for predicting malignant behavior in these tumors. August and coworkers308 reported that the presence of more than 5% MIB-1 positive cells was associated with malignant behavior in 85% of the cases in their series. These authors also noted that tumors were more likely to metastasize when CD44s was negative in the tumors. As indicated in prior studies, S-100 protein positivity had a significant (P = .02) but nonlinear association with benign tumors, and the absence of S-100 protein correlated with greater tumor weight. Cathepsin B, cathepsin D, type IV collagenase, c-Met, Bcl-2, and basic fibroblast growth factor were present in both benign and malignant tumors. Neuroblastoma
Neuroblastomas are small round blue cell tumors that may arise in the adrenal gland and in a variety of
B
Figure 10-31 A, Adrenal pheochromocytoma. Immunoperoxidase stain for chromogranin A shows strong cytoplasmic staining. B, Immunoperoxidase stain for S-100 protein demonstrates positivity in sustentacular cells.
350
Immunohistology of Endocrine Tumors
Neuroblastoma 100 90 80
Percent
70 60 50 40 30 20 10
CD
5 N 6 PG B8 4 P9 .5 NS E N CH FP RA VI SY M N CD AP 11 pC 7 E S- A 10 W 0 KE T R( C -1 (K AE D5 ER 1/ 7 )C AE AM 3 ) 5 CD .2 99
0
Figure 10-32 Distribution of markers in neuroblastoma. PGP, Protein gene product; NSE, neuron-specific enolase; NFP, neurofilament protein; CHR-A, chromogranin A; VIM, vimentin; SYNAP, synaptophysin; pCEA, polyclonal carcinoembryonic antigen; WT-1, Wilms tumor 1; KER(AE1/AE3), keratin detected with monoclonal antibodies AE1 and AE3; KER(CAM5.2), keratin detected with CAM5.2.
extraadrenal sites. The differential diagnosis is wide and includes rhabdomyosarcoma (RMS), Ewing sarcoma/ primitive neuroectodermal tumor (ES/PNET), medulloblastoma, small cell osteosarcoma, lymphoblastic lymphoma, blastematous Wilms tumor, and small cell desmoplastic tumor. Numerous markers have been used for the diagnosis of neuroblastomas that include NE markers, cytoskeletal proteins, catecholaminesynthesizing enzymes, and neuroblastoma-“specific” antibodies (Fig. 10-32).309-315 Many of these markers lack specificity or sensitivity or both as individual reagents and must be used as panels. NSE is present in 85% to 100% of cases of neuroblastoma (Fig. 10-33), and a similarly high level of
A
Figure 10-33 Metastatic neuroblastoma involving a lymph node. Immunoperoxidase stain for neuron-specific enolase is restricted to tumor cells.
positivity has been reported for PGP9.5.313 However, both of these markers may also be present in other small round blue cell tumors. Wirnsberger and colleagues313 reported that among antibodies directed against chromogranins and related proteins, monoclonal islet cell antibody HISL-19 was present in 100% of neuroblastomas, followed by chromogranin A (52%) and chromogranin A and B (45%; Fig. 10-34, A). NFP was present in 80% and was localized primarily in cell processes or nerve fibers, whereas synaptophysin was present in 75% of cases. Dopamine β-hydroxylase was present in 75% of cases. In general, reactivity for these markers was greater in well-differentiated than in poorly differentiated neuroblastomas.313 Among peptide hormones, VIP was present in 30%, and neuropeptide Y was present in 10%. CD57 was not found in any neuroblastoma but was demonstrable in 7 of 7 ganglioneuromas.313 Microtubule-associated protein 1 (MAP1) and 2 (MAP2) and β-tubulin are present in 100% of cases, but the number of cases studied to date has been small.314,315 S-100 protein is restricted in its distribution to the sustentacular (stromal) cells (see Fig. 10-34, B).
B
Figure 10-34 A, Extraadrenal neuroblastoma. Immunoperoxidase stain for chromogranin A reveals a granular staining pattern in areas of process formation. B, Extraadrenal neuroblastoma. Immunoperoxidase stain for S-100 protein shows positivity restricted to sustentacular cells.
Tumors of Specific Sites
CD99 is a useful marker for the distinction of neuroblastomas from other small round blue cell tumors.316-320 More than 100 cases of neuroblastoma have now been studied for CD99, and all have been negative. In contrast, nearly 100% of cases of ES/PNET are CD99-positive. Anti–β2-microglobulin is another marker that is negative in neuroblastoma but positive in approximately 75% of ES/PNETs.321 Worthy of note is the IHC marker NB84, a monoclonal antibody raised to neuroblastoma cells.321 Miettinen and coworkers322 studied 22 cases of undifferentiated neuroblastoma and 83 cases of differentiated neuroblastoma, a total of 105 cases, and found that 95.5% (21/22) of the former and all (83/83) of the latter (104/105; 99%) showed immunoreactivity for NB84. In addition, 80% (4/5) of ES/PNETs and 100% (3/3) of desmoplastic small round cell tumors also showed positive staining. In contrast, 7 of 39 (17.9%) cases of ES and 1 of 14 (7.1%) cases of blastematous Wilms tumors were NB84 positive. Alveolar and embryonal rhabdomyosarcomas, lymphoblastic lymphomas, and pulmonary small cell carcinomas were negative.322 However, Folpe and coworkers323 reported NB84 immunoreactivity in 3 of 13 rhabdomyosarcomas, 10 of 11 medulloblastomas, 1 of 9 esthesioneuroblastomas, and 2 of 3 small cell osteosarcomas. Bielle and coworkers324 assessed the expression of PHOX2B, a transcription factor highly specific for the peripheral autonomic nervous system in a series of 388 cases by expression microarray and in 109 cases by IHC analysis and found that PHOX2B is expressed in all peripheral neuroblastic tumors, paragangliomas, and pheochromocytomas but in no other pediatric tumors. Furthermore, they noted that PHOX2B expression was present in 6/6 cases of undifferentiated neuroblastomas but was not seen in other undifferentiated small round blue cell tumors. Expression of CD133, a putative stem cell marker, was detected in advanced stages of neuroblastoma,325 therefore this molecule may constitute a potential clinical prognostic marker in children suffering from neuroblastoma. A panel of antibodies that includes NB84, CD99, PHOX2B, cytokeratins, lymphoid, and muscle-specific markers should be used in rendering a diagnosis of neuroblastoma. Beyond Immunohistochemistry: Molecular Diagnostic Applications. The molecular aspects of neuroblastoma are covered in Chapter 22. Heritable conditions associated with the development of pheochromocytomas and paragangliomas include MEN 2A, MEN 2B, von Hippel– Lindau (VHL) disease, and neurofibromatosis type 1 (NF1).326 These syndromes result from mutations of the RET protooncogene (MEN 2A and 2B), the VHL gene (VHL syndrome), and the NF1 gene. More recently, the familial paraganglioma (PGL) syndromes have been identified as resulting from mutations in the succinic dehydrogenase (SDH) genes SDHD (formerly PGL1), succinate dehydrogenase complex subunit C (SDHC; formerly PGL3), and SDHB (formerly PGL4). Approximately 50% of tumors with SDHB mutations
351
are malignant, whereas approximately 5% or less of the other mutations are associated with malignancy. Expression-profiling studies have demonstrated different clusters of markers in tumors with a specific genetic background and in subsets of sporadic tumors. For example, chromogranin B is more highly expressed in MEN 2–associated tumors than in VHL-associated pheochromocytomas.327 Gene-profiling studies of benign and malignant pheochromocytomas have demonstrated that almost 90% of the differentially expressed genes are underexpressed in malignant versus benign tumors.328,329 These features are consistent with a less differentiated biochemical pathway in the malignant tumors, characterized by a lack of production of epinephrine and relatively high production of dopamine compared with norepinephrine.328 Several of the downregulated genes include peptidylglycine α-amidating monooxygenase and glutaminyl-peptide cyclotransferase.329 Stutterheim and coworkers330 demonstrated that PHOX2B is superior to tyrosine hydroxylase (TH) and ganglioside GD2 synthase in specificity and sensitivity for minimal residual disease in detection of neuroblastoma by using real-time quantitative PCR, and they proposed that it be included in prospective minimal residual disease studies in neuroblastoma alongside TH and other minimal residue disease markers. KEY DIAGNOSTIC POINTS Adrenocortical Tumors • Adrenocortical tumors are positive for vimentin and are variably positive for cytokeratins. • Melan-A (A103), inhibin, and calretinin are useful for the distinction of adrenal cortical carcinomas from other tumor types. • Adrenocortical tumors are often positive for synaptophysin and neurofilaments. • Adrenocortical carcinomas have a proliferative index (assessed with MIB-1) in excess of 8% and are positive for p53 in approximately 45% of cases. • Adrenocortical tumors generally overexpress IGF-2 and CDK5.
KEY DIAGNOSTIC POINTS Adrenal Medullary Tumors • Neuroblastomas and pheochromocytomas are positive for a wide spectrum of neuroendocrine markers. • Both tumor types may also contain peptide hormones. • Pheochromocytomas are positive for chromogranin and synaptophysin; the expression of synaptophysin is characteristic of adrenocortical tumors. • Malignant pheochromocytomas have a higher MIB-1 proliferative index than benign pheochromocytomas. • Gene-profiling studies have demonstrated that almost 90% of the differentially expressed genes are underexpressed in malignant pheochromocytomas compared with their benign counterparts.
352
Immunohistology of Endocrine Tumors
TABLE 10-5 Hormonal Profiles of Gastrointestinal Endocrine Tumors Product
Foregut (% +)
Midgut (% +)
Hingut (% +)
Serotonin
30
89
13
Somatostatin
80
4
63
Substance P
10
41
0
0
0
88
Glucagon
10
0
50
Calcitonin
0
11
0
Adrenocorticotrophic hormone
20
4
0
Gastrin
30
0
0
Pancreatic polypeptide
GASTROINTESTINAL ENDOCRINE CELLS
ETs (carcinoids) of the GI tract (GI ETs) have been divided into three major groups based on their origins from foregut, midgut, and hindgut derivatives. Correlation is strong among their sites of origin and the distribution patterns of peptide hormones and amines. For example, serotonin is present in 89% of midgut carcinoids, 30% of foregut carcinoids, and 13% of hindgut GI ETs (Table 10-5).331 GI ETs are typically positive for cytokeratins, and 100% exhibit positivity for CAM5.2, 80% exhibit positivity with other pancytokeratin antibodies, and approximately 40% exhibit positivity for CK20.320-329,331-334 Approximately 25% are positive for vimentin, and NFPs are present in a variable proportion of cases. GI ETs are positive for a wide variety of generic NE markers. NSE is present in almost 80% of cases, whereas PGP9.5 is present in approximately 90%.15 Synaptophysin is present in 100% of cases at all sites, and the reactivity of other NE markers differs according to the site of origin.335 Chromogranin A is present in 88% to 100% of foregut, 100% of midgut, and 24% to 40% of hindgut
A
Figure 10-35 Ileal carcinoid tumor. Immunoperoxidase stain for chromogranin A reveals positivity in tumor cell nests.
GI ETs (Figs. 10-35 and 10-36). Chromogranin B, in contrast, is present in 100% of hindgut carcinoids.34,336 neuroendocrine secretory protein 55 (NESP-55), a member of the chromogranin family, is present in approximately 10% of rectal GI ETs and is absent from GI ETs from other sites in the GI tract; however, approximately 40% of pancreatic ETs (PETs) are positive for this marker.37,38 Peptidylglycine α-amidating enzyme has been reported in 14% of gastric, 100% of ileal, and 100% of rectal GI ETs.258 NCAM is present in 76% of foregut, 58% of midgut, and 20% of hindgut tumors. Antibodies to NCAM stain both tumor cells and sustentacular elements.336 S-100 protein is present in 41% of foregut carcinoids and 50% of midgut and hindgut tumors. The pattern of staining for S-100 protein is generally similar to that observed for NCAM, with reactivities present in both tumor cells and sustentacular elements. CEA is present in approximately 40% of GI ETs with polyclonal antisera or monoclonal antibodies, whereas CD15 is present in 30% of these tumors.337-339 The monoclonal antibody CA 15.3, which identifies both carbohydrate and peptide determinants (MUC1-type
B
Figure 10-36 A, Metastatic carcinoid involving the liver. Immunoperoxidase stain for chromogranin A shows strong positivity in tumor cells. B, Metastatic carcinoid involving the liver.
Tumors of Specific Sites
353
depth of tumor invasion, suggesting that cofilin might be useful clinically as an adjunct molecular prognostic marker in evaluation of GI endocrine cell tumors.342 Beyond Immunohistochemistry: Molecular Diagnostic Applications
Few studies have been done on the molecular features of GI ETs. Allelic loss of 11q has been detected in GI ETs associated with MEN 1. LOH of 11q is also present in a subset of sporadic GI ETs.343 Mutations of the MEN1 gene are present in approximately 30% of sporadic gastrinomas and in occasional midgut and hindgut ETs. In contrast to PETs, the CpG island methylator phenotype is frequent in GI ETs; CTNNB1 catenin (cadherin-associated protein), β-1 (formerly β-catenin) exon 3 mutations are relatively common (38%), and as many as 80% show nuclear and cytoplasmic localization of the corresponding protein.344 Other studies, however, reported absence of exon 3 mutations, but nuclear β-catenin was found in 30% of cases.345 In contrast, extra-GI ETs were negative for nuclear β-catenin. PANCREATIC ENDOCRINE CELLS
M VI
PC 3
2
M PG
A
PC
Cg
NF P HC G ( )
LM
NS E W CK
100 90 80 70 60 50 40 30 20 10 0 SY N
mucin), reacts with 75% of GI ETs, whereas CA 19.9, which reacts with sialylated Lewis antigen, is negative.166 CDX-2 has also been demonstrated in variable proportions of GI neuroendocrine tumors, depending of their sites of origin (Fig. 10-37). In the study of Moskaluk and coworkers,340 73% of midgut and 44% of hindgut GI ETs showed the most extensive staining for CDX-2. Srivastava and coworkers37 reported CDX-2 positivity in nearly 100% of ileal and appendiceal GI ETs but in no gastroduodenal or rectal GI ETs. In addition, PDX-1 was present in almost 30% of PETs.38 Prostatic acid phosphatase (PAP) may be present in some GI ETs. In a study of 33 GI ETs of foregut, midgut, and hindgut origins, PAP was present in 5 of 5 hindgut tumors, whereas other carcinoids were negative.341 In contrast, prostate-specific antigen (PSA) is typically negative in these tumors. The alpha chain of hCG is also present to varying degrees in carcinoid tumors. Heitz and coworkers190 reported staining for the alpha chain in 46% of foregut and 25% of hindgut GI ETs but none of 35 midgut GI ETs. Calbindin, a 28,000-kD calciumbinding protein has been localized to subpopulations of central and peripheral nervous system neurons, distal tubular cells of the kidney, and enteric NE cells. IHC studies have demonstrated that calbindin is present in a small number of NE cells, predominantly in the appendix and small intestine, and in 100% of midgut and foregut GI ETs.196 In contrast, calbindin immunoreactivity was absent from a single case of rectal ET. High immunolabeling with cofilin, an actin-binding protein, in GI ETs appears to be associated with the
Percent
Figure 10-37 Ileal carcinoid tumor stained for CDX-2. The nuclei of the tumor cells are strongly positive.
With broad-spectrum antibodies, cytokeratin immunoreactivity is present in normal pancreatic endocrine cells and in approximately 90% of PETs, whereas CK20 is present in 12.5% of cases (Fig. 10-38).59,63,201,334,346 Vimentin has variable reactivity, and approximately 25% of cases demonstrate cytoplasmic staining. Neurofilament immunoreactivity occurs in as many as 50% of cases. Among NE markers, NSE and synaptophysin are present in all normal pancreatic endocrine cells and in virtually all PETs.347 Chromogranin A is present in approximately 75% of all PETs, and the extent of staining generally correlates with the degree of granularity, as noted in sections stained for peptide hormones.347 Calbindin and MIC2 (CD99) have been reported in a small subset of PETs.196,318 The normal islets of Langerhans contain four major cell types.348 The insulin-producing beta cells comprise 60% to 70% of the cells in the main part of the pancreas
Figure 10-38 Distribution of markers in pancreatic endocrine tumors. SYN, Synaptophysin; NSE, neuron-specific enolase; LMWCK, low-molecular-weight cytokeratin; CgA, chromogranin A; PC2, proconvertase 2; PGM, peptidylglycine α-amidating enzyme; PC3, proconvertase 3; NFP, neurofilament protein; hCG(α), human chorionic gonadotropin alpha; VIM, vimentin.
354
Immunohistology of Endocrine Tumors
and 20% to 30% of the cells in the posterior head of the gland. Glucagon-producing alpha cells constitute 15% to 20% of the cells in the main portion of the gland and less than 5% of the cells in the posterior head. Somatostatin-positive cells comprise 5% to 10% of the cells in the main portion of the gland and approximately 5% of the islet cell population in the posterior portion of the gland. Pancreatic polypeptide cells represent 70% of the islet cells in the posterior portion of the gland and 2% to 5% of the cells in the remaining islets. Approximately 10% of pancreatic endocrine cells are present in extrainsular sites, where they are distributed among ductal cells or paraductular acinar cells. Occasional serotonin-producing cells are present in large ducts and are the most likely cell of origin of true carcinoid tumors of the pancreas. Insulinomas are typically positive for insulin and proinsulin, including cases that are negative by standard histochemical stains.348,349 Approximately 50% of insulinomas are multihormonal and may contain cells that are positive for glucagon, somatostatin, pancreatic polypeptide (PP), gastrin, ACTH, or calcitonin. Considerable variation is often seen in the staining intensity for insulin, and cells that contain abundant granules ultrastructurally give the most intense immunoreactivity. Glucagonomas are identified on the basis of reactivity for the corresponding peptide. Both glicentin and glucagon-like peptides are typically present as well. Glucagonomas may also contain peptides unrelated to proglucagon, including somatostatin and insulin. Somatostatinomas are identified on the basis of their reactivity with antibodies to somatostatin.348 These tumors may also contain calcitonin, ACTH, and gastrin. Tumors of identical morphology and IHC profiles also occur within the duodenum. PP-producing tumors are generally classified among nonfunctional tumors, although these tumors may rarely be associated with the syndrome of watery diarrhea, hypokalemia, and achlorhydria (WDHA). In addition to their content of PP, these tumors may contain scattered cells that contain other hormonal peptides. Gastrinomas are characterized by varying degrees of immunoreactivity for gastrin; however, some of these tumors may be nonreactive (Fig. 10-39).348,349 In the latter instance, antibodies to different regions of the N- and C-terminal portions of the gastrin molecule may be positive. Some gastrinomas that may be entirely negative for gastrin may give positive signals for gastrin mRNA in ISH formats.350 Gastrinomas, similar to other PETs, may contain scattered cells that are positive for glucagon, PP, insulin, somatostatin, serotonin, or ACTH.351 Gastrinomas may also occur in a variety of extrapancreatic sites, including the duodenum.348 VIP-producing tumors have been associated with the syndrome of WDHA. In a series of 28 cases of WDHA studied by Solcia and colleagues,352 VIP was present in 87%, and peptide histidine methionine was present in 57% of cases. Growth hormone–releasing hormone and PP were present in 50% and 53% of cases respectively.352 In addition to PETs, ganglioneuromas and ganglioneuroblastomas have been associated with the syndrome of WDHA.
A
B Figure 10-39 A, Pancreatic gastrinoma (hematoxylin and eosin). B, Immunoperoxidase stain for gastrin shows weak cytoplasmic staining.
Rare examples of serotonin-producing ETs may occur within the pancreas. Other tumors that occur as primary PETs may produce growth hormone–releasing hormone (acromegaly), ACTH (Cushing syndrome), and PTH or PTH-like peptide (hypercalcemia). Nonfunctional PETs may contain scattered cells that are positive for a variety of hormones, most commonly PP and glucagon (Fig. 10-40). The alpha chain of hCG has been regarded as a marker of malignancy in PETs and occurs in approximately 70% of cases (Fig. 10-41).353 More recent studies, however, have demonstrated immunoreactivity for this marker in benign PETs354 and the presence of progesterone receptor protein in a significant proportion of PETs.355 The MIB-1 labeling index has been used as a predictor of survival in several studies of PETs and has been included as a criterion in the World Health Organization (WHO) classification of these tumors.356-359 Positive staining for CK19 has also been regarded as a marker of malignancy in PETs. In a study of MIB-1 and CK19 in a series of PETs, Deshpande and coworkers360 demonstrated that the expression of each marker was significant prognostically by univariate analysis. Loss of CD99 expression has also been associated with a poor prognosis in PETs.361 In a correlated study of the expression of a variety of markers and the 2004 WHO criteria, Schmitt and colleagues362 demonstrated that CK19 was
Tumors of Specific Sites
Figure 10-40 Nonfunctional pancreatic endocrine tumor. Immunoperoxidase stain for pancreatic polypeptide shows positivity in scattered individual tumor cells.
a useful prognostic marker independent of the WHO criteria. However, no prognostic significance of COX-2, p27, or CD99 expression was noted. LaRosa and coworkers363 demonstrated that CK19 expression correlated with patient survival only when detected with the RCK108 antibody and mainly in insulinomas. Moreover, they demonstrated that the MIB-1 index and the presence of metastases were the only two independent predictors of survival. In the study of Zhang and coworkers,364 PETs with CK19 and KIT expression were significantly associated with death from disease in a univariate setting, but in multivariate analysis, only WHO criteria and KIT expression were shown to be independent. An IHC classification system365 derived from a combination of KIT and CK19 expression that could predict the clinical behavior was suggested: low risk (KIT-/CK19-), intermediate risk (KIT-/CK19+), and high risk (KIT+/ CK19+). Their results indicated KIT as an independent prognostic marker for PETs.364,365 The expression of neuro-D1 and mammalian achaete-scute complex–like protein (MASH) have been
Figure 10-41 Nonfunctional pancreatic endocrine tumor. Immunoperoxidase stain for the alpha chain of human chorionic gonadotropin shows a few positive cells.
355
assessed in gastroenteropancreatic (GEP) tumors.366 These studies have demonstrated that MASH-1 is highly expressed in poorly differentiated GEP tumors, whereas neuro-D1 is present in all well-differentiated carcinomas and tumors. Interestingly, low levels of neuro-D1 expression were seen in approximately one third of poorly differentiated GEP carcinomas, and this feature was associated with a significantly shorter overall survival. In a recent study, Shin and colleagues367 found that high expressions of ataxia telangiectasia mutated gene (ATM) and cyclin B1 were related to well-differentiated PETs of the WHO classification but not to tumor-nodemetastasis (TNM) stages. After multivariate analysis, cyclin B1 was the only significant factor for survival (P = .008) and was associated with a smaller tumor size, less vascular invasion, decreased recurrence rate, and a decreased death rate. These results suggested that expression of ATM and cyclin B1 may be useful in identifying patients with poor prognosis who may benefit from close follow-up and more aggressive therapy. Beyond Immunohistochemistry: Molecular Diagnostic Applications
PETs occur with increased frequency in patients with MEN 1 and VHL syndromes and are considerably less common in association with NF1 or tuberous sclerosis.368 However, the vast majority of these tumors are sporadic, and relatively few expression-array studies of these tumors have been done. Couvelard and coworkers368 demonstrated 71 upregulated and 51 downregulated genes in malignant PETs that included genes related to angiogenesis and remodeling (CD34, cadherin-5, E-selectin, semaphorin-E, fibrillin); signal transduction via tyrosine kinase (tyrosine kinase-2, PDGFRB, Mkk4, discoidin domain receptor 1); calciumdependent cell signaling (transient receptor potential cation channel 1, calcium channel voltage-dependent β-2, neurocalcium-δ, GABA-A receptor γ-2); and responses to drugs (MDR1 and CEA-related cell adhesion molecule). By using tissue arrays, the authors confirmed the differential expression of CD34, E-selectin, Mkk4, and MDR1 in metastatic versus nonmetastatic tumors. Almost all PETs in MEN 1 demonstrate allelic loss of the MEN1 gene, whereas somatic mutations of MEN1 occur in approximately 20% of sporadic PETs.369 Mutations are present in approximately 10% of insulinomas and nonfunctioning tumors but are more common in gastrinomas, glucagonomas, and VIPomas. In contrast, almost 70% of PETs demonstrate losses at 11q13. This suggests possible haploinsufficiency of the MEN1 gene or that the other allele might be inactivated by epigenetic mechanisms. CpG island methylation in the MEN1 promoter, however, has not been identified. An alternative explanation is that this region of chromosome 11 might contain other tumor suppressor genes. Mutations of the VHL gene are uncommon in PETs despite the relatively high rates of LOH of 3p25. Moreover, p16, PTEN, K-RAS, p53, and DPC4 are only occasionally mutated in these tumors.369,370
Immunohistology of Endocrine Tumors
A CE m
CK
RP G
TH AC
(b )
N
TH AC
SY
E
100 90 80 70 60 50 40 30 20 10 0 CK
CE A
m
CT
CK
NS E CD 57 SY N AC TH (b ) G RP AC TH
Figure 10-43 Distribution of markers in typical lung carcinoid. CgA, Chromogranin A; NSE, neuron-specific enolase; SYN, synaptophysin; ACTH(b), big adrenocorticotropic hormone; GRP, gastrinreleasing peptide; ACTH, adrenocorticotropic hormone; CK, cytokeratin; CT, calcitonin; mCEA, monoclonal carcinoembryonic antigen.
NS
and IHC.372 The tumor types include typical carcinoids, atypical carcinoids, small cell carcinomas, and large cell NECs. The marker profiles for those tumors are summarized in Figures 10-43 through 10-46. Occasional pulmonary NE tumors may contain peptides such as calcitonin (Fig. 10-47), and approximately 85% of pulmonary NE tumors are reactive with antibodies to LMW cytokeratins.372,373 In the series reported by Travis and associates,372 reactivity with broad-spectrum keratin antibodies (AE1/AE3) was present in 56% of typical carcinoids, 40% of atypical carcinoids, and 100% of small cell carcinomas and large cell NECs. It has also been reported that 82% of small cell carcinomas are negative for CK7 and CK20.60 TTF-1 is present in normal pulmonary NE cells and is a useful marker for the identification of pulmonary NE tumors. However, TTF-1 is present in small cell carcinomas that arise in a variety of extrapulmonary sites (Table 10-6). Most studies report TTF-1 positivity in more than 90% of pulmonary small cell NECs. In the study of Oliveira and colleagues,374 TTF-1 was present in 95% (19/20) of well-differentiated pulmonary NE
Percent
100 90 80 70 60 50 40 30 20 10 0 Cg A
Percent
The NE cells of the lung are present as single cells and as small cell clusters that have been termed neuroepithelial bodies.371 It has been suggested that neuroepithelial bodies may have a chemoreceptor function in which single NE cells may act as paracrine elements. A variety of regulatory products that include serotonin, gastrinreleasing peptide, and calcitonin are present both in single NE cells and within neuroepithelial bodies, whereas leu-enkephalin is present only in single NE cells (Fig 10-42).371 The pulmonary NE cells may undergo a series of hyperplastic changes in response to irritation or after exposure to carcinogens. Generally, hyperplastic NE cells retain the patterns of expression of regulatory products characteristic of their normal counterparts. More severe forms of hyperplasia and dysplasia are accompanied by the production of ectopic products that include VIP and different molecular forms of ACTH. NE tumors of the lung include four major entities that can be distinguished on the basis of morphology
Figure 10-44 Distribution of markers in atypical lung carcinoid. CgA, Chromogranin A; NSE, neuron-specific enolase; SYN, synaptophysin; ACTH(b), big adrenocorticotropic hormone; ACTH, adrenocorticotropic hormone; GRP, gastrin-releasing peptide; CK, cytokeratin; mCEA, monoclonal carcinoembryonic antigen.
SY N m CE A CD 56 NS E AC T AC H TH (b ) Cg A CD 57 G RP CK 20
PULMONARY ENDOCRINE CELLS
CD
Cg
Figure 10-42 Fetal lung. Immunoperoxidase stain for gastrinreleasing peptide shows positivity in single cells.
57
100 90 80 70 60 50 40 30 20 10 0
A
Percent
356
Figure 10-45 Distribution of markers in small cell carcinoma. CK, Cytokeratin; SYN, synaptophysin; mCEA, monoclonal carcinoembryonic antigen; NSE, neuron-specific enolase; ACTH, adrenocorticotropic hormone; ACTH(b), big adrenocorticotropic hormone; CgA, chromogranin A; GRP, gastrin-releasing peptide; CK 20, cytokeratin 20.
357
TH
CT
AC
E Cg A CD 56 SY N CD 57 G AC RP TH (b )
NS
.5
P9
CE m
PG
CK
100 90 80 70 60 50 40 30 20 10 0 A
Percent
Tumors of Specific Sites
Figure 10-46 Distribution of markers in large cell neuroendocrine carcinoma. mCEA, Monoclonal carcinoembryonic antigen; PGP9.5, protein gene product 9.5; CK, cytokeratin; NSE, neuron-specific enolase; CgA, chromogranin A; SYN, synaptophysin; GRP, gastrinreleasing peptide; ACTH(b), big adrenocorticotropic hormone; CT, calcitonin; ACTH, adrenocorticotropic hormone.
tumors, including typical and atypical carcinoids, and in 80% (8/10) of metastases of these tumors. On the other hand, Du and colleagues375 reported TTF-1 positivity in 28%, 29%, and 37% of typical carcinoids, atypical carcinoids, and large cell NECs, respectively. Interestingly, TTF-1 positivity was more commonly present in peripheral carcinoids of the spindle cell type than in central carcinoids. Rarely, TTF-1 may be present in gastrointestinal and pancreatic NE tumors.376 Chromogranin A is present in 100% of typical and atypical carcinoids, in 80% of large cell NECs, and in as many as 50% of small cell carcinomas, depending on the
A
Figure 10-48 Pulmonary tumorlet. Immunoperoxidase stain for Leu-7 shows positivity in lesional cells.
antigen retrieval method and sensitivity of the detection method.372 Synaptophysin is present in 84% of typical carcinoids, 80% of atypical carcinoids, 40% of large cell NECs, and 100% of small cell carcinomas. CD57 immunoreactivity is present in 89% of typical carcinoids, 100% of atypical carcinoids, and 40% and 50% of large cell NECs and small cell carcinomas respectively. Pulmonary tumorlets are also positive for CD57 (Fig. 10-48). Monoclonal CEA (mCEA) is present in 100% of large cell NECs and small cell carcinomas, 42% of typical carcinoids, and 20% of atypical carcinoids. Jiang and colleagues377 also studied large cell NECs and confirmed the findings of Travis and coworkers.372 In addition, Jiang and coworkers377 reported positivity for
B
Figure 10-47 Pulmonary large cell neuroendocrine carcinoma. A, Hematoxylin and eosin. B, Immunoperoxidase stain for calcitonin. Many of the tumor cells are strongly positive.
358
Immunohistology of Endocrine Tumors
TABLE 10-6 Immunohistochemical Profile of Small Cell Carcinoma of Various Sites Anatomic Site/Origin
TTF-1
CK7
CK20
ER/PR
PSA
PAP
Lung
+
S
S
−
−
−
Prostate
+
S
S
−
S
S
Bladder
S
−
−
−
−
−
Breast
S
+
−
S
−
−
Thyroid*
+
?
?
−
−
−
Gastrointestinal tract
S
?
−
−
−
−
Salivary gland
−
?
S
−
−
−
Cervix
S
?
S
?
−
−
Skin (Merkel cell)
−
−
+
−
−
−
Neuroendocrine-specific markers, such as chromogranin and synaptophysin, are variably positive in all tumors regardless of site and therefore are not useful for this purpose. *Also in medullary types. +, almost always positive; S, sometimes positive; −, negative; ?, unknown; CK, cytokeratin; ER/PR, estrogen receptor/progesterone receptor; PAP, prostatic acid phosphatase; PSA, prostatic-specific antigen; TTF-1, thyroid transcription factor 1.
PGP9.5 in 100% of the cases and positivity for TUJ1 (neuron-specific class III β-tubulin) in 82%. NCAM positivity was present in 73%.377 Zhang and coworkers378 evaluated p63 expression in differentiation between small cell lung carcinomas and poorly differentiated squamous cell carcinomas (SCCs) and demonstrated p63 expression in 27 of 28 poorly differentiated SCCs, whereas most small cell lung carcinomas (26/28, 93%) had no staining for p63. Matsuki and colleagues379 analyzed and demonstrated the role of the antihistidine decarboxylase antibody as a potential marker of NE differentiation. In their series, the antibody stained most small cell lung carcinoma (18 of 23, sensitivity 0.78) and was rarely reactive with non-NE lung tumors (2 of 44; specificity, 0.95). Histidine decarboxylase was also positive for 6 of 12 large cell NECs and 4 of 7 GI small cell carcinomas. Beyond Immunohistochemistry: Molecular Diagnostic Applications
Xu and colleagues380 suggested the use of antibodies to KOC—K-homology domain–containing protein, which is overexpressed in cancer and is a member of the insulin-like growth factor (IGF) mRNA-binding protein family—in the diagnosis of high-grade NECs of the lung and in their distinction from carcinoids. In their study, all high-grade NECs of the lung had diffuse or focal cytoplasmic positivity for KOC, and the typical and atypical carcinoids were largely negative (a single atypical carcinoid with oncocytic cells showed weak cytoplasmic staining). In addition, high-grade NECs were more likely to express c-Kit and Bcl-2 than carcinoid tumors. LaPoint and collegues381 found that all highgrade NEC (small cell carcinomas and large cell NECs of the lung) coexpressed c-Kit and Bcl-2, whereas all atypical and typical carcinoids were negative for c-Kit, and only 6.3% of typical carcinoids and 16.7% of atypical carcinoids were positive for Bcl-2. These findings suggest a possible therapeutic role in targeting of these
two molecules in patients with high-grade NECs of the lung. Jiang and coworkers382 used ISH to investigate the human homolog 1 of the Drosophila neurogenic achaetescute gene (ASCL1) expression in 238 surgically resected lung carcinomas. Then, the expression was correlated with immunostaining results of NE markers, and expression was detected in 10% (2/20) of adenocarcinomas, 13.3% (4/30) of typical carcinoids, 84.6% (11/13) of atypical carcinoids, 56.7% (38/67) of large-cell NECs, and 71.8% (56/78) of small cell carcinomas, respectively, but not in any SCC (0/21) or large cell carcinoma (0/9). Moreover, ASCL1 expression closely correlated with endocrine phenotype and differentiation extent in pulmonary NE tumors and with a significantly shorter survival in small cell carcinoma patients (P = .041).382 Collapsin response–mediator proteins (CRMPs) include a family of five members that are involved in the semaphorin signaling pathway during neurogenesis. DPYSL5 (formerly CRMP5) is involved in the development of neural tissue, and antibodies to DPYSL5 protein have been used as serologic markers of small cell lung carcinoma in the setting of paraneoplastic neurologic disorders.383 DPYSL5 is expressed strongly and extensively in 98% of high-grade pulmonary NE tumors, including small and large cell NE tumors, but not in adenocarcinomas or squamous carcinomas. In contrast to the high frequency of strong positivity in high-grade pulmonary NECs, most pulmonary carcinoids and atypical carcinoids were negative or only weakly reactive for this marker.
Endocrine Tumors in Other Sites Cervix Carcinoids, atypical carcinoids, small cell carcinomas, and large cell NECs have been reported as primary
Endocrine Tumors in Other Sites
cervical tumors, small cell carcinomas being the most common.384-387 All small cell cervical carcinomas studied to date have been cytokeratin positive, and more than 90% have been positive for EMA. Reactivity for CEA with polyclonal antisera occurs in 77%. Small cell cervical carcinomas are sometimes positive for CK20 (14.3%) and often for TTF-1 (as many as 85%).110,388-391 With respect to NE markers, NSE is present in 95%, synaptophysin in 46%, chromogranin A in 43%, and CD57 in 37%. Similar or higher frequencies of positivity for NE markers have been reported in more recent studies.391-394 These tumors may also contain peptide and amine hormones that include serotonin (31%), ACTH (23%), and somatostatin (8%). Moreover, p63 immunoreactivity was demonstrated in half of small cell carcinomas studied, suggesting that this marker is not specific for squamous differentiation and perhaps is not as useful in discerning this tumor from small cell SCC as suggested elsewhere.395-397 Chromogranin A immunoreactivity has been reported to range from 38% to 100% in large cell NE cervical carcinomas.391,394 In these studies, synaptophysin staining was present in 66% to 100%, CD56 in 89%, and NSE in 50% of cases (Fig. 10-49).391,394 Occasional serotonin-positive cells were present in 50% of cases, whereas somatostatin-positive cells were present in 37%.394 In addition, 75% of cases contained CEA, as demonstrated with a monoclonal antibody,394 and TTF-1 was positive in 50% of cases.391 Stoler and associates398 demonstrated human papillomavirus 18 (HPV-18) in 78% of small cell cervical carcinomas with NE differentiation. The presence of this papilloma subtype was five times more frequent than HPV-16 in the 20 cases studied. Ishida and coworkers399 found that of 10 small cell NECs of the cervix studied, HPV-18 was detected in all 3 pure cases and in 5 of 7 mixed tumors (mixed small cell and adenocarcinomas). No other HPV types were detected, even though types 6, 11, 16, 31, 33, 42, 52, and 58 were also investigated. Other studies have confirmed the high frequency of HPV-18 in these tumors.393,400 Unlike small cell carcinomas, large cell NECs of the uterine cervix appear to be associated with HPV-16
Figure 10-49 Large cell (neuroendocrine) carcinoma of the cervix. Immunoperoxidase stain for neuron-specific enolase shows positivity in tumor cells.
359
infection.401,402 In addition, overexpression of p16, a cyclin-dependent kinase inhibitor associated with HPV infection, has been noted in almost all cases.391,393,400 Effective treatment options for primary cervical NE tumors are lacking, and despite multiple studies, reliable prognostic and predictive factors have not been identified.403,404 Tangitgamol and colleagues404 only found expression of ERBB2 (formerly HER-2/neu) to be significantly associated with survival of patients with cervical small cell carcinomas and large cell NECs. Patients whose tumors lacked ERBB2 expression had significantly shorter survival than those with ERBB2–positive tumors. Immunostains for epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2), and estrogen and progesterone receptors did not show consistent expression related to survival in these patients. In contrast, Straughn and coworkers392 did not find any ERBB2 expression in any of the cervical small cell NECs studied. Zarka and coworkers405 studied four cases of cervical small cell carcinoma by IHC and found that all tumors exhibited a unique profile with E-cadherin, P-cadherin, N-cadherin, Bcl-2, and p53. The only commonality among these tumors was the absence of N-cadherin immunoreactivity. The authors suggested that because 65% of small cell carcinomas from other sites reportedly express for N-cadherin, that this differential staining pattern could be used to distinguish primary small cell carcinoma of the cervix from metastatic disease.
Prostate Normal prostatic epithelium contains subpopulations of NE cells that can be identified on the basis of their content of NSE, chromogranin, peptide hormones, and serotonin.406-408 These cells are involved in regulating epithelial cell growth and differentiation in an androgenindependent manner.409 Prostatic neoplasms with NE differentiation include carcinoids, small cell carcinomas, and the “usual” adenocarcinomas with subpopulations of NE cells. “Pure” NE neoplasms, such as carcinoids and small cell carcinomas, are rare and comprise less than 5% of all prostatic malignancies.389,410-416 Small cell carcinomas of the prostate are TTF-1 positive in approximately 50% to 83% of cases (see Table 10-6),110,389,417,418 whereas more than 200 conventional prostatic adenocarcinomas with high Gleason scores were negative for this marker.413,417 CD44 appears to be a useful marker to discriminate prostatic small cell NECs from Gleason 5–pattern adenocarcinomas and small cell carcinomas of other origins.419 More recently, negative PSA and positive CD56 and TTF-1 staining were found to be the most helpful markers in this distinction among the immunostains studied.414 However, the largest series of prostatic small cell carcinomas (44 cases) recently reported that P501S and prostate-specific membrane antigen were better in this regard than PSA. With that said, the study also reported that the majority of cases studied (60%) were negative for all three markers.418 The origin of prostatic carcinomas with NE cells remains unclear, although some studies support the
360
Immunohistology of Endocrine Tumors
concept of transdifferentiation of exocrine tumor cells to cells with an NE phenotype. Activation of the MAPK/ ERK pathway appears to be one of the major mechanisms for the transdifferentiation of prostatic carcinoma cells into cells with NE differentiation.
Skin
Figure 10-50 Metastatic prostatic adenocarcinoma with neuroendocrine cells. Immunoperoxidase stain for chromogranin A shows positivity in a few tumor cells.
concept that they arise from multipotential prostatic stem cells as evidenced by the coexpression of both NE and prostate-specific markers.411,414,415 Moreover, NE cells from normal and hyperplastic prostates express bcl-2. There is a positive correlation between the expression of bcl-2 and NE markers in prostatic carcinomas with NE differentiation. Bcl-2 was also found to be positive in all 18 cases of prostatic small cell carcinoma studied.417 In addition, α-methylacyl-CoA (AMACR) is expressed in the NE components of carcinomas with NE cells but not in normal NE cells.409 NE differentiation in conventional prostatic carcinomas occurs in approximately 10% of cases, where at least focally, prominent collections of neoplastic NE cells may be evident.410 The presence of NE differentiation may be more prognostically significant in androgenindependent tumors and metastatic tumors than in hormone-sensitive and locally recurrent tumors.407 Androgen withdrawal contributes to increased NE differentiation in prostatic carcinomas. In adenocarcinomas with NE cells, 54% contain serotonin, 46% are positive for NSE, 65% are positive for chromogranin (Fig. 10-50), and 22% are positive for hCG. Somatostatin, calcitonin, and ACTH are present in less than 5% of cases.416 Schmid and coworkers408 demonstrated that the pattern of chromogranin distribution is correlated with tumor grade. Grade I tumors show positive reactions for chromogranins A and B and secretogranin II, with colocalization of all three products in the majority of NE cells. In grade II and grade III tumors, chromogranin B is the predominant granin. Scattered NE cells have been demonstrated in up to 88% of cases of high-grade prostatic intraepithelial neoplasia.416 The highest proportion of cases are immunoreactive for serotonin (73%), NSE (67%), chromogranin (62%), and hCG (30%).
Merkel cell (NE) carcinoma of the skin is an uncommon entity first described as trabecular carcinoma.421 These tumors are uniformly positive for broad-spectrum cytokeratins and stain positively for CK20 in 97% of cases, with a dotlike pattern of reactivity (Fig. 10-51).334,388 This high frequency of CK20 immunoreactivity has been confirmed in many other studies.422-425 Other small cell malignancies that may exhibit CK20 positivity include pulmonary small cell carcinomas (2%), small cell cervical carcinomas (9%), and small cell carcinomas of salivary gland origin (60%). Nagao and coworkers426 recently reported that 73% (11/15) of cases of small cell carcinoma that originated in the salivary gland were CK20 positive, and almost all had a paranuclear dotlike pattern of reactivity. Cheuk and colleagues423 reported lack of CK20 immunoreactivity in small cell carcinomas from various sites that included the GI tract, pancreas, prostate, bladder, thymus, and orbit with the exception of two cases that originated in the cervix/vagina. CK7 has been reported to be positive in 25% of Merkel cell carcinoma cases, but none of the cases was positive for CK5/6 or CK17,427 whereas 78% percent of Merkel cell carcinomas are positive for EMA.428 Microtubuleassociated protein (MAP) is also present in these tumors,429 but TTF-1 is typically negative.110 Virtually all Merkel cell carcinomas are positive for NSE, and chromogranins A and B are found in 72% and 100% of the tumors, respectively.430 CD56 is also highly expressed in these tumors.431 Secretoneunin, which is derived from secretogranin II, is present in 22% of cases, and synaptophysin is present in 39%.432 Merkel cell tumors are variably positive for CD99, and in the series reported by Nicholson and associates,425 12 of 30 cases (40%) were positive for this marker. In nine cases that were consistent with Merkel cell carcinoma clinically, CD99 was positive, and CK20 was negative.425 Several studies
MOLECULAR APPROACHES
The results of molecular studies have demonstrated identical allelic profiles in NE and nonendocrine cells of prostatic carcinomas.420 These findings support the
Figure 10-51 Merkel cell carcinoma. Immunoperoxidase stain for cytokeratin 20 shows a dotlike pattern of staining.
Endocrine Tumors in Other Sites
have reported the expression of terminal deoxynucleotidyl transferase (TdT) in as many as 70% of Merkel cell carcinomas.433,434 In addition to TdT, Pax-5, a B-cell specific activation protein, is commonly expressed in these tumors.435 Some studies have shown CD117 (KIT receptor) positivity in as many as 95% of cases.436-438 Pulmonary small cell carcinomas may also be positive for CD117.436 Nuclear localization of E-cadherin immunoreactivity has been reported in Merkel cell carcinomas and may have diagnostic utility for this entity.439,440 An additional approach to the distinction of Merkel cell tumors from small cell pulmonary carcinomas involves the use of antibodies to MASH. Although more than 80% of small cell pulmonary carcinomas are MASH positive, only 1 of 30 Merkel cell carcinomas was positive for this marker.441 Merkel cell carcinomas also express the K homology domain–containing protein KOC, similar to other high-grade NE malignancies.442 MOLECULAR APPROACHES AND THERANOSTICS
Recent identification of Merkel cell polyomavirus DNA and Merkel cell polyomavirus large T-antigen expression in a proportion of Merkel cell carcinomas has suggested viral-induced oncogenesis.443-446 Subsequent studies have demonstrated IHC expression by using CM2B4, a monoclonal antibody that recognizes a Merkel cell polyomavirus–associated T antigen, in the majority (63% to 77%) of Merkel cell carcinomas, whereas no staining was seen in non–Merkel cell carcinomas and combined (NE and non-NE elements) Merkel cell carcinomas.446,447 Positivity for CD117 in these tumors has led to investigations of the efficacy of imatinib (Gleevec), a specific inhibitor of tyrosine kinases.448 This agent has been shown to decrease proliferation of Merkel cell carcinoma cells in vitro.449
Breast NE tumors of the breast include carcinoids and NECs of the small cell type (Fig. 10-52).450-453 These tumors,
A
361
as well as breast carcinomas with focal NE differentiation, are thought by some to arise from NE cells that form a small, intrinsic component of the normal breast epithelium; others claim that there are no such indigenous cell types in the breast.454,455 According to the WHO’s Classification of Tumours of the Breast (2012), primary mammary carcinomas with NE features are recognized as a special type of invasive breast carcinoma.456 This category includes NE tumor (well differentiated), NEC (small cell carcinoma), and carcinoma with NE differentiation. Carcinomas with NE differentiation determined by histochemical and IHC studies can be seen in up to 30% of invasive carcinomas of no special type and other special types, in particular mucinous carcinoma and solid papillary carcinoma.456 Small cell carcinomas are typically positive for cytokeratins that include AE1/AE3 (91%), CAM5.2 (82%), CK7 (78%), and CK19 (78%), but stains for CK20 are negative (see Table 10-6).457 These tumors may not exhibit a typical NE phenotype, and immunoreactivity for various markers has been reported as variable. It should be stressed, however, that the diagnosis of mammary small cell carcinoma is not contingent upon the demonstration of NE differentiation by IHC as is true for small cell carcinomas of other sites, such as the lung.457 Among NE markers, NSE is present in 90% to 100%, whereas synaptophysin and chromogranin occur in 56% and 41%, of cases respectively (Fig. 10-52, B); CD56 and CD57 have been noted in 78% and 43% of cases respectively. Estrogen and progesterone receptors are expressed in the majority of well-differentiated tumors and in more than half of the poorly differentiated examples.457 Immunoreactivity for calcitonin occurs in 27% of cases, whereas stains for gastrin-releasing peptide and serotonin have been reported in 39% and 14% of cases respectively. Estrogen and progesterone receptor positivity have been reported in 54% and 45% respectively. Bcl-2 is consistently positive, but stains for HER-2/neu are negative.457 In one study, CD99 was reported in 100% (3/3) of cases of small cell carcinomas, and cell membrane immunoreactivity for E-cadherin was reported in 92% (11/12) of reported cases.458-460
B
Figure 10-52 A, Small cell (neuroendocrine) carcinoma of the breast (hematoxylin and eosin). B, Immunoperoxidase stain for chromogranin A shows moderately intense cytoplasmic staining.
362
Immunohistology of Endocrine Tumors
MOLECULAR APPROACHES
Weigelt and colleagues461 analyzed 113 special types of breast cancer, including NECs, by gene-expression profiling that included hierarchical clustering analysis in an attempt to refine breast cancer classification and improve patient stratification. Results from this study included the identification of a family of cancers with endocrine/ NE features that exist within the larger group of “luminal” cancers. The luminal type is part of three major subtypes, the other being basal-like and HER-2/neu–positive, which comprise a widely applied molecular classification of breast cancers by gene-expression profiling that are distinct in their transcriptomic features and patient outcomes.461-463 The results of Weigelt and colleagues suggest that the panel of subtypes as defined by WHO criteria can be condensed, and thus simplified, based on their molecular profiles. Ideally, this would lead to refinement in prognostication and development of tailored therapies for breast cancer patients.461,464
Thymus Thymic NE tumors include carcinoids, atypical carcinoids, and small cell carcinomas. Morphologically, individual tumors often show admixtures of these growth patterns.465-467 They are typically positive for cytokeratins AE1/AE3 and CAM5.2465-467 and express a wide variety of NE markers that include NSE (100%), synaptophysin (81%), chromogranins (75%), and CD57 (67.5%). They may also be positive for glycoprotein and peptide hormones and amines, including α-hCG (100%), β-hCG (37.5%), somatostatin (36%), ACTH (32%), cholecystokinin (18%), calcitonin (11%), serotonin (5%), and CGRP (25%).336,467-471 In contrast to pulmonary NE tumors, thymic NE tumors are negative for TTF-1.374,375 More recently described thymic epithelial markers CD205 (DEC-205) and FOXN1 were negative in two thymic NECs in contrast to thymomas and thymic carcinomas.472
The chromosomal aberrations detected in the single MEN 1–associated case were dissimilar from those of the sporadic cases. Additionally, no evidence of 11q13 deletion was found on the locus of the MEN1 gene; this is in marked contrast to the high frequency of allelic losses on 11q in both sporadic and MEN-associated NE tumors in other (foregut) sites.2 These findings support the fundamental molecular divergence between thymic and other foregut NE tumors. Theoretically, molecular characterization could be used in a diagnostic setting to distinguish a primary thymic NE tumor from a metastasis of a nonthymic foregut primary. Rieker and coworkers474 evaluated 10 thymic NE tumors (5 atypical carcinoids, 4 typical carcinoids, 1 spindle cell carcinoid) by IHC and comparative genomic hybridization. Chromosomal imbalances were detected in 8 of 10 cases with the most frequent gains on chromosome Xp and 7p, 7q, 11q, 12q, and 20q. Losses were most frequently found in 6q, 6p, 4q, 3p, 10q, 11q, and 13q. These results demonstrated a degree of overlap with chromosomal imbalances commonly observed in advanced thymomas, suggesting a genetic/ evolutionary relationship between thymic NE and non-NE tumors.
Summary Even though the range of NE neoplasms in various sites has common themes, the immunohistologic profiles tend to be unique to the various anatomic sites. The challenge remains to recognize tumors by standard histologic stains, along with clinical data, and to apply appropriate panels of IHC as described earlier to arrive at a correct diagnosis. Genomic and theranostic applications of NE tumors are taking on a new, important role in patient management, and molecular data may be extremely useful to enhance diagnostic accuracy and prognostic assessment.
MOLECULAR APPROACHES
Acknowledgements
Few studies have investigated the molecular profiles of thymic NE tumors. Using comparative genomic hybridization, Pan and colleagues473 studied 11 tumors (10 sporadic; 1 MEN 1 associated). Genomic alterations were identified in nine cases with gains in chromosomes X, 8, 18, and 20p and losses in chromosomes 6,13q, 13p9q, and 11q.
We thank Ms. Joanne Harker for her help in the preparation of this manuscript. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Erickson LA, Lloyd RV: Practical markers used in the diagnosis of endocrine tumors. Adv Anat Pathol. 11:175–189, 2004. 2. DeLellis RA: Endocrine tumors. In Colvin RB, Bhan AK, McCluskey RT, editors: Diagnostic Immunopathology, New York, 1995, Raven Press, pp 551–578. 3. DeLellis RA, Wolfe HJ: Analysis of gene expression in endocrine cells. In Fenoglio-Preiser CM, Williman CL, editors: Molecular Diagnostics in Pathology, Baltimore, 1991, Williams & Wilkins, pp 299–323. 4. Lloyd RV: Practical markers used in the diagnosis of NE tumors. Endocr Pathol. 14:293–301, 2003. 5. Portela-Gomes GM, Stridsberg M, Grimelius L, et al: Expression of five different somatostatin receptor subtypes in endocrine cells of the pancreas. Appl Immunohistochem. 8:126–132, 2000. 6. DeLellis RA, Tischler AS: The dispersed NE cell system. In Kovacs K, Asa SL, editors: Functional Endocrine Pathology, Oxford, 1998, Blackwell Science, pp 529–549. 7. Lauweryns JM, Van Ranst L: Immunocytochemical localization of aromatic l–amino acid decarboxylase in human, rat and mouse bronchopulmonary and gastrointestinal endocrine cells. J Histochem Cytochem. 36:1181–1186, 1988. 8. Lloyd RV, Sisson JC, Shapiro B, et al: Immunohistochemical localization of epinephrine, norepinephrine, catecholaminesynthesizing enzymes and chromogranin in NE cells and tumors. Am J Pathol. 125:45–54, 1986. 9. Lloyd RV, Jin L, Qian X, et al: Analysis of the chromogranin A post-translational cleavage product pancreastatin and the prohormone convertases PC2 and PC3 in normal and neoplastic human pituitaries. Am J Pathol. 146:1188–1198, 1995. 10. Scopsi L, Gullo M, Rilke F, et al: Proprotein convertases (PC1/ PC3 and PC2) in normal and neoplastic tissues: Their use as markers of NE differentiation. J Clin Endocrinol Metab. 80:294– 301, 1995. 11. Scopsi L, Lee R, Gullo M, et al: Peptidylglycine, a amidating monooxygenase in NE tumors: Its identification, characterization, quantification and relation to the grade of morphologic differentiation, amidated peptide content and granin immunocytochemistry. Appl Immunohistochem. 6:120–132, 1998. 12. Schmechel D, Marangos PJ, Brightman M: Neurone-specific enolase is a molecular marker for peripheral and central NE cells. Nature. 276:834–836, 1978. 13. Haimoto H, Takahashi Y, Koshikawa T, et al: Immunohistochemical localization of gamma enolase in normal human tissues other than nervous and NE tissue. Lab Invest. 52:257–263, 1985. 14. Schmechel DE: Gamma submit of the glycolytic enzyme enolase: Nonspecific or neuron specific? Lab Invest. 52:239–242, 1985. 15. Rode J, Dhillon AP, Doran JF, et al: PGP 9.5, a new marker for human NE tumors. Histopathology. 9:147–158, 1985. 16. Li GL, Farooque M, Holtz A, et al: Expression of the ubiquitin carboxyl-terminal hydrolase PGP 9.5 in axons following spinal cord compression trauma. APMIS. 105:384–390, 1997. 17. Wilson PO, Barber PC, Hamid QA, et al: The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol. 69:91–104, 1988. 18. Bordi C, Pilato FP, D’Adda T: Comparative study of several NE markers in pancreatic endocrine tumors. Virchows Arch A Pathol Anat Histopathol. 413:387–398, 1988. 19. Tezel E, Hibi K, Nagasaka T, et al: PGP9.5 as a prognostic factor in pancreatic cancer. Clin Cancer Res. 6:4764–4767, 2000. 20. Mendelsohn G: Histaminase localization in medullary thyroid carcinoma and small cell lung carcinoma. In DeLellis RA, editor: Diagnostic Immunohistochemistry, New York, 1981, Masson, pp 299–312. 21. Savin S, Cvejic D, Isic T, et al: Thyroid peroxidase immunohistochemistry in differential diagnosis of thyroid tumors. Endocr Pathol. 17:53–56, 2006. 22. Sasano H, Mason JI, Sasano N: Immunohistochemical analysis of cytochrome P450 17 alpha in human adrenocortical disorders. Hum Pathol. 20:113–117, 1989. 23. Sasano H, Okamoto M, Sasano N: Immunohistochemical study of cytochrome P-450 11B hydroxylase in human adrenal cortex with- mineralo- and glucocorticoid excess. Virchows Arch A Pathol Anat Histopathol. 413:313–318, 1988.
362.e1
24. Sasano H, Okamoto M, Mason JI, et al: Immunohistochemical studies of steroidogenic enzymes (aromatase, 17 alphahydroxylase and cholesterol side chain cleavage cytochrome P450) in sex cord stromal tumors of the ovary. Hum Pathol. 20:452–457, 1989. 25. Nagasaki S, Suzuki T, Miki Y, et al: 17B-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis. Cancer Res. 69:(4):1392–1399, 2009. 26. Kulke MH, Hornick JL, Frauenhoffer C, et al: O6-Methylguanine DNA Methyltransferase Deficiency and Response to Temozolomide-Based Therapy in Patients with Neuroendocrine Tumors. Clin Cancer Res. 15(1):338–345, 2009. 27. Lloyd RV, Wilson BS: Specific endocrine tissue marker defined by a monoclonal antibody. Science. 222:628–630, 1983. 28. O’Connor DT, Burton D, Deftos LJ: Chromogranin A: Immunohistology reveals its universal occurrence in normal polypeptide hormone producing endocrine glands. Life Sci. 33:1657–1663, 1983. 29. Wilson BS, Lloyd RV: Detection of chromogranin in NE cells with a monoclonal antibody. Am J Pathol. 115:458–468, 1984. 30. Hagn C, Schmid KW, Fischer-Colbrie R, et al: Chromogranin A, B, and C in human adrenal medulla and endocrine tissues. Lab Invest. 55:405–411, 1986. 31. Huttner WB, Gerdes H-H, Rosa P: Chromogranins/ secretogranins—widespread constituents of the secretory granule matrix in endocrine cells and neurons. In Langley K, Gratzl M, editors: Markers for Neural and Endocrine Cells: Molecular and Cell Biology, Weinhein, 1991, VCH. 32. Taupenot L, Harper KL, O’Connor DT: The Chromogranin– Secretogranin Family. N Engl J Med. 348:1134–1149, 2003. 33. Portela-Gomes MG, Hacker GW, Weitgasser R: NE cell markers for pancreatic islets and tumors. Appl Immunohistochem Mol Morphol. 12:183–192, 2004. 34. Fahrenkamp AG, Wibbeke C, Winde G: Immunohistochemical distribution of chromogranins A and B and secretogranin II in NE tumors of the gastrointestinal tract. Virchows Arch. 426:361– 367, 1995. 35. Schmid KW, Kroll M, Hittmair A, et al: Chromogranin A and B in adenomas of the pituitary: An immunohistochemical study of 42 cases. Am J Surg Pathol. 15:1072–1077, 1991. 36. Fischer-Colbrie R, Eder S, Lovisetti-Scamihorn P, et al: NE secretory protein-55: a novel marker for the constitutive secretory pathway. Ann NY Acad Sci. 971:317–322, 2002. 37. Srivastava A, Padilla O, Fischer-Collbrie R, et al: NE secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids. Am J Surg Pathol. 28:1371–1378, 2004. 38. Srivastava A, Hornick JL: Immunohistochemical staining for CDX2, PDX-1, NESP-55 and TTF-7 can help distinguish gastrointestinal carcinoid tumors from pancreatic endocrine and pulmonary carcinoid tumors. Am J Surg Pathol. 2008. American Journal of Surgical Pathology. 33(4):626–632, 2009. 39. Gould VE, Lee I, Wiedenmann B, et al: Synaptophysin: A novel marker for neurons, certain NE cells and their neoplasms. Hum Pathol. 17:979–983, 1986. 40. Komminoth P, Roth J, Schroder S, et al: Overlapping expression of immunohistochemical markers and synaptophysin mRNA in pheochromocytomas and adrenocortical carcinomas: Implications for the differential diagnosis of adrenal gland tumors. Lab Invest. 72:424–431, 1995. 41. Portela-Gomes GM, Lukinius GM, Grimelius L: Synaptic vesicle protein 2: A new NE cell marker. Am J Pathol. 157:1299–1309, 2000. 42. Bumming P, Nilsson O, Ahlman H, et al: Gastrointestinal stromal tumors regularly express synaptic vesicle proteins: evidence of a NE phenotype. Endocr Relat Cancer. 14:835–863, 2007. 43. Jakobsen AM, Anderson P, Saglik G, et al: Differential expression of vesicular monoamine transporter (VMAT) 1 and 2 in gastrointestinal endocrine tumors. J Pathol. 195:463–472, 2001. 44. Gut A, Kiraly CE, Fukuda M, et al: Expression and localization of synaptotagmin isoforms in endocrine beta cells: their function in insulin exocytosos. J Cell Sci. 114;1709–1716, 2001.
362.e2
Immunohistology of Endocrine Tumors
45. Adolfsen B, Saraswati S, Yoshihara M, et al: Synaptotagmins are trafficked to distinct subsellular domains including the post synaptic compartment. J Cell Bol. 166:249–260, 2004. 46. Regazzi R, Wolheim CB, Lang J, et al: VAMP-2 and cellubrevin are expressed in pancreatic beta cells and are essential for Ca(2+) but not for GTP gamma S-induced insulin secretion. EMBO J. 14:2723–2730, 1995. 47. Grabowski P, Schönfelder J, Ahnert-Hilger G, et al: Heterogeneous expression of NE marker proteins in human undifferentiated carcinoma of the colon and rectum. Ann NY Acad Sci. 1014:270–274, 2008. 48. Arber DA, Weirs LM: CD57: A review. Appl Immunohistochem. 3:137–152, 1995. 49. McGarry RC, Helfand SL, Quarles RH, et al: Recognition of the myelin associated glycoprotein by the monoclonal antibody HNK-1. Nature. 306:376–378, 1983. 50. Tischler AS, Mobtaker H, Mann K, et al: Anti-lymphocyte antibody Leu 7 (HNK-1) recognizes a constituent of NE granule matrix. J Histochem Cytochem. 34:1213–1216, 1986. 51. Ghali VS, Jimenez EJS, Garcia RL: Distribution of Leu 7 antigen (HNK-1) in thyroid tumors: Its usefulness as a diagnostic marker for follicular and papillary carcinomas. Hum Pathol. 23:21–25, 1992. 52. Langley K, Gratzl M: Neural cell adhesion molecule (NCAM) in neural and endocrine cells. In Langley K, Gratzl M, editors: Markers for Neural and Endocrine Cells: Molecular and Cell Biology. Diagnostic Applications, Weinheim, 1991, VCH, pp 133–177. 53. Kapur P, Rakheja D: Immunohistochemical expression of neural cell adhesion molecule in Wilms tumors, nephrogenic rests, and fetal and postnatal renal cortices. Pediatr Dev Pathol. 14(1):16– 18, 2011. 54. Shipley WR, Hammer RD, Lennington WJ, et al: Paraffin immunohistochemical detection of CD56, a useful marker for neural cell adhesion molecule in normal and neoplastic fixed tissues. Appl Immunohistochem. 5:87–93, 1997. 55. Jin L, Hemperly JJ, Lloyd RV: Expression of neural cell adhesion molecule in normal and neoplastic human NE tissues. Am J Pathol. 138:961–969, 1991. 56. Komminoth P, Roth J, Saremaslani P, et al: Polysialic acid of the neural cell adhesion molecule in the human thyroid: A marker for medullary thyroid carcinoma and primary C-cell hyperplasia: An immunohistochemical study on 79 thyroid lesions. Am J Surg Pathol. 18:399–411, 1994. 57. Komminoth P, Roth J, Lackie PM, et al: Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids. Am J Pathol. 139:297–304, 1991. 58. Fuchs E, Weber K: Intermediate filaments: Structure, dynamics, function and disease. Ann Rev Biochem. 63:345–382, 1994. 59. Hoefler H, Denk H, Lackinger E, et al: Immunocytochemical demonstration of intermediate filament cytoskeleton proteins in human endocrine tissues and (neuro-) endocrine tumors. Virchows Arch A Pathol Anat Histopathol. 409:609–626, 1986. 60. Wang NP, Zee S, Zarbo RJ, et al: Coordinate expression of cytokeratins 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem. 3:99–107, 1995. 61. Shen WJ, Zaidi SK, Patel S, et al: Ablation of vimentin results in defective steroidogenesis. Endocrinology. 153(7):3249–3257, 2012. 62. Trojanowski JQ, Lee VM, Schlaepfer WW: An immunohistochemical study of human central and peripheral nervous system tumors, using monoclonal antibodies against neurofilaments and glial filaments. Hum Pathol. 15:248–257, 1984. 63. Perez MA, Saul SH, Trojanowski JQ: Neurofilament and chromogranin expression in normal and neoplastic NE cells of the human gastrointestinal tract and pancreas. Cancer. 65:1219– 1227, 1990. 64. Kulig E, Lloyd RV: Transcription factors and endocrine disease. Endocr Pathol. 1:245–250, 1996. 65. Gradwohl G, Dierich A, LeMeur M, et al: Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci USA. 97(4):1607–1611, 2000. 66. Viger RS, Guittot MS, Anttonen M, et al: Role of the GATA Family of Transcription Factors in Endocrine Development, Function, and Disease. Mol Endocrinol. 22(4):781–798. 67, 2008.
67. Reubi JC, Kappeler A, Waser B, et al: Immunohistochemical localization of somatostatin receptors sst2A in human tumors. Am J Pathol. 153:233–245, 1998. 68. Erickson LA, Jin L, Wollen P, et al: Parathyroid hyperplasia, adenomas and carcinomas: differential expression of p27 Kip1 protein. AM J Surg Pathol. 23:288–295, 1999. 69. LaRosa S, Sessa F, Capella C, et al: Prognostic criteria in non-functioning pancreatic endocrine tumors. Virchows Arc. 429:323–333, 1996. 70. DeLellis RA: Proliferation markers in NE tumors: useful or useless? A critical reappraisal. Verh Dtsch Ges Pathol. 81:53–61, 1997. 71. Vosse BA, Seelentag W, Bachmann A, et al: Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin complex system and the EnVision + system. Appl Immunohistochem Mol Morphol. 15:103–107, 2007. 72. Asa SL: Atlas of Tumor Pathology: Tumors of the Pituitary Gland, Washington, D.C., 1998, Armed Forces Institute of Pathology. 73. Girod C, Trouillar J, Dubois MP: Immunocytochemical localization of S100 protein in stellate cells (folliculo-stellate cells) of the anterior lobe of the normal human pituitary. Cell Tissue Res. 241:505–511, 1985. 74. Mete O, Asa SL: Clinicopathological correlations in pituitary adenomas. Brain Pathol. 22:443–453, 2012. 75. Lloyd RV, Cano M, Rosa P, et al: Distribution of chromogranin A and secretogranin I (chromogranin B) in NE cells and tumors. Am J Pathol. 130:296–304, 1988. 76. Hofler H, Walter GF, Denk H: Immunohistochemistry of folliculo-stellate cells in normal human adenohypophyses and in pituitary adenomas. Acta Neuropathol (Berl). 65:35–40, 1994. 77. Scheitauer BW, Kovacs K, Horvath E, et al: Pituitary carcinoma. In DeLellis RA, Lloyd RV, Heitz P, et al, editors: Tumours of the Endocrine Organs. WHO Classification of Tumours, Lyon, 2004, IRC, pp 36–39. 78. Thappar K, Kovacs K, Scheithauer BW, et al: Proliferative activity and invasiveness among pituitary adenomas and carcinomas: an analysis using the MIB-1 antibody. Neurosurg. 38:99–106, 1996. 79. Thappar K, Schaithauer BW, Kovacs K, et al: p53 expression in pituitary adenomas and carcinomas: correlation with invasiveness and tumor growth fractions. Neurosurg. 38:765–771, 1996. 80. Asa SL, Ezzat S: Genetics and proteomics of pituitary tumors. Endocrine. 28 (1): 43–37, 2005. 81. Wierinckx A, Auger C, Devauchelle P, et al: A diagnostic marker set for invasion, proliferation and aggressiveness of prolactin pituitary tumors. Endocr Relat Cancer. 14:887–900, 2007. 82. Riss D, Jim L, Qian X, et al: Differential expression of galectin-3 in pituitary tumors. Cancer Res. 63:2251–2255, 2003. 83. Righi A, Jin L, Zhang S, et al: Identification and consequences of galectin-3 expression in pituitary tumors. Mol Cell Endocrinol. 326:8–14, 2010. 84. Wang EL, Qian ZR, Rahman MM, et al: Increased expression of HMGA1 correlates with tumour invasiveness and proliferation in human pituitary adenomas. Histopathol. 56:501–509, 2010. 85. Asa SL: Practical pituitary pathology: what does the pathologist need to know? Arch Pathol Lab Med. 132:1231–1240, 2008. 86. Burger PC, Scheithauer BW: Atlas of Tumor Pathology: Tumors of the Central Nervous System. Series 4, Washington, D.C., 2007, American Registry of Pathology in collaboration with Armed Forces Institute of Pathology. 87. Coca S, Vaquero J, Escandon J, et al: Immunohistochemical characterization of pineocytomas. Clin Neuropathol. 11:298– 303, 1992. 88. Hayashi K, Hoshida Y, Horie Y, et al: Immunohistochemical study on the distribution of a and b subunits of S100 protein in brain tumors. Acta Neuropathol (Berl). 81:657–663, 1991. 89. Korf HW, Klein DC, Zigler JS, et al: S-antigen-like immunoreactivity in a human pineocytoma. Acta Neuropathol (Berl). 69:165–167, 1986. 90. Perentes E, Rubinstein LJ, Herman MM, et al: S-antigen immunoreactivity in human pineal glands and pineal parenchymal tumors: A monoclonal antibody study. Acta Neuropathol (Berl). 71:224–227, 1986. 91. Arivazhagan A, Anandh B, Santash V, et al: Pineal parenchymal tumors-utility of immunohistochemical markers in prognostication. Clin Neuropathol. 27:325–333, 2008.
References 92. Santagata S, Maire CL, Idbaih A, et al: CRX is a diagnostic marker of retinal and pineal lineage tumors. PLoS ONE. 4:e7932, 2009. 93. Terry J, Calicchio ML, Rodriguez-Galindo C, et al: Immunohistochemical expression of CRX in extracranial malignant small round cell tumors. Am J Surg Pathol. 36:1165–1169, 2012. 94. Bocker W, Dralle H, Dorn G: Thyroglobulin: An immunohistochemical marker in thyroid disease. In DeLellis RA, editor: Diagnostic Immunohistochemistry, New York, 1981, Masson, pp 37–60. 95. Sambade C, Franssila K, Cameselle-Teijeiro J, et al: Hyalinizing trabecular adenoma: A misnomer for a peculiar tumor of the thyroid gland. Endocr Pathol. 2:83–91, 1991. 96. Hirokawa M, Carney JA: Cell membrane and cytoplasmic staining for MIB-1 in hyalinizing trabecular adenoma of the thyroid. Am J Surg Pathol. 24:575–578, 2000. 97. Leonardo E, Volante M, Barbareschi M, et al: Cell membrane reactivity of MIB-1 antibody to Ki-67 in human tumors: fact or artifact? Appl Immunohistochem Mol Morphol. 15:220–223, 2007. 98. Berge-Lefranc JL, Cartouzou G, DeMicco C, et al: Quantification of thyroglobulin messenger RNA by in-situ hybridization in differentiated thyroid cancers: Difference between welldifferentiated and moderately differentiated histologic types. Cancer. 56:345–350, 1985. 99. Keen CE, Szakacs S, Okon E, et al: CA125 and thyroglobulin staining in papillary carcinomas of thyroid and ovarian origin is not entirely specific for site of origin. Histopathology. 34:113– 117, 1999. 100. Carcangiu ML, Zampi G, Rosai J: Poorly differentiated (“insular”) thyroid carcinoma: A reinterpretation of Lang hans’ “wuchernde Struma.” Am J Surg Pathol. 8:655–668, 1984. 101. Ordonez NG, El-Naggar AK, Hickey RC, et al: Anaplastic thyroid carcinoma: Immunocytochemical study of 32 cases. Am J Clin Pathol. 96:15–24, 1991. 102. Carcangiu ML, Steeper T, Zampi G, et al: Anaplastic thyroid cancer: A study of 70 cases. Am J Clin Pathol. 83:135–158, 1985. 103. Kawaoi A, Okano T, Nemoto N, et al: Simultaneous detection of thyroglobulin, (Tg) thyroxine (T4) and tri-iodothyronine (T3) in nontoxic thyroid tumors by the immunoperoxidase method. Am J Pathol. 108:39–49, 1982. 104. DeMicco C, Ruf J, Chrestian MA, et al: Immunohistochemical study of thyroid peroxidase in normal, hyperplastic, and neoplastic human thyroid tissues. Cancer. 67:3036–3041, 1991. 105. Katoh R, Kawaoi A, Miyagi E, et al: Thyroid transcription factor 1 in normal, hyperplastic and neoplastic thyroid follicular thyroid cells examined by immunohistochemistry and nonradioactive in situ hybridization. Mod Pathol. 13:570–576, 2000. 106. Lau SK, Luthringer DJ, Eisen RN: Thyroid transcription factor-1: a review. Appl Immunohistochem Mol Morphol. 10:97–102, 2002. 107. Ordonez NG: Thyroid transcription factor 1 is a marker of lung and thyroid carcinomas. Adv Anat Pathol. 7:123–127, 2000. 108. Kaufmann O, Dietel M: Thyroid transcription factor-1 is the superior immunohistochemical marker for pulmonary adenocarcinomas and large cell carcinomas compared to surfactant proteins A and B. Histopathology. 36:8–16, 2000. 109. Byrd-Gloster AL, Khoor A, Glass LF, et al: Differential expression of thyroid transcription factor-1 in small cell lung carcinoma and Merkel cell tumor. Hum Pathol. 31:58–62, 2000. 110. Agoff SN, Lamps LW, Philip AT, et al: Thyroid transcription factor-1 is expressed in extrapulmonary small cell carcinomas but not in other extrapulmonary NE tumors. Mod Pathol. 13: 238–242, 2000. 111. Nonaka D, Tang Y, Chiriboga L, et al: Diagnostic utility of thyroid transcription factors PAX8 and TTF2 (Fox E1) in thyroid epithelial neoplasms. Mod Pathol. 21:192–200, 2008. 112. Henzen-Logmans SC, Mullink H, Ramaekers FC, et al: Expression of cytokeratins and vimentin in epithelial cells of normal and pathologic thyroid tissue. Virchows Arch A Pathol Anat Histopathol. 410:347–354, 1987. 113. Miettinen M, Franssila K, Lehto V-P, et al: Expression of intermediate filament proteins in thyroid gland and thyroid tumors. Lab Invest. 50:262–270, 1984. 114. Viale G, Dell’Orto P, Coggi G, et al: Co-expression of cytokeratins and vimentin in normal and diseased thyroid glands: Lack
362.e3
of diagnostic utility of vimentin immunostaining. Am J Surg Pathol. 13:1034–1040, 1989. 115. Schelfhout LJDM, van Muijen GN, Fleuren GJ: Expression of keratin 19 distinguishes papillary thyroid carcinoma from follicular carcinomas and follicular thyroid adenoma. Am J Clin Pathol. 92:654–658, 1989. 116. Raphael SJ, McKeown-Eyssen G, Asa SL: High molecular weight cytokeratin and cytokeratin 19 in the diagnosis of thyroid tumors. Mod Pathol. 7:295–300, 1994. 117. Fonseca E, Nesland JM, Hoie J, et al: Pattern of expression of intermediate cytokeratin filaments in the thyroid gland: An immunohistochemical study of simple and stratified epithelialtype cytokeratins. Virchows Arch. 430:239–245, 1997. 118. Miettinen M, Kovatich AJ, Karkkainen P: Keratin subsets in papillary and follicular thyroid lesions: A paraffin section analysis with diagnostic implications. Virchows Arch. 431:407–413, 1997. 119. Kragsterman B, Grimelius L, Wallin G, et al: Cytokeratin 19 expression in papillary thyroid carcinoma. Appl Immunohistochem. 7:181–185, 1999. 120. Baloch ZW, Abraham S, Roberts S, et al: Differential expression of cytokeratins in follicular variant of papillary carcinoma: An immunohistochemical study and its diagnostic utility. Hum Pathol. 30:1166–1171, 1999. 121. Fonseca E, Nesland J, Sobrinho-Simoes M: Expression of stratified epithelial cytokeratins in hyalinizing trabecular adenoma supports their relationship with papillary carcinoma of the thyroid. Histopathology. 31:330–335, 1997. 122. Hirokawa M, Carney JA, Ohtsuki Y: Hyalinizing trabecular adenoma and papillary carcinoma of the thyroid express different cytokeratin patterns. Am J Surg Pathol. 24:877–881, 2000. 123. Liberman E, Weidner N: Papillary and follicular neoplasms of the thyroid gland: Differential immunohistochemical staining with high molecular weight keratin and involucrin. Appl Immunohistochem. 8:42–48, 2000. 124. Sahoo S, Hoda SA, Rosai J, et al: Cytokeratin 19 immunoreactivity in the diagnosis of papillary thyroid carcinoma: A note of caution. Am J Clin Pathol. 116:695–702, 2001. 125. Soares P, Cameselle-Teijeiro J, Sobrinho-Simoes M: Immunohistochemical detection of p53 in differentiated, poorly differentiated and undifferentiated carcinomas of the thyroid. Histopathology. 24:205–210, 1994. 126. Holm R, Nesland JM: Retinoblastoma and p53 tumor suppressor gene protein expression in carcinomas of the thyroid gland. J Pathol. 172:267–272, 1994. 127. Tallini G, Santoro M, Helie M, et al: RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes. Clin Cancer Res. 4:287–294, 1998. 128. Papotti M, Volante M, Guiliano A, et al: RET/PTC activation in hyalinizing trabecular tumors of the thyroid. Am J Surg Pathol. 24:1615–1621, 2000. 129. Cheung CC, Boerner SL, MacMillan CM, et al: Hyalinizing trabecular tumor of tthe thyroid: A variant of papillary carcinoma proved by molecular genetics. Am J Surg Pathol. 24:1622– 1626, 2000. 130. LiVolsi VA: Hyalinizing trabecular tumor of the thyroid: Adenoma, carcinoma, or neoplasm of uncertain malignant potential? Am J Surg Pathol. 24:1683–1684, 2000. 131. Nikiforov YE, Ohori NP: Papillary carcinoma (Chapter 11). In Nikiforov YE, Biddinger PW, Thompson LDR, editors: Diagnostic Pathology and Molecular Genetics of the Thyroid, ed 2, Philadelphia, 2012, Wolters Kluwer/Lippincott Williams and Wilkins, pp 183–246. 132. Kroll TG, Sarraf P, Pecciarini L, et al: PAX8-PPAR gamma 1 fusion oncogene in human thyroid carcinoma. Science. 289:1357– 1360, 2000. 133. Nikiforov YE, Ohori NP: Follicular carcinoma (Chapter 10). In Nikiforov YE, Biddinger PW, Thompson LDR, editors: Diagnostic Pathology and Molecular Genetics of the Thyroid, ed 2, Philadelphia, 2012, Wolters Kluwer/Lippincott Williams and Wilkins, pp 152–182. 134. Nikiforova MN, Biddinger PW, Candill CM, et al: PAX8-PPAR gamma rearrangement in thyroid tumors: RT-PCR and immunohistochemical analyser. Am J Surg Pathol. 26:1016–1011-23, 2002.
362.e4
Immunohistology of Endocrine Tumors
135. Marques AR, Espadinha C, Catarino AL, et al: Expression of PAX8-PPAR gamma/rearrangements in both follicular thyroid carcinoma and adenoma. J Clin Endocrinol Metab. 87:3947– 3952, 2002. 136. Cheung L, Messina M, Gill A, et al: Detection of the PAX8PPAR gamma fusion oncogene in both folllicular thyroid carcinomas and adenomas. J Clin Endocrinol Metab. 88:354–357, 2003. 137. Gustafson KS, LiVolsi VA, Furth EE, et al: Peroxisome proliferator-activated receptor gamma expression in follicular patterned thyroid lesions. Caveats for the use of immunohistochemical studies. Am J Clin Pathol. 120:175–181, 2003. 138. Koporek O, Kornauth C, Capper D, et al: Immunohistochemical detection of the BRAF V600E-mutated protein in papillary thyroid carcinoma. Am J Surg Pathol. 36:844–850, 2012. 139. Bullock M, O’Neill C, Chou A, et al: Utilization of a MAB for BRAFV600E detection in papillary thyroid carcinoma. Endocr Relat Cancer. 19:779–784, 2012. 140. Sack MJ, Astengo-Osuna C, Lin BT, et al: HBME-1 immunostaining in thyroid fine needle aspirations: A useful marker in the diagnosis of carcinoma. Mod Pathol. 10:668–674, 1997. 141. Miettinen M, Karkkainen P: Differential HBME-1 reactivity in benign vs. malignant thyroid tissue is helpful in the diagnosis of thyroid tumors. [abstract] Mod Pathol. 5:50A, 1996. 142. Mase T, Funahashi H, Koshikawa T, et al: HBME-1 immunostaining in thyroid tumors especially in follicular neoplasms. Endocr J. 50:173–177, 2003. 143. Casey MB, Lohse CM, Lloyd RV: Distinction between papillary thyroid hyperplasia and papillary thyroid carcinoma by immunohistochemical staining for cytokeratin 19, galectin-3 and HBME-1. Endocr Pathol. 14:55–60, 2003. 144. Cheung CC, Ezzat S, Freeman JL, et al: Immunohistochemical diagnosis of papillary thyroid carcinoma. Mod Pathol. 14:338– 342, 2001. 145. Choi YL, Kim MK, Suh JW, et al: Immunoexpression of HBME-1, high molecular weight cytokeratin, cytokeratin 19, thyroid transcription factor-1 and E-cadherin in thyroid carcinoma. J Korean Med Sci. 20:853–859, 2005. 146. Fischer S, Asa S: Application of immunohistochemistry to thyroid neoplasms. Arch Pathol Lab Med. 132:359–372, 2008. 147. Xu XC, el-Naggar AK, Lotan R: Differential expression of galectin-1 and galectin-3 in thyroid tumors. Potential diagnostic implications. Am J Pathol. 147:815–822, 1995. 148. Fernandez PL, Merino MJ, Gomez M, et al: Galectin-3 and laminin expression in neoplastic and non-neoplastic thyroid tissue. J Pathol. 181:80–86, 1997. 149. Orlandi F, Saggiorato E, Pivano G, et al: Galectin-3 is a presurgical marker of human thyroid carcinoma. Cancer Res. 58:3015– 3020, 1998. 150. Herrmann ME, LiVolsi VA, Pasha TL, et al: Immunohistochemical expression of galectin 3 in benign and malignant lesions. Arch Pathol Lab Med. 126:710–713, 2002. 151. Bartolazzi A, Gasbarri A, Papotti M, et al: Application of an immunodiagnostic method for improving pre-operative diagnosis of nodular thyroid lesions. Lancet. 357:1644–1650, 2001. 152. Volante M, Bozzalla-Cassione F, DePompa R, et al: Galectin-3 and HBME-1 expression in oncocytic cell tumors of the thyroid. Virchows Arch. 445:183–188, 2004. 153. Wiseman SM, Melck A, Masoudi H, et al: Molecular phenotype of thyroid tumors identifiers: a marker panel for differentiated thyroid cancer diagnosis. Am Surg Oncol. 15:2811–2826, 2008. 154. Griffith OL, Chiu CG, Gown AM, et al: Biomarker panel diagnosis of thyroid cancer: a critical review. Expert Rev Anticancer Ther. 8:1399–1413, 2008. 155. Prasad ML, Pellegata NS, Kloos RT, et al: CITED1 expression suggests papillary thyroid carcinoma in high throughput tissue microarray-based study. Thyroid. 14:169–175, 2004. 156. Huang Y, Prasad M, Lemon WJ, et al: Gene expression in papillary thyroid carcinoma reveals highly consistent profiles. Proc Nat’l Acad Sci USA. 98:15044–15049, 2001. 157. Prasad ML, Pellegata NS, Huang Y, et al: Galectin-3, fibronectin-1, CITED1, HBME-1 and cytokeratin-19 immunohistochemistry is useful for the differential diagnosis of thyroid tumors. Mod Pathol. 18:48–57, 2005.
158. Sconamiglio T, Hyjek E, Kao J, et al: Diagnostic usefulness of HBME-1, galectin-3, CK19 and CITED1 and evaluation of their expression in encapsulated lesions with questionable features of papillary thyroid carcinoma. Am J Clin Pathol. 126:700–708, 2006. 159. Scheumman GF, Hoang-Vu C, Cetin Y, et al: Clinical significance of E-cadherin as a prognostic marker in thyroid carcinoma. J Clin Endocrinol Metab. 80:2168–2172, 1995. 160. Garcia-Rostan G, Camp RL Herreo A, et al: Beta catenin dysregulation in thyroid neoplasms: down regulation, aberrant nuclear expression, and CTNNB1 exon 3 mutations are markers for aggressive tumor phenotypes and poor prognosis. Am J Pathol. 158:987–996, 2001. 161. Wiseman SM, Griffith OL, Deen S, et al: Identification of molecular markers altered during transformation of differentiated into anaplastic thyroid carcinoma. Arch Surg. 142:717–729, 2007. 162. Dasovic-Knezevic M, Bormer O, Holm R, et al: Carcinoembryonic antigen in medullary thyroid carcinoma: An immunohistochemical study applying six novel monoclonal antibodies. Mod Pathol. 2:610–617, 1989. 163. Schroder S, Schwarz W, Rehpenning W, et al: Prognostic significance of Leu M1 immunostaining in papillary carcinomas of the thyroid gland. Virchows Arch A Pathol Anat Histopathol. 411:435–439, 1987. 164. Loy TS, Darkow GV, Spollen LE, et al: Immunostaining for Leu 7 in the diagnosis of thyroid carcinoma. Arch Pathol Lab Med. 118:172–174, 1994. 165. Ostrowski ML, Brown RW, Wheeler TM, et al: Leu 7 immunoreactivity in cytologic specimens of thyroid lesions with an emphasis on follicular neoplasms. Diagn Cytopathol. 12:297– 302, 1995. 166. Gatalica Z, Miettinen M: Distribution of carcinoma antigens CA19-9 and CA15-3: An immunohistochemical study of 400 tumors. Appl Immunohistochem. 2:205–211, 1994. 167. Alves P, Soares P, Fonseca E, et al: Papillary thyroid carcinoma overexpresses fully and underglycosylated mucins together with native and sialylated simple mucin antigens and histo-blood group antigens. Endocrine Pathol. 10:315–324, 1999. 168. Lee HM, Baek SK, Kwon SY, et al: Cyclooxygenase 1 and 2 expression in the human thyroid gland. Eur Arch Otorhinolaryngolo. 263:199–204, 2006. 169. Garcia-Gonzalez M, Abdulkader I, Boquete AV, et al: Cyclooxygenase 2 in normal hyperplastic and neoplastic follicular cells of the human thyroid gland. Vichows Arch. 447:12–17, 2005. 170. Ito Y, Yoshida H, Nakano K, et al: Cyclooxygenase-2 expression in thyroid neoplasms. Histopathol. 42:492–497, 2003. 171. Bur M, Shiraki W, Masood S: Estrogen and progesterone receptor detection in neoplastic and non-neoplastic thyroid tissue. Mod Pathol. 6:469–472, 1993. 172. McLaren KM, Cossar DW: The immunohistochemical localization of S-100 in the diagnosis of papillary carcinoma of the thyroid. Hum Pathol. 27:633–636, 1996. 173. Papotti M, Gugliotta P, Forte G, et al: Immunocytochemical identification of oxyphilic mitochondrion rich cells. Appl Immunohistochem. 2:261–267, 1994. 174. Nylander K, Vojtesek B, Nenutil R, et al: Differential expression of p63 isoforms in normal tissues and neoplastic cells. J Pathol. 198:417–427, 2002. 175. Hunt JL, LiVolsi VA, Barnes EL: p63 expression in sclerosing mucoepidermoid carcinomas with eosinophilia arising in the thyroid. Mod Pathol. 17:526–529, 2004. 176. Morari EC, Silva JR, Guilhen AC, et al: Muc1 expression may help characterize thyroid nodules but does not predict patients’ outcomes. Endocr Pathol. 21:242–249, 2010. 177. Bieche I, Ruffet E, Zweibaum A, et al: MUC1 mucin gene, transcripts, and protein in adenomas and papillary carcinomas of the thyroid. Thyroid. 7:725–731, 1997. 178. Nipp M, Elsner M, Balluff B, et al: S100-A10, thioredoxin, and S100-A6 as biomarkers of papillary thyroid carcinoma with lymph node metastasis identified by MALDI imaging. J Mol Med (Berl). 90:163–174, 2012. 179. DeLellis RA, Wolfe HJ: The pathology of the human calcitonin C- cell. Pathol Annu. 16:25–52, 1981. 180. Sikri KL, Varndell IM, Hamid QA, et al: Medullary carcinoma of the thyroid: An immunocytochemical and histochemical
References study of 25 cases using 8 separate markers. Cancer. 56:2481– 2491, 1985. 181. Perry A, Molberg K, Albores-Saavedra J: Physiologic versus neoplastic C-cell hyperplasia of the thyroid: Separation of distinct histologic and biologic entities. Cancer. 77:750–756, 1996. 182. Holm R, Sobrinho-Simoes M, Nesland JM, et al: Medullary carcinoma of the thyroid gland: An immunocytochemical study. Ultrastruct Pathol. 8:25–41, 1985. 183. Uribe M, Fenoglio-Preiser CM, Grimes M, et al: Medullary carcinoma of the thyroid gland: Clinical, pathological and immunohistochemical features with a review of the literature. Am J Surg Pathol. 9:577–594, 1985. 184. Zajac JD, Penschow J, Mason T, et al: Identification of calcitonin and calcitonin gene related peptide messenger RNA in medullary thyroid carcinoma by hybridization histochemistry. J Clin Endocrinol Metab. 62:1037–1043, 1986. 185. Franc B, Rosenberg-Bourgin M, Caillou B, et al: Medullary thyroid carcinoma: Search for histological predictors of survival (109 proband case analysis). Hum Pathol. 29:1078–1084, 1998. 186. Eusebi V, Damiani S, Riva C, et al: Calcitonin free oat cell carcinoma of the thyroid gland. Virchows Arch A Pathol Anat Histopathol. 417:267–271, 1990. 187. Steenbergh PH, Hoppener JW, Zandberg J, et al: Calcitonin gene related peptide coding sequence is conserved in the human genome and is expressed in medullary thyroid carcinoma. J Clin Endocrinol Metab. 59:358–360, 1984. 188. Scopsi L, Ferrari C, Pilotti S, et al: Immunocytochemical localization and identification of prosomatostatin gene products in medullary carcinoma of human thyroid gland. Hum Pathol. 21:820–830, 1990. 189. Sunday ME, Wolfe HJ, Roos BA, et al: Gastrin releasing peptide gene expression in developing, hyperplastic and neoplastic thyroid C-cells. Endocrinology. 122:1551–1558, 1988. 190. Heitz PU, von Herbay G, Kloppel G, et al: The expression of subunits of human chorionic gonadotropin (hCG) by nontrophoblastic, nonendocrine and endocrine tumors. Am J Clin Pathol. 88:467–472, 1987. 191. DeLellis RA, Moore FM, Wolfe HJ: Thyroglobulin immunoreactivity in human medullary thyroid carcinoma. Lab Invest. 48:20A, 1983. 192. Holm R, Sobrinho-Simoes M, Nesland JM, et al: Concurrent production of calcitonin and thyroglobulin by the same neoplastic cells. Ultrastruct Pathol. 10:241–248, 1986. 193. Ljungberg O, Bondeson L, Bondeson AG: Differentiated thyroid carcinoma, intermediate type: A new tumor entity with features of follicular and parafollicular cell carcinomas. Hum Pathol. 15:218–228, 1984. 194. Volante M, Papotti M, Roth J, et al: Mixed medullary follicular thyroid carcinoma: Molecular evidence for a dual origin of tumor components. Am J Pathol. 155:1499–1509, 1999. 195. Schmid KW, Fischer-Colbrie R, Hagn C, et al: Chromogranin A and B and secretogranin II in medullary carcinomas of the thyroid. Am J Surg Pathol. 11:551–556, 1987. 196. Katsetos CD, Jami MM, Krishna L, et al: Novel immuno histochemical localization of 28000 molecular weight (Mr) calcium binding protein (Calbindin-D28k) in enterochromaffin cells of the human appendix and NE tumors (carcinoids and small cell carcinomas) of the midgut and foregut. Arch Pathol Lab Med. 118:633–639, 1994. 197. Viale G, Roncalli M, Grimelius L, et al: Prognostic value of bcl-2 immunoreactivity in medullary thyroid carcinoma. Hum Pathol. 26:945–950, 1995. 198. DeLellis RA, Rule AH, Spiler I, et al: Calcitonin and carcinoembryonic antigen as tumor markers in medullary thyroid carcinoma. Am J Clin Pathol. 70:587–594, 1978. 199. Schroder S, Kloppel G: Carcinoembryonic antigen and nonspecific cross reacting antigen in thyroid cancer: An immunocytochemical study using polyclonal and monoclonal antibodies. Am J Surg Pathol. 11:100–108, 1987. 200. Mendelsohn G, Wills SA Jr, Baylin SB: Relationship of tissue carcinoembryonic antigen and calcitonin to tumor virulence in medullary thyroid carcinoma: An immunohistochemical study in early, localized and virulent disseminated stages of disease. Cancer. 54:657–662, 1984.
362.e5
201. Kimura N, Nakazato Y, Nagura H, et al: Expression of intermediate filaments in NE tumors. Arch Pathol Lab Med. 114:506–510, 1990. 202. Nikiforov YE: Thyroid carcinoma: molecular pathways and therapeutic targets. Mod Pathol. 21(Suppl 2):S37–S43, 2008. 203. Nikiforova MN, Nikiforov YE: Molecular genetics of thyroid cancer; implications for diagnostic treatment and prognosis. Expert Rev Mol Diagn. 8:830–895, 2008. 204. DeLellis RA: Pathology and genetics of thyroid carcinoma. J Surg Oncol. 94:662–669, 2006. 205. Ciampi R, Nikiforov YE: RET/PTC rearrangements and BRAF mutations in thyroid tumorigenesis. Endocrinol. 148:936–941, 2007. 206. Nikiforov Y: Genetic alterations involved in the transition from well to poorly differentiated and anaplastic carcinoma. Endocr Pathol. 15:319–328, 2004. 207. Grieco M, Santoro M, Berlingieri MT, et al: PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell. 60:557– 563, 1990. 208. Nikiforov YE, Rowland JM, Bove KE, et al: Destinct pattern of ret oncogene rearrangements in morphological variants of radiation induced and sporadic thyroid papillary carcinomas in children. Cancer Res. 57:1690–1694, 1997. 209. Pierotti MA, Borgarzone I, Borello MG, et al: Cytogenetics and molecular genetics of carcinomas arising from thyroid epithelial follicular cells. Genes Chromosomes Cancer. 16:1–14, 1996. 210. Kimura ET, Nikiforova MN, Zhu Z, et al: High prevalence of BRAP mutations in thyroid cancer. Genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signalling pathway in papillary thyroid carcinoma. Cancer Res. 63:1454–1457, 2003. 211. Soares P, Trovisco V, Rocha AS, et al: BRAF mutations and RET/ PTC rearrangements all alternative events in the etiopathogenesis of PTC. Oncogene. 22:4578–4580, 2003. 212. Nikiforova MN, Kimura ET, Gandhi M, et al: BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinoma. J Clin Endocrinol Metab. 88:5399–5404, 2003. 213. Zhu Z, Gandhi M, Nikiforova MN, et al: Molecular profile and clinical-pathological features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations. Am J Clin Pathol. 120:71–77, 2003. 214. Esapa CT, Johnson SJ, Kendall-Taylor P, et al: Prevalence of Ras mutations in thyroid neoplasia. Clin Endocrinol (Oxf). 50:529– 523, 1999. 215. Abu-Amero KK, Alzahrani AS, Zou M, et al: High frequency of somatic mitochondrial DNA mutations in human thyroid carcinomas and complex 1 respiratory defect in thyroid cancer cell lines. Oncogene. 24:1455–1460, 2005. 216. Botelho MV, Capela J, Soares P, et al: Somatic and germline mutation in GRIM-19, a dual function gene involved in mitochondrial metabolism and cell death, is linked to mitochondrionrich (Hurthle cell) tumours of the thyroid. Br J Cancer. 92:1817–1818, 2005. 217. Smallridge R, Marlow L, Copland J: Anaplastic thyroid cancer: molecular pathogenesis and emerging therapies. Endocr Relat Cancer. 2008 (epub). 218. Eng C: RET proto-oncogene in the development of human cancer. J Clin Oncol. 17:J:380–393, 1999. 219. Nikiforov YE: Molecular analysis of thyroid tumors. Mod Pathol. 24: S34–S43, 2011. 220. Nikiforova M, Nikiforov YE: Molecular testing of thyroid FNA samples (Chapter 21). In Nikiforov YE, Biddinger PW, Thompson LDR, editors: Diagnostic Pathology and Molecular Genetics of the Thyroid, ed 2, Philadelphia, 2012, Wolters Kluwer/Lippincott Williams and Wilkins, pp 425–429. 221. Finley DJ, Arora N, Zhu B, et al: Molecular profiling distinguishes papillary carcinoma from benign thyroid nodules. J Clin Endocrinol Metab. 89:314–3223, 2004. 222. Giordano TJ, Kuick R, Thomas DG, et al: Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, RET/PTC mutations-specific gene expression profiles discovered by DNA microarray analysis. Oncogene. 24:6646–6656, 2005.
362.e6
Immunohistology of Endocrine Tumors
223. Frattini M, Ferrario C, Bressan P, et al: Alternative mutations of BRAF, RET and NTRK1 are associated with similar but distinct gene expression patterns in papillary thyroid cancer. Oncogene. 23:7426–7440, 2004. 224. Alexander EK, Kennedy GC, Baloch ZW, et al: Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med. 367:705–715, 2012. 225. Ball DW: Medullary thyroid carcinoma: therapeutic targets and molecular markers. Current Opinions in Oncology. 19:18–23, 2007. 226. Miettinen M, Clark R, Lehto VP, et al: Intermediate filament proteins in parathyroid glands and parathyroid adenomas. Arch Pathol Lab Med. 109:986–989, 1985. 227. Futrell JM, Roth SI, Su SP, et al: Immunocytochemical localization of parathyroid hormone in bovine parathyroid glands and human parathyroid adenomas. Am J Pathol. 94:615–622, 1979. 228. Tomita T: Immunocytochemical staining patterns for parathyroid hormone and chromogranin in parathyroid hyperplasia, adenoma and carcinoma. Endocr Pathol. 10:145–156, 1999. 229. Stork PJ, Herteaux C, Frazier R, et al: Expression and distribution of parathyroid hormone and parathyroid hormone messenger RNA in pathological conditions of the parathyroid gland. Lab Invest. 61:169–174, 1992. 230. Weber CJ, Russell J, Chryssochoos JT, et al: Parathyroid hormone content distinguishes true normal parathyroids from parathyroids of patients with primary hyperparathyroidism. World J Surg. 20:1010–1015, 1996. 231. Danks JA, Ebeling PR, Hayman J, et al: Parathyroid hormone related protein: Immunohistochemical localization in cancers and in normal skin. J Bone Miner Res. 4:273–278, 1989. 232. Schmid KW, Morgan JM, Baumert M, et al: Calcitonin and calcitonin gene related peptide mRNA detection in a population of hyperplastic parathyroid cells, also expressing chromogranin B. Lab Invest. 73:90–95, 1995. 233. Kahn A, Tischler AS, Patwardhan NA, et al: Calcatonin immunoreactivity in neoplastic and hyperplastic parathyroid glands. Endocr Pathol. 14:249–250, 2003. 234. Hellman P, Karlsson-Parra A, Klareskog L, et al: Expression and function of a CD4 like molecule in parathyroid tissue. Surgery. 120:985–992, 1996. 235. McGregor DK, Khurana KK, Cao C, et al: Diagnosing primary and metastatic renal cell carcinoma: the use of the monoclonal antibody “Renal Cell Carcinoma Marker.” Am J Surg Pathol. 25:1485–1492, 2001. 236. Bakshi N, Kunju LP, Giordano T, et al: Expression of renal carcinoma antigen in renal epithelial and non-renal tumors: diagnostic implications. Appl Immunohistochem Mol Morphol. 15:310–315, 2007. 237. Gokden N, Gokden M, Phan DC, et al: The utility of PAX-2 in distinguishing metastatic clear cell renal carcinoma from its morphologic mimics: an immunohistochemical study with renal cell carcinoma marker. Am J Surg Pathol. 32:1462–1467, 2008. 238. Vasef MA, Brynes RK, Sturm M, et al: Expression of cyclin D1 in parathyroid carcinomas, adenomas and hyperplasias: A paraffin immunohistochemical study. Mod Pathol. 12:412–416, 1999. 239. Abbona GC, Papotti M, Gasparri G, et al: Proliferative activity in parathyroid tumors as detected by Ki-67 immunostaining. Hum Pathol. 26:135–138, 1995. 240. Vargas MP, Vargas HI, Kleiner DE, et al: The role of prognostic markers (MIB-1, RB, bcl-2) in the diagnosis of parathyroid tumors. Mod Pathol. 10:12–17, 1997. 241. Cryns VL, Thor A, Xu H-J, et al: Loss of the retinoblastoma tumor suppressor gene in parathyroid carcinoma. N Engl J Med. 330:757–761, 1994. 242. Farnebo F, Auer G, Farnebo LO, et al: Evaluation of retinoblastoma and Ki-67 immunostaining as diagnostic markers of benign and malignant parathyroid disease. World J Surg. 23:68–74, 1999. 243. Kayath MJ, Martin LC, Vieira JG, et al: A comparative study of p53 immunoexpression in parathyroid hyperplasias secondary to uremia, primary hyperplasias, adenomas and carcinomas. Eur J Endocrinol. 139:78–83, 1998. 244. Pearce SH, Trump D, Wooding C, et al: Loss of heterozygosity studies at the retinoblastoma and breast cancer susceptibility
BRCA2 loci in pituitary, parathyroid, pancreatic and carcinoid tumors. Clin Endocrinol (Oxf). 45:195–200, 1996. 245. Cetani F, Pardi E, Viacava P, et al: A reappraisal of the Rb1 gene abnormalities in the diagnosis of parathyroid carcinoma. Clin Endocrinol (Oxf). 60:99–106, 2004. 246. Shattuck TM, Kim TS, Costa J, et al: Mutational analysis of RB and BRCA2 as candidate tumor suppressor genes in parathyroid carcinomas. Clin Endocrinol (Oxf). 59:180–189, 2003. 247. Carpten JD, Robbins CM, Villablanca A, et al: HRPT2 encoding parafibromin is mutated in hyperparathyroidism-jaw tumor syndrome. Nat Genet. 32:676–680, 2002. 248. Teh BT, Sweet KM, Morrison CD: Hyperparathyroidism-jaw tumor syndrome. In DeLellis RA, Lloyd RV, Heitz PN, et al, editors: Pathology and Genetics of Tumours of Endocrine Organs. (WHO Classification), Lyon, France, 2004, IARC Press, pp 228–229. 249. Howell VM, Haven CJ, Kahnoski K, et al: HRPT2 mutations are associated with malignancy in sporadic parathyroid tumors. J Med Genet. 40:657–663, 2003. 250. Shattuck TM, Valimaki S. Obara T, et al: somatic and germline mutations of the HRPT2 gene in sporadic parathyroid carcinoma. N Engl J Med. 349:1722–1729, 2003. 251. Tan MH, Morrison C, Wang P, et al: Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clin Cancer Res. 10:6629–6637, 2004. 252. Juhlin C, Larsson C, Yakoleva T, et al: Loss of parafibromin expression in a subset of parathyroid adenomas. Endocrin-Related Cancer. 13:509–523, 2006. 253. Gill AJ, Clarkson A, Gimm O, et al: Loss of nuclear expression of parafibromin distinguishes parathyroid carcinomas and hyperparathyroidism-jaw tumor associated adenomas from sporadic parathyroid adenomas and hyperplasias. Am J Surg Pathol. 30:1140–1149, 2006. 254. Cetani F, Ambrogini E, Viacava P, et al: Should parafibromin staining replace HRPT2 gene analysis as an additional tool for histologic diagnosis of parathyroid carcinoma? Europ J Endocrinol. 156:547–554, 2007. 255. Erovic BM, Harris L, Jamali M: Biomarkers of parathyroid carcinoma. Endocr Pathol. 23:221–231, 2012. 256. Juhlin CC, Villablanca A, Sandelin K, et al: Parafibromin immunoreactivity; its use as an additional diagnostic marker for parathyroid tumor classification. Endocrin Related Cancer. 14:501–512, 2007. 257. Tominaga Y, Tsuzuki T, Matsuoka A, et al: Expression of parafibromin in distant metastatic parathyroid tumors in patients with advanced secondary hyperparathyroidism due to chronic kidney disease. World J Surg. 32:815–821, 2008. 258. Mangray S, Kurek KC, Sabo E, et al: Immunohistochemical expression of parafibromin is of limited value in distinguishing parathyroid carcinoma from adenoma. Mod Pathol. 21:108A (abstract), 2008. 259. Mangray S, DeLellis RA: Parafibromin s a tool for the diagnosis of Parathyroid tumors (letter). Adv Anat Pathol. 15:179, 2008. 260. Schroder S, Niendorf A, Achilles E, et al: Immunocytochemical differential diagnosis of adrenocortical neoplasms using the monoclonal antibody D11. Virchows Arch A Pathol Anat Histopathol. 417:89–96, 1990. 261. Schroder S, Padberg BC, Achilles E, et al: Immunocytochemistry in adrenocortical tumors: A clinicopathological study of 72 neoplasms. Virchows Arch A Pathol Anat Histopathol. 420:65–70, 1992. 262. Tartour E, Caillou B, Tenenbaum F, et al: Immunohistochemical study of adrenocortical carcinoma: Predictive value of the D11 monoclonal antibody. Cancer. 72:3296–3303, 1993. 263. Sasano H, Shizawa S, Suzuki T, et al: Transcription factor adrenal 4 binding protein as a marker of adrenocortical malignancy. Hum Pathol. 26:1154–1156, 1995. 264. Busam KJ, Iversen K, Coplan KA, et al: Immunoreactivity for A103, an antibody to melan-A (Mart-1) in adrenocortical and other steroid tumors. Am J Surg Pathol. 22:57–63, 1998. 265. Renshaw AA, Granter SR: A comparison of A103 and inhibin reactivity in adrenal cortical tumors: Distinction from hepatocellular carcinoma and renal tumors. Mod Pathol. 11:1160–1164, 1998.
References 266. Loy TS, Phillips RW, Linder CL: A103 immunostaining in the diagnosis of adrenal cortical tumors: an immunohistochemical study of 316 cases. Arch Pathol Lab Med. 126:170–172, 2002. 267. Ghorab Z, Jorda M, Ganjei P, et al: Melan A (A103) is expressed in adrenocortical neoplasms but not in renal cell and hepatocellular carcinomas. Appl Immunohistochem Mol Morphol. 11:330– 333, 2003. 268. Shin SJ, Hoda RS, Ying L, et al: Diagnostic utility of the monoclonal antibody A103 in fine-needle aspiration biopsies of the adrenal. Am J Clin Pathol. 113:295–302, 2000. 269. Pelkey TJ, Frierson HF, Mills SE, et al: The a submit of inhibin in adrenal cortical neoplasia. Mod Pathol. 11:516–524, 1998. 270. Fetsch PA, Powers CN, Zakowski M, et al: Anti-alpha inhibin: Marker of choice for the consistent distinction between adrenocortical carcinoma (ACC) and renal cell carcinoma (RCC) in fine needle aspirations (FNA). Cancer. 87:168–172, 1999. 271. McCluggage WG, Maxwell P, Patterson A, et al: Immunohistochemical staining of hepatocellular carcinoma with monoclonal antibody against inhibin. Histopathology. 30:518–522, 1997. 272. Munro LM, Kennedy A, McNicol AM: The expression of inhibin/ activin subunits in the human adrenal cortex and its tumors. J Endocrinol. 161:341–347, 1999. 273. McCluggage WG, Maxwell P: Adenocarcinoma of various sites may exhibit immunoreactivity with anti-inhibin antibodies. Histopathology. 35:216–220, 1999. 274. Brown FM, Gaffey TA, Wold LE, et al: Myxoid neoplasms of the adrenal cortex: a rare histologic variant. Am J Surg Pathol. 24:396–410, 2000. 275. Arola J, Liu J, Heikkila P, et al: Expression of inhibin alpha in adrenocortical tumors reflects the hormonal status of the neoplasm. J Endocrinol. 165:223–229, 2000. 276. Cho EY, Ahn GH: Immunoexpression of inhibin α-subunit in adrenal neoplasms. Appl Immunohistochem Mol Morphol. 9:222– 228, 2001. 277. Vargas MP, Vargas HI, Kleiner DE, et al: Adrenocortical neoplasms: role of prognostic markers MIB-1, p53 and RB. Am J Surg Pathol. 21:556–562, 1997. 278. Schmitt A, Saremaslani P, Schmid S, et al: IGF II and MIB-1 immunohistochemistry is helpful in the differentiation of benign from malignant adrenocortical tumors. Histopathol. 49:298–307, 2006. 279. Miettinen M: NE differentiation in adrenocortical carcinoma: New immunohistochemical findings supported by electron microscopy. Lab Invest. 66:169–174, 1992. 280. Sasaki K, Desimone M, Rao HR, et al: Adrenocortical carcinosarcoma: a case report and review of the literature. Diagn Pathol. 5:51, 2010. doi:10.1186/1746-1596-5-51. 281. Chetty R, Pillay P, Jaichand V: Cytokeratin expression in adrenal phaeochromocytomas and extra-adrenal paragangliomas. J Clin Pathol. 51:477–478, 1998. 282. Fogt F, Vortmeyer AO, Poremba C, et al: Bcl-2 expression in normal adrenal glands and in adrenal neoplasms. Mod Pathol. 11:716–720, 1998. 283. Zhang PJ, Genega EM, Tomaszewski JE, et al: The role of calretinin, inhibin, melan-A, bcl-2, and c-kit in differentiating adrenal cortical and medullary tumors: an immunohistochemical study. Mod Pathol. 16:591–597, 2003. 284. Jorda M, De Madeiros B, Nadji M: Calretinin and inhibin are useful in separating adrenocortical neoplasms from pheochromocytomas. Appl Immunohistochem Mol Morphol. 10:67–70, 2002. 285. Miettinen M, Lehto V-P, Virtanen I: Immunofluorescence microscopic evaluation of the intermediate filament expression of the adrenal cortex and medulla and their tumors. Am J Pathol. 118:360–366, 1985. 286. Gaffey MJ, Traweek ST, Mills SE, et al: Cytokeratin expression in adrenocortical neoplasia: An immunohistochemical and biochemical study with implications for the differential diagnosis of adrenocortical, hepatocellular and renal cell carcinoma. Hum Pathol. 23:144–153, 1992. 287. Wick MR, Cherwitz DL, McGlennen RC, et al: Adrenocortical carcinoma: An immunohistochemical comparison with renal cell carcinoma. Am J Pathol. 122:343–352, 1986. 288. Cote RJ, Cardon-Cardo C, Reuter VE, et al: Immunopathology of adrenal and renal cortical tumors: Coordinated change in
362.e7
antigen expression is associated with neoplastic conversion in the adrenal cortex. Am J Pathol. 136:1077–1084, 1990. 289. Unger PD, Hoffman K, Thung SN, et al: HMB-45 reactivity in adrenal pheochromocytomas. Arch Pathol Lab Med. 116:151– 153, 1992. 290. Caya JG: HMB-45 reactivity in adrenal pheochromocytomas. Arch Pathol Lab Med. 118:1169, 1994. 291. Soon PS, McDonald KL, Robinson BG, et al: Molecular markers and the pathogenesis of adrenocortical cancer. Oncologist. 13:548–561, 2008. 292. Velazquez-Fernandez D, Laurell C, Geli J, et al: Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy. Surgery. 138:1087–1094, 2005. 293. Giordano TJ, Thomas DG, Kuiche R, et al: Distinct transcriptional profiles of adrenocortical tumors uncovered by microarray analysis. Am J Pathol. 162:521–531, 2003. 294. West AN, Neale GA, Pounds S, et al: Gene expression profiling of childhood adrenocortical tumors. Cancer Res. 67:600–608, 2007. 295. Marx C, Bornstein SR, Wolkersdorfer GW, et al: Releveane of major histocompatibility complex class II expression as a hallmark for the cellular differentiation in the human adrenal cortex. J Clin Endocrinol Metab. 82:2136–2140, 1997. 296. Kuruba R, Gallagher SF: Current management of adrenal tumors. Curr Opin Oncol. 20:34–36, 2008. 297. Lloyd RV, Shapiro B, Sisson JC, et al: An immunohistochemical study of pheochromocytomas. Arch Pathol Lab Med. 108:541– 544, 1984. 298. Lloyd RV, Blaivas M, Wilson BS: Distribution of chromogranin and S100 protein in normal and abnormal adrenal medullary tissues. Arch Pathol Lab Med. 109:633–635, 1985. 299. Verhofstad AAJ, Steinbusch HWM, Joosten JWJ, et al: Immunocytochemical localization of nonadrenaline, adrenaline and serotonin. In Polak JM, Van Noordens S, editors: Immunohistochemistry: Practical Applications in Pathology and Biology, Bristol, England, 1983, Wright-PSG, pp 143–168. 300. Trojanowski JQ, Lee VM: Expression of neurofilament antigens by normal and neoplastic human adrenal chromaffin cells. N Engl J Med. 313:101–104, 1985. 301. Wang BA, Gabrilove L, Pertsemlidis D, et al: Oncocytic Pheochromocytoma with Cytokeratin Reactivity: A Case Report with Immunohistochemical and Ultrastructural Studies. Int J Surg Pathol. 5(1-2):61, 1997. 302. Li M, Wening BM: Adrenal Oncocytic Pheochromocytoma. Am J Surg Pathol. 24:1552–1557, 2000. 303. Grignon DJ, Ro JY, MacKay B, et al: Paraganglioma of the urinary bladder: Immunohistochemical, ultrastructural and DNA flow cytometric studies. Hum Pathol. 22:1162–1169, 1991. 304. Johnson TL, Zarbo RJ, Lloyd RV, et al: Paragangliomas of the head and neck: Immunohistochemical NE and intermediate filament typing. Mod Pathol. 1:216–223, 1988. 305. Salim SA, Milroy C, Rode J, et al: Immunocytochemical characterization of NE tumors of the larynx. Histopathology. 23:69–73, 1993. 306. Helman LJ, Cohen PS, Averbuch SD, et al: Neuropeptide Y expression distinguishes malignant from benign pheochromocytoma. J Clin Oncol. 7:720–725, 1989. 307. Clarke MR, Weyant RJ, Watson CG, et al: Prognostic markers in pheochromocytoma. Hum Pathol. 29:522–526, 1998. 308. August C, August K, Schroeder S, et al: CGH and CD44/MIB-1 immunohistochemistry are helpful to distinguish metastasized sporadic pheochromocytomas. Mod Pathol. 17:1119–1128, 2004. 309. Triche TJ, Askin F: Neuroblastoma and the differential diagnosis of small-, round-, blue-cell tumors. Hum Pathol. 14:569–595, 1983. 310. Hachitanda Y, Tsuneyoshi M, Enjoji M: An ultrastructural and immunohistochemical evaluation of cytodifferentiation in neuroblastic tumors. Mod Pathol. 2:13–19, 1989. 311. Pagani A, Forni M, Tonini GP, et al: Expression of members of the chromogranin family in primary neuroblastomas. Diagn Mol Pathol. 1:16–24, 1992. 312. Carter RL, Al-Sams SZ, Corbett RP, et al: A comparative study of immunohistochemical staining for neuron-specific enolase, protein gene product 9.5 and s-100 in neuroblastoma, Ewing’s
362.e8
Immunohistology of Endocrine Tumors
sarcoma and other round cell tumors in children. Histopathology. 16:461–467, 1990. 313. Wirnsberger GH, Becker H, Ziervogel K, et al: Diagnostic immunohistochemistry of neuroblastic tumors. Am J Surg Pathol. 16:49–57, 1992. 314. Franquemont DW, Mills SE, Lack EE: Immunohistochemical detection of neuroblastomatous foci in composite adrenal pheochromocytoma-neuroblastoma. Am J Clin Pathol. 102:163– 170, 1994. 315. Argani P, Erlandson RA, Rosai J: Thymic neuroblastoma in adults: Report of three cases with special emphasis on its association with the syndrome of inappropriate secretion of antidiuretic hormone. Am J Clin Pathol. 108:537–543, 1997. 316. Fellinger EJ, Garin-Chesa P, Triche TJ, et al: Immunohistochemical analysis of Ewing’s sarcoma cell surface antigen p30/32 MIC2. Am J Pathol. 39:317–325, 1991. 317. Stevenson AJ, Chatten J, Bertoni F, et al: CD99 (p30/32mic2) neuroectodermal/Ewing’s sarcoma antigen as an immunohistochemical marker: Review of more than 600 tumors and the literature experience. Appl Immunohistochem. 2:231–240, 1994. 318. Weidner N, Tjoe J: Immunohistochemical profile of monoclonal antibody 013: Antibody that recognizes glycoprotein p 30/32mic2 and is useful in diagnosing Ewing’s sarcoma and peripheral neuroepithelioma. Am J Surg Pathol. 18:486–494, 1994. 319. Scotlandi K, Serra M, Manara MC, et al: Immunostaining of the p30/32mic2 antigen and molecular detection of EWS rearrangements for the diagnosis of Ewing’s sarcoma and peripheral neuroectodermal tumor. Hum Pathol. 27:408–416, 1996. 320. Hess E, Cohen C, DeRose PB, et al: Nonspecificity of p30/32mic2 immunolocalization with the 013 monoclonal antibody in the diagnosis of Ewing’s sarcoma: Application of an algorithmic immunohistochemical analysis. Appl Immunohistochem. 5:94– 103, 1997. 321. Pappo AS, Douglass ED, Meyer WH, et al: Use of HBA 71 and anti-B2-microglobulin to distinguish peripheral neuroepithelioma from neuroblastoma. Hum Pathol. 24:880–885, 1993. 322. Miettinen M, Chatten J, Paetau A, et al: Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors. Am J Surg Pathol. 22:327–332, 1998. 323. Folpe AL, Patterson K, Gown AM: Antineuroblastoma antibody NB-84 also identifies a significant subset of other small blue round cell tumors. Appl Immunohistochem. 5:239–245, 1997. 324. Bielle F, Fréneaux P, Jeanne-Pasquier C, et al: PHOX2B immunolabeling a novel tool for the diagnosis of undifferentiated neuroblastomas among childhood small round blue-c ell tumors. Am J Surg Pathol. 36(8):1141–1149, 2012. 325. Mehrazma M, Madjd Z, Kalantari E, et al: Expression of Stem Cell Markers, CD133 and CD44, in Pediatric Solid Tumors: A Study Using Tissue Microarray. Fetal Pediatr Pathol. 2012. [Epub ahead of print]. 326. Tischler AS: Pheochromocytoma and extra-adrenal paraganglioma: update. Arch Pathol Lab Med. 132:1272–1284, 2008. 327. Brouwers FM, Glasker S, Nave AF, et al: Proteomic profiling of von Hippel Lindau syndrome and multiple endocrine neoplasia type 2 pheochromocytomas reveal different expression of chromogranin B. Endocr Relat Cancer. 14:463–471, 2007. 328. Brouwers FM, Elkahloun AGS, Munson PJ, et al: Gene expression profiling of benign and malignant pheochromocytoma. Ann NY Acad Sci. 1073:541–556, 2006. 329. Thouënnon E, Elkahloun A, Guillemot J, et al: Insights into the pathophysiology of pheochromocytoma malignancy. J Clin Endocrinol Metab. 92:4865–4487, 2007. 330. Stutterheim J, Gerritsen A, Zappeij-Kannegieter L, et al: PHOX2B is a novel and specific marker for minimal residual disease testing in neuroblastoma. Clin Oncol. 26(33):5443– 5449, 2008. 331. Dayal Y: Endocrine cells of the gut and their neoplasms. In Norris HT, editor: Pathology of the Colon, Small Intestine and Anus, New York, 1991, Churchill Livingstone, pp 305–366. 332. Moll R, Franke WW: Cytoskeletal differences between human NE tumors: A cytoskeletal protein of molecular weight 46000 distinguishes cutaneous from pulmonary NE tumors. Differentiation. 30:165–175, 1985.
333. Burke AP, Sobin LH, Federspiel BH, et al: Appendiceal carcinoids: Correlation of histology and immunohistochemistry. Mod Pathol. 2:630–637, 1989. 334. Miettinen M: Keratin 20: Immunohistochemical marker for gastrointestinal, urothelial, and Merkel cell carcinomas. Mod Pathol. 8:384–388, 1995. 335. Al-Khafaji B, Noffsinger AE, Miller MA, et al: Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors. Hum Pathol. 29:992–999, 1998. 336. Kimura N, Pilichowska M, Okamoto H, et al: Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors. Mod Pathol. 13:140–146, 2000. 337. Thomas RM, Baybick JH, Elsayed AM, et al: Gastric carcinoids: An immunohistochemical and clinicopatho logic study of 104 patients. Cancer. 73:2053–2058, 1994. 338. Machlouf HR, Burke AP, Sobin LH: Carcinoid tumors of the ampulla of Vater: A comparison with duodenal carcinoid tumors. Cancer. 85:1241–1249, 1999. 339. Sheibani K, Battifora H, Burke JS, et al: Leu-M1 antigen in human neoplasms: An immunohistologic study of 400 cases. Am J Surg Pathol. 10:227–236, 1986. 340. Moskaluk CA, Zheng H, Powell SM, et al: CDX2 protein expression in normal and malignant human tissue: an immunohistochemical survey using tissue microarrrays. Mod Pathol. 16:913–919, 2003. 341. Azumi N, Traweek ST, Battifora H: Prostatic acid phosphatase in carcinoid tumors: Immunohistochemical and immunoblot studies. Am J Surg Pathol. 15:785–790, 1991. 342. Yan B, Yap CT, Wang S, et al: Cofilin immunolabelling correlates with depth of invasion in gastrointestinal endocrine cell tumors. Acta Histochem. 112(1):101–106, 2010. 343. Zikusoka MN, Kidd M, Eick G, et al: The molecular genetics of gastroenteropancreatic NE tumors. Cancer. 104:2292–2309, 2005. 344. Fujimori M, Ikeda S, Shimizu Y, et al: Accumulation of betacatenin gene in gastrointestinal carcinoid tumor. Cancer Res. 61:6656–6659, 2001. 345. Su MC, Wang CC, Chen CC, et al: Nuclear translocation of beta-catenin and APC mutation in gastrointestinal carcinoid tumor. Ann Surg Oncol. 13:1604–1609, 2006. 346. Shah IA, Schlageter M-O, Netto D: Immunoreactivity of neurofilament proteins in NE neoplasms. Mod Pathol. 4:215–219, 1991. 347. Chejfec G, Falkmer S, Grimelius L, et al: Synaptophysin: A new marker for pancreatic NE tumors. Am J Surg Pathol. 11:241–247, 1987. 348. Solcia E, Capella C, Kloppel G: Atlas of Tumor Pathology: Tumors of the Pancreas, Washington, D.C., 1997, Armed Forces Institute of Pathology. 349. Heitz PU, Kasper M, Polak JM, et al: Pancreatic endocrine tumors: Immunocytochemical analysis of 125 tumors. Hum Pathol. 13:263–271, 1982. 350. Perkins PL, McLeod MK, Jin L, et al: Analysis of gastrinomas by immunohistochemistry and in-situ hybridization histochemistry. Diagn Mol Pathol. 1:155–164, 1992. 351. Le Bodic M-F, Heyman M-F, Lecomete M, et al: Immuno histochemical study of 100 pancreatic tumors in 28 patients with multiple endocrine neoplasia type I. Am J Surg Pathol. 20:1378– 1384, 1996. 352. Solcia E, Capella C, Riva C, et al: The morphology and NE profile of pancreatic epithelial VIPomas and extrapancreatic, VIP producing neurogenic tumors. Ann NY Acad Sci. 527:508–517, 1988. 353. Heitz PU, Kasper M, Kloppel G, et al: Glycoprotein-hormone alpha-chain production by pancreatic endocrine tumors: A specific marker for malignancy: Immunocytochemical analysis of tumors of 155 patients. Cancer. 51:277–282, 1983. 354. Graeme-Cook F, Nardi G, Compton CC: Immunocytochemical staining for human chorionic gonadotropin subunits does not predict malignancy in insulinomas. Am J Clin Pathol. 93:273– 276, 1990. 355. Viale G, Doglioni C, Gambacorta M, et al: Progesterone receptor immunoreactivity in pancreatic endocrine tumors: An
References immunocytochemical study of 156 NE tumors of the pancreas, gastrointestinal and respiratory tracts and skin. Cancer. 70:2268– 2277, 1992. 356. Pelosi G, Bresaola E, Bogina G, et al: Endocrine tumors of the pancreas: Ki-67 immunoreactivity on paraffin sections is an independent predictor for malignancy: a comparative study with proliferating-cell nuclear antigen and progesterone receptor protein immunostaining, mitotic index and other clinicopathological variables. Hum Pathol. 27:1124–1134, 1996. 357. Heitz PU, Komminoth P, Perren A, et al: Pancreatic endocrine tumours: an introduction. In Delellis RA, Lloyd RV, Heitz PU, et al, editors: Pathology and Genetics of Tumours of Endocrine Organs. WHO Classification of Tumours, Lyon, 2004, IARC Press, pp 177–182. 358. Klimstra DS, Modlin IR, Coppola D, et al: The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems. Pancreas. 39(6):707–712, 2010. 359. Rindi G, Arnold R, Bosman FT, et al: Nomenclature and classification of neuroendocrine neoplasms of the digestive system. In Bosman TF, Carneiro F, Hruban RH, et al, editors: WHO Classification of Tumours of the Digestive System, ed 4, Lyon, 2010, International Agency for Research on cancer (IARC), p 13. 360. Deshpande V, Fernandez-del Castillo C, Muzikansky A, et al: Cytokeratin 19 is a powerful predictor of survival in pancreatic endocrine tumors. Am J Surg Pathol. 28:1145–1153, 2004. 361. Goto A, Niki T, Terrado Y, et al: Prevalence of CD99 expression in pancreatic endocrine tumors (PETs). Histopathol. 45:384– 392, 2004. 362. Schmitt AM, Anlauf M, Rousson V, et al: WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors. Am J Surg Pathol. 31:1677–1682, 2007. 363. LaRosa S, Rigoli E, Uccella S, et al: Prognostic and biological significance of cytokeratin 19 in pancreatic endocrine tumors. Histopathol. 50:597–606, 2007. 364. Zhang L, Smyrk TC, Oliveira AM, et al: KIT is an independent prognostic marker for pancreatic endocrine tumors: a finding derived from analysis of islet cell differentiation markers. Am J Surg Pathol. 33(10):1562–1569, 2009. 365. Zhang L, Lohse CM, Dao LN, et al: Proposed histopathologic grading system derived from a study of KIT and CK19 expression in pancreatic endocrine neoplasm. Hum Pathol. 42(3):324– 331, 2011. 366. Shida T, Furuya M, Kishimoto T, et al: The expression of neuroD1 and MASH1 in the gastroenteropancreatic NE tumors. Mod Pathol. 21:1363–1370, 2008. 367. Shin JU, Lee CH, Lee KT, et al: Prognostic significance of ATM and cyclin B1 in pancreatic neuroendocrine tumor. Tumour Biol. 33(5):1645–1651, 2012. 368. Couvelard A, Hu J, Steers G, et al: Identification of potential therapeutic targets by gene expression profiling in pancreatic endocrine tumors. Gastroenterol. 131:1597–1610, 2006. 369. Perren A, Anlauf M, Komminoth P: Molecular profiles of pancreatic endocrine tumors. Virchows Arch. 451(Supl 1):S39–S46, 2007. 370. Shirahama S, Matsuyoshi J, Yamamoto S, et al: Alteration of the K-ras and p16/CDKN2 gene in endocrine pancreatic carcinomas. Nihon Rinsho. 62(5):889–893, 2004. 371. Gould VE, Linnoila RI, Memoli VA, et al: NE components of the bronchopulmonary tract: Hyperplasias, dysplasias and neoplasias. Lab Invest. 49:519–537, 1983. 372. Travis WD, Linnoila ID, Tsokos MG, et al: NE tumors of the lung with proposed criteria for large cell NE carcinoma: An ultrastructural, immunohistochemical and flow cytometric study of 35 cases. Am J Surg Pathol. 15:529–553, 1991. 373. Blobel GA, Gould VE, Moll R, et al: Co-expression of NE markers and epithelial cytoskeletal proteins in bronchopulmonary NE neoplasms. Lab Invest. 52:39–51, 1985. 374. Oliveira AM, Tazelaar HD, Myers JL, et al: Thyroid transcription factor-1 distinguishes metastatic pulmonary from well differentiated NE tumors of other sites. Am J Surg Pathol. 25:815–819, 2001. 375. Du EZ, Goldstraw P, Zacharias J, et al: TTF-1 expression is specific for lung primary in typical and atypical carcinoids: TTF-1 positive carcinoids are predominantly in peripheral location. Hum Pathol. 35:825–831, 2004.
362.e9
376. Cai YC, Banner B, Glickman J, et al: Cytokeratin 7 and 20 and thyroid transcription factor-1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors. Hum Pathol. 32:1087–1093, 2001. 377. Jiang S-X, Kameya T, Shoji M, et al: Large cell NE carcinoma of the lung: A histological and immuno histochemical study of 22 cases. Am J Surg Pathol. 22:526–537, 1998. 378. Zhang H, Liu J, Cagle PT, et al: Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach. Mod Pathol. 18:111– 118, 2005. 379. Matsuki Y, Tanimoto A, Hamada T, et al: Histidine decarboxylase expression as a new sensitive and specific marker for small cell lung carcinoma. Mod Pathol. 16(1):72–78, 2003. 380. Xu H, Bourne PA, Spaulding BO, et al: High-grade neuroendocrine carcinomas of the lung express K homology domain containing overexpressed in cancer by carcinoid tumors do not. Human Pathol. 34:555–563, 2007. 381. LaPoint RJ, Bourne PA, Wang HL, et al: Coexpression of c-kit and bcl-2 in small cell carcinoma and large cell neuroendocrine carcinoma of the lung. Appl Immunohistochem Mol Morph. 15:401–406, 2007. 382. Jiang SX, Kameya T, Asamura H, et al: hASH1 expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors. Mod Pathol. 17(2):222–229, 2004. 383. Meyronet D, Massoma P, Thivolet F, et al: Extensive expression of a collapsin response mediation protein 5 (CRMP5) is a specific marker of high grade lung NE carcinoma. Am J Surg Pathol. 32:1699–1708, 2008. 384. Gersell DJ, Mazoujian G, Mutch DG, et al: Small cell undifferentiated carcinoma of the cervix: A clinicopathologic, ultrastructural and immunocytochemical study of 15 cases. Am J Surg Pathol. 12:684–698, 1988. 385. Abeler VM, Holm R, Nesland JM, et al: Small cell carcinoma of the cervix: A clinicopathologic study of 26 patients. Cancer. 73:672–677, 1994. 386. Gilks CB, Young RH, Gersell DJ, et al: Large cell NE carcinoma of the uterine cervix: A clinicopathologic study of 12 cases. Am J Surg Pathol. 21:905–914, 1997. 387. Sukpan K, Settakorn J, Khunamornpong S, et al: Expression of surviving, CD117 and C-erB-2 in neuroendocrine carcinoma of the uterine cervix. Int J Gynecol Cancer. 21:911–917, 2011. 388. Chan JK, Suster S, Wenig B, et al: Cytokeratin 20 immunoreactivity distinguishes Merkel cell (primary cutaneous NE) carcinomas and salivary gland small cell carcinomas from small cell carcinomas of various sites. Am J Surg Pathol. 21(2):226–234, 1997. 389. Ordonez NG: Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol. 24:1217–1223, 2000. 390. Li D-J, Zhuang Y, Li Y-F, et al: A clinicopathological aspect of primary small-cell carcinoma of the uterine cervix: a singlecentre study of 25 cases. J Clin Pathol. 64:1102, 2011. 391. McCluggage WG, Kennedy K, Busam KJ: An immunohistochemical study of cervical neuroendocrine carcinomas: neoplasms that are commonly TTF-1 positive and which may express CK20 and p63. Am J Surg Pathol. 34:525–532, 2010. 392. Straughn JM, Richter HE, Conner MG, et al: Predictors of outcome in small cell carcinoma of the cervix-a case series. Gynecol Oncol. 83:216–220, 2001. 393. Masumoto N, Fujii T, Ishikawa M, et al: p16 overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix. Hum Pathol. 34:778–783, 2003. 394. Conner MG, Richter H, Moran CA, et al: Small cell carcinoma of the cervix: a clinicopathologic and immunohistochemical study of 23 cases. Ann Diagn Pathol. 6:345–348, 2002. 395. Houghton O, McCluggage WG: The expression and diagnostic utility of p63 in the female genital tract. Adv Anat Pathol. 16(5):316–321, 2009. 396. Wang TY, Chen BF, Yang YC, et al: Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: a study of 250 cases. Hum Pathol. 32:479– 486, 2001.
362.e10
Immunohistology of Endocrine Tumors
397. Lin Z, Liu M, Li Z, et al: DeltaNp63 protein expression in uterine cervical and endometrial cancers. J Cancer Res Clin Oncol. 132:811–816, 2006. 398. Stoler MH, Mills SE, Gersell DJ, et al: Small cell NE carcinoma of the cervix: A human papillomavirus 18 associated cancer. Am J Surg Pathol. 15:28–32, 1991. 399. Ishida GM, Kato N, Hayasaka T, et al: Small cell NE carcinomas of the uterine cervix: a histological, immunohistochemical, and molecular genetic study. Int J Gynecol Pathol. 23:366–372, 2004. 400. Wang HL, Lu DW: Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix. Am J Surg Pathol. 28:901–908, 2004. 401. Grayson W, Rhemtula HA, Taylor LF, et al: Detection of human papillomavirus in large cell NE carcinoma of the uterine cervix: a study of 12 cases. J Clin Pathol. 55:108–114, 2002. 402. Matthews-Greer J, Dominguez-Malagon H, Herrera GA, et al: Human papillomavirus typing of rare cervical carcinomas. Arch Pathol Lab Med. 128:553–556, 2004. 403. Wang K-L, Yang Y-C, Wang T-Y, et al: NE carcinoma of the uterine cervix: a clinicopathologic retrospective study of 31 cases with prognostic implications. J Chemotherapy. 18:209– 216, 2006. 404. Tangitgamol S, Ramirez PT, Sun CC, et al: Expression of HER-2/ neu, epidermal growth factor receptor, vascular endothelial growth factor, cyclooxygenase-2, estrogen receptor, and progesterone receptor in small cell and large cell NE carcinoma of the cervix: a clinicopathologic and prognostic study. Int J Gynecol Cancer. 15:646–656, 2005. 405. Zarka TA, Han AC, Edelson MI, et al: Expression of cadherins, p53 and bcl-2 in small cell carcinomas of the cervix: potential tumor suppressor role for N-cadherin. Int J Gynecol Cancer. 13:240–243, 2003. 406. diSant’Agnese PA, de Mesy Jensen KL, Churukian CJ, et al: Human prostatic endocrine-paracrine (APUD) cells: Distributional analysis with a comparison of serotonin and neuron specific enolase immunoreactivity and silver stains. Arch Pathol Lab Med. 109:607–612, 1985. 407. diSant’Agnese PA: NE differentiation in prostatic carcinoma: An update. Prostate (Suppl). 8:74–79, 1998. 408. Schmid KW, Helpap B, Totsch M, et al: Immunohistochemical localization of chromogranins A and B and secretogranin II in normal, hyperplastic and neoplastic prostate. Histopathology. 24:233–239, 1994. 409. Yuan TC, Veeraman S, Lin MF: NE-like prostate cancer cells: NE trans differentiation of prostate adenocarcinoma cells. Endocr Relat Cancer. 14:531–547, 2007. 410. di Sant’ Agnese PA: Divergent NE differentiation in prostatic carcinoma. Sem Diagn Pathol. 17:4967–4161, 2000. 411. Ghannoum JE, DeLellis RA, Shin SJ: Primary carcinoid tumor of the prostate with concurrent adenocarcinoma: a case report. Int J Surg Pathol. 12(2):167–170, 2004. 412. Azumi N, Shibuya H, Ishikura M: Primary prostatic carcinoid with intracytoplasmic prostatic acid phosphatase and prostate specific antigen. Am J Surg Pathol. 8:545–550, 1984. 413. Goldstein NS: Immunophenotypic characterization of 225 prostate adenocarcinomas with intermediate or high Gleason scores. Am J Clin Pathol. 117:471–477, 2002. 414. Kawai S, Hiroshima K, Tsukamoto Y, et al: Small cell carcinoma of the prostate expressing prostate-specific antigen and showing syndrome of inappropriate secretion of antidiuretic hormone: an autopsy case report. Pathol Int. 53:892–896, 2003. 415. Azumi N, Shibuya H, Ishikura M: Primary prostatic carcinoid tumor with intracytoplasmic prostatic acid phosphatase and prostate-specific antigen. Am J Surg Pathol. 8:545–550, 1984. 416. Bostwick DG, Dousa MK, Crawford BG, et al: NE differentiation in prostatic intraepithelial neoplasia and adenocarcinoma. Am J Surg Pathol. 18:1240–1246, 1994. 417. Yao JL, Madeb R, Bourne P, et al: Small cell carcinoma of the prostate: an immunohistochemical study. Am J Surg Pathol. 30:705–712, 2006. 418. Wang W, Epstein JI: Small cell carcinoma of the prostate: a morphologic and immunohistochemical study of 95 cases. Am J Surg Pathol. 32:65–71, 2008.
419. Simon RA, diSant’Agnes PA, Huang L-S, et al: CD44 expression is a feature of prostatic small cell carcinoma and distinguishes it from its mimickers. Hum Pathol. 40:252–258, 2009. 420. Sauer CG, Roemar A, Grobholz R: Genetic analysis of NE tumor cells in prostate carcinoma. Prostate. 66:227–234, 2006. 421. Gould VE, Moll R, Moll I, et al: NE (Merkel) cells of the skin: Hyperplasias, dysplasias and neoplasms. Lab Invest. 52:334–353, 1985. 422. Leech SN, Kolar AJO, Barrett PD, et al: Merkel cell carcinoma can be distinguished from metastatic small cell carcinoma using antibodies to cytokeratin 20 and thyroid transcription factor 1. J Clin Pathol. 54:727–729, 2001. 423. Cheuk W, Kwan MY, Suster S, et al: Immunostaining for thyroid transcription factor 1 and cytokeratin 20 aids the distinction of small cell carcinoma from Merkel cell carcinoma, but not pulmonary from extrapulmonary small cell carcinomas. Arch Pathol Lab Med. 125:228–231, 2001. 424. Hanly AJ, Elgart GW, Jorda M, et al: Analysis of thyroid transcription factor-1 and cytokeratin 20 separates Merkel cell carcinoma from small cell carcinoma of lung. J Cutan Pathol. 27:118–120, 2000. 425. Nicholson SA, McDermott MB, Swanson PE, et al: CD99 and cytokeratin 20 in small cell and basaloid tumors of the skin. Appl Immunohistochem Mol Morphol. 8:37–41, 2000. 426. Nagao T, Gaffey TA, Olsen KD, et al: Small cell carcinoma of the major salivary glands: clinicopathologic study with emphasis on cytokeratin 20 immunoreactivity and clinical outcome. Am J Surg Pathol. 28(6):762–770, 2004. 427. Jensen K, Kohler S, Rouse RV: Cytokeratin staining in Merkel cell carcinoma: an immunohistochemical study of cytokeratins 5/6, 7, 17, and 20. Appl Immunohistochem Mol Morph. 8:310– 315, 2000. 428. Drijkoningen M, de Wolf-Peeters C, van Limbergen E, et al: Merkel cell tumor of the skin: An immunohistochemical study. Hum Pathol. 17:301–307, 1986. 429. Liu Y, Mangini J, Saad R, et al: Diagnostic value of microtubuleassociated protein-2 in merkel cell carcinoma. Appl Immunohistochem Mol Morphol. 11:326–329, 2003. 430. Sibley RK, Dahl D, Primary NE: (Merkel cell?) carcinoma of the skin. II: An immunohistochemical study of 21 cases. Am J Surg Pathol. 9:109–116, 1985. 431. Dinh V, Feun L, Elgart G, et al: Merkel cell carcinomas. Hematol Oncol Clin North Am. 21(3);527–544, 2007. 432. Brinkschmidt C, Stolze P, Fahrenkamp AG, et al: Immuno histochemical demonstration of chromogranin A, chromogranin B and secretoneunin in Merkel cell carcinoma of the skin: An immunohistochemical study suggesting two types of Merkel cell carcinoma. Appl Immunohistochem. 3:37–44, 1995. 433. Sur M, AlArdati H, Ross C, et al: Tdt expression in Merkel cell carcinoma: potential diagnostic pitfall with blastic hematological malignancies and expanded immunohistochemical analysis. Mod Pathol. 20:1113–1120, 2007. 434. Buresh CT, Oliai BR: Reactivity with TdT in Merkel cell carcinoma: a potential diagnostic pitfall. Am J Clin Pathol. 129:894– 898, 2008. 435. Dong HY, Liu W, Cohen P, et al: B-cell activation protein encoded by the PAX-5 gene is commonly expressed in Merkel cell carcinoma and small cell carcinomas. Am J Surg Pathol. 29:687–692, 2005. 436. Yang DT, Holden JA, Florell SR: CD117, CK20, TTF-1, and DNA topoisomerase II-α antigen expression in small cell tumors. J Cutan Pathol. 31:254–261, 2004. 437. Su LD, Fullen DR, Lowe L, et al: CD117 (kit receptor) expression in Merkel cell carcinoma. Am J Dermatopathol. 24:289– 293, 2002. 438. Strong S, Shalders K, Carr R, et al: KIT receptor (CD117) expression in Merkel cell carcinoma. Br J Dermatol. 150:384– 385, 2004. 439. Han AC, Soler AP, Tang C-K, et al: Nuclear localization of E-cadherin expression in Merkel cell carcinoma. Arch Pathol Lab Med. 124:1147–1151, 2000. 440. Tanaka Y, Sano T, Qian ZR, et al: Expression of adhesion molecules and cytokeratin 20 in merkel cell carcinomas. Endocr Pathol. 15:117–129, 2004.
References 441. Ralston J, Chireboga L, Nonaka D: Mash1: A useful marker in differentiating pulmonary small cell carcinoma from Merkel cell carcinomas. Mod Pathol. 21:1257–1362, 2009. 442. Pryor JG, Simon R, Bourne PA, et al: Merkel cell carcinoma expresses K homology domain-containing protein over expressed in cancer similar to other high grade neuroendocrine carcinomas. Hum Pathol. 40:238–243, 2009. 443. Feng H, Shuda M, Chang Y, et al: Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 319:1096– 1100, 2008. 444. Kassem A, Schöpflin A, Diaz C, et al: frequent detection of Merkel cell polyomavirus in human Merkel cell carcinomas and identification of a unique deletion in the VP1gene. Cancer Res. 68:5009–5013, 2008. 445. Duncavage EJ, Le B-M, Wang D, et al: Merkel cell polyomavirus: a specific marker for merkel cell carcinoma in histologically similar tumors. Am J Surg Pathol. 33:1771–1777, 2009. 446. Busam KJ, Jungbluth AA, Rekthman N, et al: Merkel cell polyomavirus expression in merkel cell carcinomas and its absence in combined tumors and pulmonary neuroendocrine carcinomas. Am J Surg Pathol. 33:1378–1385, 2009. 447. Ly TY, Walsh NM, Pasternak S: The spectrum of markel cell polyomavirus expression in merkel cell carcinoma, in a variety of cutaneous neoplasms, and in neuroendocrine carcinomas from different anatomical sites. Hum Pathol. 43:557–566, 2012. 448. Kondapalli L, Soltani K, Lacouture ME: The promise of molecular targeted therapies: protein kinase inhibitors in the treatment of cutaneous malignancies. J Am Acad Dermatol. 53:291–302, 2005. 449. Fenig E, Nordenberg J, Beery E, et al: Combined effect of aloeemodin and chemotherapeutic agents on the proliferation of an adherent variant cell line of Merkel cell carcinomas. Oncol Rep. 11:213–217, 2004. 450. Maluf HM, Koerner FC: Carcinomas of the breast with endocrine differentiation: A review. Virchows Arch. 425:449–457, 1994. 451. Papotti M, Gherardi G, Eusebi V, et al: Primary oat cell (NE) carcinoma of the breast: Report of four cases. Virchows Arch A Pathol Anat Histopathol. 420:103–108, 1992. 452. Adegbola T, Connolly CE, Mortimer G: Small cell neuroendocrine carcinoma of the breast: a report of 3 cases and review of the literature. J Clin Pathol. 58:775–778, 2005. 453. Francois A, Chatikhine VA, Chevallier B, et al: NE primary small cell carcinoma of the breast: Report of a case and review of the literature. Am J Clin Oncol. 18:133–138, 1995. 454. Sapino A, Righi L, Cassoni P, et al: Expression of the NE phenotype in carcinomas of the breast. Semin Diagn Pathol. 17(2):127–137, 2000. 455. Viacava P, Castagna M, Bevilacqua G: Absence of NE cells in fetal and adult mammary gland: are NE breast tumors real NE tumours? Breast. 4:143–146, 1995. 456. Lakhani SR, Ellis IO, Schnitt SJ, et al: WHO Classification of Tumours of the Breast. WHO, Classification of Tumours, Volume 4, Lyon, 2012, IARC Press.
362.e11
457. Shin SJ, DeLellis RA, Ying BA, et al: Small cell carcinoma of the breast: A clinico-pathological and immunohistochemical study of 9 patients. Am J Surg Pathol. 24:1231–1238, 2000. 458. Bergman S, Hoda SA, Geisinger KR, et al: E-cadherin-negative primary small cell carcinoma of the breast: report of a case and review of the literature. Am J Clin Pathol. 121:1170121, 2004. 459. Hoang MP, Maitra A, Gazdar AF, et al: Primary mammary small cell carcinoma: a molecular analysis of 2 cases. Human Pathol. 32:753–757, 2001. 460. Yamasaki T, Shimazaki H, Aida S, et al: Case report: primary small cell (oat cell) carcinoma of the breast: report of a case and review of the literature. Pathol Int. 50:914–918, 2000. 461. Weigelt B, Horlings HM, Kreike B, et al: Refinement of breast cancer classification by molecular characterization of histological special types. J Pathol. 216:141–150, 2008. 462. Perou CM, Sorlie T, Eisen MB, et al: Molecular portraits of human breast tumours. Nature. 406:747–752, 2000. 463. Sorlie T, Perou CM, Tibshirani R, et al: Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 98:10869–10874, 2001. 464. Reis-Filho JS, Lakhani SR: Breast cancer special types: why bother? J Pathol. 2008. [Epub ahead of print]. 465. Klemm KM, Moran CA: Primary NE carcinoma of the thymus. Semin Diagn Pathol. 16:32–41, 1999. 466. Moran CA, Suster S: Thymic NE carcinomas with combined features ranging from well-differentiated (carcinoid) to small cell carcinoma: A clinicopathologic and immunohistochemical study of 11 cases. Am J Clin Pathol. 113:345–350, 2000. 467. De Montpreville VT, Macchiarini P, Dulmet E: Thymic NE carcinoma (carcinoid): A clinicopathologic study of fourteen cases. J Thorac Cardiovasc Surg. 111:134–141, 1996. 468. Kaufmann O, Dietel M: Expression of thyroid transcription factor-1 in pulmonary and extrapulmonary small cell carcinomas and other NE carcinomas of various primary sites. Histopathol. 36:415–420, 2000. 469. Moran CA, Suster S: NE carcinomas (carcinoid tumor) of the thymus. A clinicopathologic analysis of 80 cases. Am J Clin Pathol. 114:100–110, 2000. 470. Hishima T, Fukayama M, Hayashi Y, et al: NE differentiation in thymic epithelial tumors with special reference to thymic carcinoma and atypical thymoma. Hum Pathol. 29:330–338, 1998. 471. Goto K, Kodama T, Matsuno Y, et al: Clinicopathologic and DNA cytometric analysis of carcinoid tumors of the thymus. Mod Pathol. 14:985–994, 2001. 472. Nonaka D, Henley JD, Chiriboga L, et al: Diagnostic utility of thymic epithelial markers CD205 (DEC205) and Foxn1 in thymic epithelial neoplasms. Am J Surg Pathol. 31:1038–1044, 2007. 473. Pan C-C, Jong Y-J, Chen Y-J: Comparative genomic hybridization analysis of thymic NE tumors. Modern Pathol. 18:358–364, 2005. 474. Reiker RJ, Aulmann S, Penzel R, et al: Chromosomal imbalances in sporadic NE tumours of the thymus. Cancer Lett. 223:169– 174, 2004.
C H A P T E R 1 1
IMMUNOHISTOLOGY THE MEDIASTINUM
OF
ANNIKKA WEISSFERDT, CESAR A. MORAN
Overview 363 Biology of Antigens and Antibodies 363 Normal Thymus 363 Thymic Epithelial Neoplasms 365 Mediastinal Neuroendocrine Neoplasms 371 Mesothelial Neoplasms of the Mediastinum 373 Mesenchymal Neoplasms of the Mediastinum 373 Mediastinal Germ Cell Tumors 379 Histiocytic Tumors of the Mediastinum 380 Lymphoproliferative Disorders of the Mediastinum 380 Prognostic Markers for Mediastinal Neoplasms 385 Summary 385
Overview The mediastinum is a unique space in the thoracic cavity. It contains the heart, large vessels, trachea, esophagus, thymus, lymph nodes, nerves, and connective tissues. These tissues can give rise to myriad pathologic processes that include neoplastic and nonneoplastic diseases. The neoplastic processes can include proliferations of all cell lineages, including epithelial, mesenchymal, lymphoid, and germ cell tumors (GCTs). The tissue diagnosis of mediastinal diseases is often challenging for pathologists because of its relative infrequency and also because of the fact that diagnostic material is often composed of small mediastinoscopic biopsies. In this context, immunohistochemical (IHC) stains play an important role in identifying the correct disease process. It should be noted, however, that the results of IHC studies should always be interpreted in association with the morphologic, clinical, and radiologic features and that the combination of all these most likely permits
arriving at the correct interpretation. This chapter aims to provide a practical approach to the use of IHC in the setting of mediastinal tumors and their differential diagnosis.
Biology of Antigens and Antibodies Apart from thymic epithelial neoplasms, most other tumors are not unique to the mediastinum, and their immunophenotype is likely to be discussed in other chapters of this book. Based on this fact, it must be noted that no specific antibodies distinguish neoplasms that originate in mediastinal structures from those that are metastatic to the mediastinum. Similar conclusions apply to thymic epithelial neoplasms. These tumors share many of their IHC properties with other, nonmediastinal tumors, which complicates the assessment of these neoplasms. Early assumptions that thymic epithelial cells could be targeted by using antibodies to thymus-specific hormones, especially thymosin, have been questioned; today, these antibodies play no practical role in the surgical pathology laboratory.1-5 More recently, a relatively new antibody, Pax-8, has been studied in thymic epithelial neoplasms and has been found to be expressed in a large number of these tumors.6 Although Pax-8 is also expressed immunohistochemically in a high percentage of tumors of thyroid, renal, and müllerian origin and therefore lacks specificity for thymic epithelial tumors, the use of this antibody in the differential diagnosis of these tumors is still beneficial, especially to distinguish them from metastatic lung tumors.
Normal Thymus The thymic gland is a lymphoepithelial organ composed of an admixture of epithelial and lymphoid components. The thymus can be subdivided into lobules, and each lobule contains two histologically distinct compartments, the cortex and the medulla. The cortex consists of closely packed T lymphocytes (thymocytes) and 363
364
Immunohistology of the Mediastinum
a sparse epithelial cell component; the medulla comprises a large number of epithelial cells and few lymphocytes. The epithelial cells form a distinct, reticular, meshlike network and are present throughout the cortex and medulla. They play a pivotal role in the maturation of T cells in the thymus through the secretion of humoral substances and via direct contact with the thymic lymphocytes. Thymic epithelial cells can be broadly divided into four types based on slight variations in their ultrastructural, histologic, and IHC appearance: 1) subcapsular cortical epithelium, 2) inner cortical epithelium, 3) medullary epithelium, and 4) Hassall corpuscles. The latter are medullary epithelial cells arranged concentrically into round structures that can exhibit keratinization or calcification. Early reports have focused on the differential expression of keratin subclasses in the different types of thymic epithelial cells. Although broad-spectrum cytokeratins (CKs) 8 and 19 were found to be positive in all epithelial subsets (Fig. 11-1, A-B),5,7,8 CK 13/16 preferentially labeled subcortical, medullary, and some inner cortical
epithelial cells. Similar results for CK13 only have also been observed. In addition, CK18 has been found to be positive in a subset of medullary epithelial cells only.8 More recent studies concentrated on the immunophenotype of Hassall corpuscles have revealed positive reactions with low- and high-molecular-weight cytokeratins (HMWCKs), with significantly greater reactivity for the latter.9 In our own experience, CK5/6 is another member of the keratin family that consistently is found to be positive in thymic epithelial cells regardless of their subtype. Apart from keratins, other widely used markers that show diffuse reactivity in thymic epithelial cells are p63 and Pax-8 (see Fig. 11-1, C).6,10,11 Among negative markers of normal thymic epithelial cells, compared with thymic epithelial neoplasms, is c-Kit.12 Of note, some markers not normally associated with epithelial differentiation may be positive in a subset of thymic epithelial cells. These include D2-40 in subcapsular and corticomedullary epithelial cells,13 Bcl-2 in medullary epithelial cells,14 and S-100 protein in cells with a dendritic appearance within Hassall corpuscles.9
A
B
C
D
Figure 11-1 A, Normal thymic remnant tissue shows the typical biphasic composition of the thymic gland: epithelial cells and thymocytes (hematoxylin and eosin). B, Pancytokeratin immunostain decorates the epithelial cells in normal thymic tissue, including Hassall corpuscles. C, Nuclear Pax-8 staining of the epithelial cell component in normal thymic tissue. D, TdT decorates immature T lymphocytes, primarily in the cortical areas of normal thymic tissue.
Thymic Epithelial Neoplasms
Thymic T lymphocytes (“thymocytes”) occur in the different compartments at different stages of maturation. In the subcapsular and cortical zones, thymic lymphoblasts and smaller thymocytes undergo mitosis. As these cells mature, they migrate and move into the medulla. During this process, the IHC phenotype of the lymphocytes changes from an immature type in the cortex to that approaching a mature T-cell type in the medulla (see Fig. 11-1, D).5,15,16 Another cellular constituent of the normal thymus worth noting from an IHC point of view is the presence of B lymphocytes in the normal thymus. B lymphocytes in the form of lymphoid follicles with germinal centers are primarily seen in patients with myasthenia gravis or another autoimmune disease but may also be present in the thymus of healthy individuals.5 Scattered, isolated B cells can be found distributed along the fibrous septa, the perivascular spaces, or in the medulla, where they are concentrated around Hassall corpuscles.17 These B lymphocytes can be identified by using antibodies directed against CD19, CD20, and CD22.1719 Pax-8 has also been shown to decorate the B lymphocytes that populate the thymus, however, this has recently been shown to be due to cross reactivity with Pax-5.6,20 The normal thymic gland contains a number of other cell types that include connective tissue elements, inflammatory cells, myoid cells, neuroendocrine cells, and germ cells, the IHC phenotype of which corresponds to the tumors derived from these tissues; these will be discussed in the following sections.
Thymic Epithelial Neoplasms Thymic epithelial neoplasms can be divided into thymomas and thymic carcinomas. Thymomas are tumors of low-grade malignant potential that have retained the organotypical features of the normal thymic gland, whereas thymic carcinomas are overt malignant tumors characterized by invasive growth, cytologic atypia, and loss of an organotypical appearance. The heterogeneity of these tumors has been the source for the proposal of several classification systems, however, none of these schemas has gained unanimous approval.21-25 For practical purposes, thymic epithelial neoplasms can be divided into thymoma (World Health Organization [WHO] types A, AB, B1, and B2), atypical thymoma (WHO type B3), and thymic carcinoma. Tables 11-1 and 11-2 summarize the most pertinent IHC findings for thymic epithelial neoplasms.
Thymoma Immunohistochemically, the epithelial cells in thymoma can be targeted with a range of epithelial antibodies that include pancytokeratin (AE1/AE3), low-molecularweight cytokeratin (LMWCK; CAM5.2), HMWCK (34βE12), and CK5/6 (Fig. 11-2, A-B).7,26,27 Contrary to that, the reaction pattern with BerEP4 and MOC-31 is more variable with reported positive staining in up to 20% and 31% of cases, respectively.27 Two markers that
365
TABLE 11-1 Immunohistochemical Phenotype of Conventional Thymoma Compared with Spindle Cell Thymoma Conventional Thymoma*
Spindle Cell Thymoma
CK
+
+
CAM5.2
+
+
34βE12
+
+
CK5/6
+
+
p63
+
+
Pax-8
+
+
FOXN1
+
+
CD205
+
+
c-Kit
N
N
CD5
S
N
CK7
S
+
TTF-1
N
S
CD20
N
S
Synaptophysin
N
S
Calretinin
N
S
Bcl-2
N
+
SMA
N
S
Antibody
*World Health Organization type B thymomas. +, Positive; S, sometimes positive; N, negative; CK, cytokeratin; SMA, smooth muscle actin; TTF-1, thyroid transcription factor 1.
TABLE 11-2 Immunophenotype of Thymic Epithelial Neoplasms Antibody
Thymoma
Thymic Carcinoma
CK
+
+
CAM5.2
+
+
34βE12
+
+
CK5/6
+
+
p63
+
+
Pax-8
+
+
FOXN1
+
+
CD205
+
+
c-Kit
N
+
CD5
S
+
CK7
S
+
TTF-1
S
N
NK
N
Napsin A
+, Positive; S, sometimes positive; N, negative; NK, not known; CK, cytokeratin; TTF-1, thyroid transcription factor 1.
366
Immunohistology of the Mediastinum
A
B
C
D
Figure 11-2 A, Thymoma rich in lymphocytes obscures the epithelial cell component (hematoxylin and eosin). B, Pancytokeratin highlights the epithelial cells in thymoma. Note the characteristic lacelike interdigitating pattern of keratin staining. C, Pax-8 decorates the epithelial cells in thymoma in a nuclear fashion. D, Immature T lymphocytes with nuclear expression of TdT in thymoma.
show consistent immunoreactivity in thymic epithelial cells are Pax-8 and p63 (see Fig. 11-2, C). In the reported series, these markers have shown diffuse and strong nuclear reactivity in up to 100% of the cells.6,10,11,27 In 2007, Nonaka and colleagues28 applied two novel markers, FOXN1 and CD205, to a series of thymic epithelial neoplasms and found that these markers decorated thymic epithelial cells in 89% of spindle cell thymomas (WHO type A) and in as many as 100% of WHO type AB and B1 through B3 thymomas. Interestingly, D2-40 positivity can be seen in as many as 70% of thymomas, especially in those types that are rich in epithelial cells (WHO types B2 and B3).13,29 Two markers that often show positive staining in thymic carcinomas are c-Kit and CD5.12,30,31 When applied to thymomas, however, c-Kit is expressed only in as many as 4% of cases, whereas CD5 can be seen in as many as 28% of thymomas.12,27,28,32 The lymphocytes in thymoma are typically of an immature T-cell type and express leukocyte common antigen (LCA), CD3, CD1a, terminal deoxynucleotidyl transferase (TdT), and CD99 (see Fig. 11-2, D).6,15,33 In a similar fashion to normal thymic tissue, CD20-positive B lymphocytes can also be found in
thymomas, either scattered or in the form of germinal centers.34 Although spindle cell and mixed types (WHO types A and AB) generally show a staining pattern similar to the other types (Fig. 11-3, A-B), two recent studies have shown that the epithelial cells in these tumors can show expression of markers not usually associated with these neoplasms: as many as 90% of cases may show positivity for CD20, and CK7 reactivity is seen in 83% of spindle cell thymomas (see Fig. 11-3, C).33,35 In addition, focal expression of calretinin, synaptophysin, Bcl-2, and smooth muscle actin (SMA) is not unusual in spindle cell thymomas (see Fig. 11-3, D-E).35 Interestingly, although generally absent in the other types of thymoma,27 thyroid transcription factor 1 (TTF-1) has been demonstrated in isolated cases of type A and A B thymomas (see Fig. 11-3, F).35,36 Thymomas, especially those with a rich lymphocytic component (types B1 and B2) are sometimes difficult to distinguish from lymphoblastic lymphoma, especially on small mediastinoscopic biopsies. In this scenario, the most useful immunostain is a pancytokeratin. Thymomas will typically display a finely arborized network of interconnecting epithelial cell processes between the
Thymic Epithelial Neoplasms
A
B
C
E
367
D
F
Figure 11-3 A, Spindle cell thymoma is characterized by a bland proliferation of epithelial spindle cells with scanty lymphocytes (hematoxylin and eosin). B, The spindled epithelial cells in spindle cell thymoma are highlighted by nuclear Pax-8 expression. C, Prominent cytokeratin 7 staining in spindle cell thymoma. D, Synaptophysin staining is not an unusual occurrence in spindle cell thymoma. E, Prominent Bcl-2 expression by spindle cell thymoma. F, Focal staining for TTF-1 can be seen in spindle cell thymoma.
lymphocytes, manifesting a prominent lacelike pattern that is not seen in lymphoblastic lymphoma (see Fig. 11-2, B).37-39 This pattern is essential for the differential diagnosis, because lymphomas of the thymus may contain entrapped nonneoplastic thymic epithelial cells that are visible on keratin stains, but they should lack the interconnected pattern seen in thymoma.40
THYMOMA VARIANTS
Several thymomas defy classification as one of the conventional types proposed by the WHO,25 and these deserve special mention, not only for their distinct morphologic appearance but also because familiarity with their IHC phenotype is of utmost importance.
368
Immunohistology of the Mediastinum
Micronodular Thymoma with Lymphoid Hyperplasia
This variant was first described by Suster and Moran41 in 1999 and represents a subtype of spindle cell thymoma (WHO type A). It is characterized by a micronodular arrangement of neoplastic spindle-shaped thymic epithelial cells embedded in a lymphoid stroma, showing prominent follicular hyperplasia with the presence of germinal centers. As with the epithelial cells in conventional thymoma, those in micronodular thymoma are characterized by a CK-, CAM5.2-, AE1/AE3positive immunophenotype with occasional focal staining also for Bcl-2.41,42 More importantly, the lymphoid stroma can be composed of a mix of mature B (LCA+, CD20+) and T (CD3+, CD5+) lymphocytes along with scattered immature T cells (CD1a+, CD99+).33,41,42 Adenomatoid Spindle Cell Thymoma
Another variant of spindle cell (type A) thymoma is referred to as adenomatoid spindle cell thymoma.43 This tumor has a prominent adenomatoid appearance with a reticular or microvesicular growth pattern that can easily be confused with other tumors such as adenomatoid, smooth muscle, or yolk sac tumors. Contrary to these neoplasms, however, the spindle cells will express epithelial markers such as CAM5.2 and epithelial membrane antigen (EMA), whereas α-fetoprotein (AFP), SMA, desmin, and S-100 are negative. Care should be taken when applying calretinin to the panel of immunomarkers, because this stain may show focal staining in the epithelial cells of adenomatoid spindle cell thymoma.43 Spindle Cell Thymoma with Papillary or Pseudopapillary Features
Another subtype of spindle cell (type A) thymoma is the recently described spindle cell thymoma with papillary or pseudopapillary features.44 A tumor composed of spindled thymic epithelial cells with areas composed of papillary or pseudopapillary structures, this variant runs the risk of being mistaken for primary papillary thymic carcinoma, papillary thyroid carcinoma, or vascular neoplasms. The use of IHC markers in the former case will be futile, because both neoplasms express similar immunomarkers, and the pathologist will have to return to morphology to make the distinction. In the latter cases, however, the use of CD31 and thyroglobulin can help distinguish among these neoplasms, because spindle cell thymoma with papillary or pseudopapillary features will be negative for these markers and will stain with CK and CK5/6 instead. Thymoma with Pseudosarcomatous Stroma
Thymoma with pseudosarcomatous stroma is an unusual histologic variant of thymoma with a striking biphasic appearance.45 These tumors are composed of anastomosing islands of polygonal thymic epithelial cells separated by a cellular spindle cell proliferation devoid of any cytologic atypia. The biphasic composition of these tumors may simulate carcinosarcoma or sarcomatoid carcinoma. In keeping with conventional thymoma, the epithelial component of these tumors shows reactivity
for CK, CAM5.2, EMA, AE1/AE3, p63, and E-cadherin; the spindle cells show a more variable immunophenotype and, in addition to vimentin, may show focal staining with SMA or even with epithelial markers.45-48 The scarce lymphoid component is identical to that seen in conventional thymoma and consists primarily of lymphocytes positive for CD3, CD5, CD99, and TdT.45,47 Thymoma with Signet-Ring Cell–Like Features
This thymoma is composed entirely of epithelial cells with cytoplasmic vacuolation, reminiscent of signet-ring cells, and a scarce lymphocytic infiltrate.49,50 Thymomas with signet-ring cell–like features can demonstrate reactivity for CK, AE1/AE3, CAM5.2, and CK5/6 within the epithelial cells and are reported to be negative for markers such as AFP, TTF-1, calretinin, CD31, S-100, vimentin, and carcinoembryonic antigen (CEA). Some of the latter reagents can help distinguish this unusual type of thymoma from its closest mimics, which include yolk sac tumor, metastatic carcinoma, and vascular neoplasms.49,50 Ancient (“Sclerosing”) Thymoma
The use of IHC stains in this type of thymoma is rather limited, because the epithelial component may be rather scarce. Most important in this scenario is the use of a CK stain to identify the epithelial nature of the tumor, because the differential diagnosis primarily includes keratin-negative lesions such as sclerosing mediastinitis, solitary fibrous tumor, or Hodgkin lymphoma.51,52 Rhabdomyomatous Thymoma
Rarely, thymomas present with a prominent myoid component, which may lead to diagnostic difficulties. In these thymomas, the two distinct cell populations, epithelial and myoid, will be intermixed, and whereas the one component can be targeted with stains such as AE1/AE3, CK, and BerEP4, the other will react with myoglobin, desmin, and vimentin.53,54 Awareness of this unusual tumor is important so as not to mistake it for a tumor of pure skeletal muscle lineage or carcinosarcoma. Plasma Cell–Rich Thymoma
Plasma cell–rich thymoma, as the name implies, is characterized by abundant stromal plasma cells of a polyclonal nature.55 Although plasma cells may be present in the normal thymus, their occurrence in thymomas is extremely rare and is often associated with autoimmune disorders.55-57 As in conventional thymoma, CK will positively stain the epithelial cells of this thymoma, whereas kappa and lambda stains can be used to assess clonality in the plasma cell component.55 Desmoplastic Spindle Cell Thymoma
Thymomas characterized by abundant stromal changes, such as hyalinized or myxoid collagen admixed with a young fibroblastic proliferation, are called desmoplastic spindle cell thymoma.58 CK, CK5/6, vimentin, Pax-8, and Bcl-2 have been shown to be expressed in the epithelial cell component of these tumors, but no reactivity for
Thymic Epithelial Neoplasms
markers such as desmin, EMA, SMA, S-100, and CD34 has been identified, distinguishing these thymomas from other tumors such as solitary fibrous tumor or synovial sarcoma.
KEY DIAGNOSTIC POINTS Thymoma Variants • Nodular thymoma with lymphoid hyperplasia • Adenomatoid spindle cell thymoma • Spindle cell thymoma with papillary or pseudopapillary features • Thymoma with pseudosarcomatous stroma • Thymoma with signet-ring cell–like features • Ancient (“sclerosing”) thymoma • Rhabdomyomatous thymoma • Plasma cell–rich thymoma • Desmoplastic spindle cell thymoma
369
Primary Thymic Carcinoma Thymic carcinomas are rare tumors, with an incidence of less than 1% of all adult cancers. These tumors represent a very heterogeneous group of tumors that displays a wide morphologic spectrum. No specific histologic features set these tumors apart from neoplasms of other organ systems, and metastatic disease to the mediastinum must be excluded clinically before a diagnosis of primary thymic carcinoma can be rendered. Although the majority of thymic carcinomas are of squamous cell, high-grade, undifferentiated, and lymphoepithelioma-like carcinoma types,59 various other types of thymic carcinoma have been described and will be discussed separately below. Thymic carcinoma demonstrates an epithelial phenotype characterized by diffuse immunoreactivity for CK5/6, CK7, and p63 (Fig. 11-4, A and B).27,60-65 In addition, two newer markers, FOXN1 and Pax-8, have recently been shown to be expressed by the majority of thymic carcinomas, making these markers a useful tool in the differential diagnosis of these tumors, most
A
B
C
D
Figure 11-4 A, Thymic carcinoma shows unequivocal malignant cytologic features (hematoxylin and eosin). B, Strong diffuse expression of pancytokeratin in thymic carcinoma. C, Nuclear expression of FOXN1 in thymic carcinoma. D, Strong and diffuse nuclear reactivity for Pax-8 in thymic carcinoma.
370
Immunohistology of the Mediastinum
E
G
importantly for non–small cell lung cancer (see Fig. 11-4, C and D).6,28,59 Contrary to thymoma, thymic carcinomas often show reactivity for c-Kit (see Fig. 11-4, E).12,66,67 In the past, CD5 had received a lot of attention because of its supposed high expression in thymic carcinoma cells and lack thereof in many other neoplasms.27,31,66,68,69 More recently, however, application of this antibody to a large series of thymic carcinomas resulted in less than 40% of positive tumor cases (see Fig. 11-4, F).59 In addition, CD5 has been shown to be expressed by a range of other neoplasms, including atypical thymoma (WHO type B3), which limits the use of this antibody for the diagnosis of thymic carcinoma.30,65,66 CD205, a promising new marker for thymic epithelial cells in thymoma, has shown rather variable results in thymic carcinoma with staining rates of less than 10% to 59% of tumor cells.28,59 It is important to note that thymic carcinomas can be positive for calretinin in a significant number of cases; this should be kept in mind when the differential diagnosis includes mesothelioma (see Fig. 11-4, G).27,59 Three additional markers deemed useful for distinguishing thymoma from thymic carcinoma are glucose transporter 1 (GLUT-1), L-type amino acid transporter 1 (LAT1), and mucin 1 (MUC1); these markers show significantly higher expression in thymic carcinomas than in
F
Figure 11-4, cont’d E, Membranous staining for c-Kit is a common finding in thymic carcinoma. F, Some cases of thymic carcinoma show a positive immunoreaction with CD5. G, Focal calretinin reactivity can sometimes be identified in thymic carcinoma.
thymomas.67,70,71 Of note, TTF-1, napsin, and CD30 have been consistently negative in thymic carcinoma.27,64,65,68,72,73 KEY DIAGNOSTIC POINTS Thymic Carcinoma • Pax-8, c-Kit, and CD5 may have utility in distinguishing thymic carcinoma from other thoracic carcinomas.
THYMIC CARCINOMA VARIANTS
Certain thymic carcinoma variants deserve special mention because of their unusual morphology and/or IHC phenotype. Micronodular Thymic Carcinoma with Lymphoid Hyperplasia
The counterpart to micronodular thymoma with lymphoid hyperplasia is a recently described variant of thymic carcinoma that displays overt cytologic atypia in the epithelial cells.74 In a fashion similar to micronodular thymoma, micronodular thymic carcinoma contains
Mediastinal Neuroendocrine Neoplasms
a prominent lymphoid stroma composed of a mix of mature B (CD20+) and T (CD3+/TdT−) cells. Kappa and lambda stains failed to demonstrate a monoclonal proliferation.74 Hepatoid Thymic Carcinoma
A very rare type of thymic carcinoma, hepatoid thymic carcinoma, contains large polygonal tumor cells that strongly resemble hepatocytes.73 The tumor cells are reported to express CK7, CK19, α1-antitrypsin, α1antichymotrypsin, and HepPar1 in the absence of staining for AFP, placental alkaline phosphatase (PLAP), or CD5 among others. Intratumoral CD1a-positive or TdT-positive lymphocytes were not identified.73 Papillary Thymic Carcinoma
On a morphologic basis, papillary thymic carcinoma is a tumor that must be distinguished from more common tumors, such as metastatic papillary thyroid carcinoma or papillary adenocarcinoma of the lung. Cases of papillary thymic carcinoma show reactivity for CEA, BerEP4, and CD15 and show variable staining for calretinin and CD5.75,76 Unlike thyroid or lung carcinomas of the papillary type, thyroglobulin and SP-A are reported to be negative in these tumors.75,76 Mucinous Adenocarcinoma of the Thymus
Primary mucinous adenocarcinomas of the thymus may be difficult to distinguish from metastatic mucinous carcinoma, primarily in lesions that originate from the colon or the lung. Immunohistochemically, most mucinous carcinomas of the thymus are positive for CK and CK7 and can show variable staining for CK20, CD5, and CDX-2. Among markers consistently negative are TTF-1, surfactant protein alpha, napsin, and Pax-8.59,77-80
371
Salivary Gland–Type Carcinomas of the Thymus
Two tumors that are most commonly seen in the salivary glands but that can also arise in other organ systems, including the thymic gland, are referred to as mucoepidermoid carcinoma and adenoid cystic carcinoma. It has to be emphasized that IHC stains do not generally play a major role in the diagnosis of these neoplasms. However, thymic mucoepidermoid carcinomas have been reported to express CK, CK7, and CK20 with variable staining for CD5 also, whereas TTF-1, synaptophysin, chromogranin A, S-100, CD99, and MUC2 have been negative.28,84 Thymic adenoid cystic carcinomas are even rarer than thymic mucoepidermoid carcinomas, however, the limited literature on this subject reveals positive staining of the tumor cells for CK5/6, p63, and 34βE12 along with focal synaptophysin reactivity. On the other hand, CD5, c-Kit, chromogranin A, and CD56 appear to be uniformly negative in these tumors.85,86 KEY DIAGNOSTIC POINTS Thymic Carcinoma Variants These thymic carcinoma variants require special clinicopathologic correlation because of limited use of immunohistochemistry in their diagnosis: • Micronodular thymic carcinoma with lymphoid hyperplasia • Hepatoid thymic carcinoma • Papillary thymic carcinoma • Mucinous adenocarcinoma of the thymus • Thymic carcinoma with rhabdoid features • Thymic clear cell carcinoma • Spindle cell thymic carcinoma • Salivary gland–type carcinomas of the thymus
Thymic Carcinoma with Rhabdoid Features
An extremely rare variant of thymic carcinoma is characterized by prominent rhabdoid cytoplasmic inclusions. In the few examples described for this tumor, reactivity of the tumor cells for CK and FOXN1 and focal staining for CK7 and vimentin have been described.59,72 Thymic Clear Cell Carcinoma
Clear cell carcinomas of the thymus have only rarely been described. They generally show a positive reaction for CK (both low- and high-molecular-weight keratins) and variable staining for CD5, CEA, and EMA, whereas Pax-8, FOXN1, p63, CK5/6, and TTF-1 have been negative.59,81,82 Spindle Cell Thymic Carcinoma
Spindle cell thymic carcinoma is a distinct subtype of thymic carcinoma characterized by its spindle cell morphology, and it can be easily mistaken for a soft tissue sarcoma. Unlike sarcoma, however, the spindle cells are positive for CAM5.2, whereas EMA and vimentin may show a variable staining result. Markers like actin, desmin, S-100 protein, human melanoma black 45 (HMB-45), CD34, CD5, and CD99 are usually negative.63,83
Mediastinal Neuroendocrine Neoplasms Primary neuroendocrine neoplasms of the mediastinum include neuroendocrine carcinomas of the thymic gland, paragangliomas, and parathyroid tumors. Table 11-3 summarizes the IHC features of mediastinal neuroendocrine tumors.
Thymic Neuroendocrine Carcinomas Primary neuroendocrine carcinomas of the thymus are rare neoplasms that account for approximately 2% to 4% of anterior mediastinal neoplasms.87,88 Clinically, a subset of cases can be associated with endocrine abnormalities or Cushing syndrome.89 Histologically, the tumors can be divided into low-grade neuroendocrine carcinoma (carcinoid tumor; Fig. 11-5, A), intermediategrade neuroendocrine carcinoma (atypical carcinoid), and high-grade neuroendocrine carcinoma (small cell carcinoma).89 Of note, these tumors can show a spectrum of distinct morphologic growth patterns that include spindle cell,90 oncocytic,91 and angiomatoid
372
Immunohistology of the Mediastinum
TABLE 11-3 Immunophenotype of Mediastinal Neuroendocrine Neoplasms Antibody
Neuroendocrine Carcinoma of Thymus
Mediastinal Paraganglioma
Parathyroid Tumors
Cytokeratin
+
N
+
Synaptophysin
+
+
+
Chromogranin
+
+
+
CD56
+
+
+
CD57
+
+
+
S-100 protein
N
+ (sustentacular cells)
N
Parathyroid hormone
N
N
+
+, Positive; S, sometimes positive; N, negative.
types92 and thymic carcinoid with mucinous stroma.93 Immunohistochemically, these tumors show a similar phenotype independent of grade of differentiation or histologic subtype. Neuroendocrine carcinomas of the thymus show consistent positivity for pancytokeratin (often in a paranuclear pattern in the high-grade type), CAM5.2, synaptophysin, chromogranin A, neuronspecific enolase (NSE), CD56, and CD57 (see Fig. 11-5, B).89,90,92-96 Furthermore, even the lesions that have not
A
C
produced a clinical endocrinopathy can show variable levels of specific peptide hormones such as adrenocorticotropic hormone (ACTH), somatostatin, serotonin, or calcitonin.97,98 Currently, no reliable markers are available to distinguish between primary (thymic) and metastatic neuroendocrine carcinomas to the mediastinum; TTF-1 can be seen in as many as 96% of pulmonary neuroendocrine carcinomas,99,100 but no meaningful data are available on the expression of this marker in
B
Figure 11-5 A, Low-grade neuroendocrine carcinoma (carcinoid tumor) of the thymus (hematoxylin and eosin). B, Thymic neuroendocrine carcinoma positive for chromogranin A. C, Pax-8 expression can be seen in 50% of thymic neuroendocrine carcinomas.
Mesenchymal Neoplasms of the Mediastinum
primary thymic neuroendocrine carcinomas. On the other hand, we recently observed staining for Pax-8 in as many as 50% of thymic neuroendocrine carcinomas (unpublished data; see Fig. 11-5, C). Closer exploration of this marker in neuroendocrine tumors of the thymus and other locations may reveal the usefulness of the marker in the differential diagnosis of these tumors.
Mediastinal Paragangliomas Intrathoracic paragangliomas can arise in the posterior mediastinum along the vertebral column and also occur in the anterior and middle mediastinal compartments. They account for less than 10% of mediastinal neuroendocrine tumors.101 Both functional paragangliomas, which produce catecholamines, and nonfunctional types can be found in the mediastinum.101 The neuroendocrine nature of these tumors is reflected in the expression of neuroendocrine IHC markers such as chromogranin, synaptophysin, CD56, and NSE. Usually, S-100 protein highlights the sustentacular cells that scaffold the typical Zellballen growth of the tumor cells. In addition, immunostaining for vimentin, corticotrophin, somatostatin, glucagon, and calcitonin has also been described.102 An important feature of paragangliomas is the complete lack of cytokeratin staining, which makes this marker a useful tool in the separation of these tumors from neuroendocrine carcinomas.101-104
Parathyroid Tumors of the Mediastinum Primary parathyroid tumors—parathyroid adenomas and parathyroid carcinomas—that occur in the mediastinal compartment are unusual, and these tumors are most likely derived from ectopic parathyroid tissue.105,106 Clinically, the majority of patients come to medical attention with hyperparathyroidism characterized by hypercalcemia and hypophosphatemia, especially in cases of parathyroid carcinoma. Histologically, these tumors may bear a considerable resemblance to thymic neuroendocrine carcinomas in some cases, and paraganglioma may also enter the differential diagnosis.89,101,105,106 Parathyroid tumors share the expression of neuroendocrine markers (chromogranin A and synaptophysin) and show reactivity for cytokeratin in a similar fashion to neuroendocrine carcinomas.89 What sets them apart from their closest mimics is the positivity for parathyroid hormone, which is restricted to parathyroid tumors in this context.89,107
Mesothelial Neoplasms of the Mediastinum Neoplasms of mesothelial differentiation that arise in the mediastinum include the benign adenomatoid tumor and malignant mesothelioma. These tumors either have their origin in the pericardium or they arise from the hilar reflections of the pleural surfaces.108,109 In the case of malignant mesothelioma, the whole histologic spectrum seen in the pleural cavities can also be
373
identified in the mediastinum, including epithelioid, biphasic, and sarcomatoid forms. The immunophenotype of adenomatoid tumors and malignant mesothelioma is virtually identical and does not differ significantly from that of their corresponding lesions in other sites. Adenomatoid tumors and epithelioid malignant mesothelioma show fairly consistent reactivity for pancytokeratin, CAM5.2, CK5/6, calretinin, Wilms tumor 1 (WT1), and D2-40.109-115 More variable reactivity has been noted for CK7, thrombomodulin, Hector Battifora mesothelial cell 1 (HBME-1), and vimentin.108,109,111,116,117 Sarcomatoid mesotheliomas can lack expression of these mesothelial markers and often only stain with cytokeratin. To increase sensitivity, the use of calretinin in combination with D2-40 has been advocated in this context.110,118 It should be noted, however, that a subset of thymic epithelial lesions can show focal calretinin positivity.27,35,59 In this scenario, Pax-8 may be of aid, being expressed by the majority of thymomas and thymic carcinomas but not mesotheliomas.6,59,119 Specialized markers of carcinomatous differentiation—such as CEA, CD15, p63, TTF-1, MOC-31, and BerEP4—are absent in adenomatoid tumors and malignant mesothelioma.108,109,111-114,120
Mesenchymal Neoplasms of the Mediastinum Fibrous, Fibrohistiocytic, and Myofibroblastic Mediastinal Proliferations Mesenchymal neoplasms of fibrous, fibrohistiocytic, and myofibroblastic origin that have been described in the mediastinum include fibromatosis (desmoid tumor), sclerosing mediastinitis (idiopathic fibroinflammatory lesion of the mediastinum), solitary fibrous tumor, synovial sarcoma, inflammatory pseudotumor (inflammatory myofibroblastic tumor), and malignant fibrous histiocytoma (pleomorphic sarcoma). Fibromatosis or desmoid tumor is a low-grade soft tissue lesion composed of bland fibroblastic/myofibroblastic cells that show locally infiltrative growth and a high propensity for recurrence.121 The recognition that these tumors are driven by β-catenin mutations has led to the discovery that in addition to rather nonspecific reactivity for vimentin and SMA, the cells of fibromatosis show distinct nuclear and cytoplasmic staining for β-catenin, as opposed to mere cytoplasmic staining in a range of other fibroblastic or myofibroblastic lesions.121-124 Importantly for the differential diagnosis, markers like CD34, c-Kit, and desmin are usually negative.121,124 A morphologically similar appearing lesion is sclerosing mediastinitis, a fibrosing process thought to be inflammatory in etiology, composed of bland spindle cells in a collagenous stroma and mixed inflammatory cells.125 The spindle cells in sclerosing mediastinitis show no distinct IHC phenotype, show only variable reactivity with vimentin and SMA, and are negative for CD34.125,126 Solitary fibrous tumors most commonly involve the pleura but may occasionally also arise in the mediastinum.127 These tumors are characterized by a spindle
374
Immunohistology of the Mediastinum
cell proliferation growing in a “patternless pattern” with variable collagen deposition and a prominent hemangiopericytoma-like vasculature (Fig. 11-6, A). Most importantly, these tumors show rather consistent reactivity for CD34, Bcl-2, CD99, and vimentin; variable reactivity for desmin; and lack expression of cytokeratin, EMA, S-100 protein, SMA, and CD31 (see Fig. 11-6, B and C).127-129 Synovial sarcoma is a distinctive soft tissue neoplasm that most commonly occurs in the extremities of young adults and rarely in the mediastinum.130,131 These tumors can present in the mediastinum either as anterior or posterior (paravertebral) mediastinal masses, and they are spindle cell tumors that can occur in this location as monophasic (pure spindle cells) or biphasic neoplasms (mixed spindle and epithelial cells; Fig. 11-7, A).131 Similar to their soft tissue counterparts, immunohistochemically, mediastinal synovial sarcomas are characterized by strong positivity for vimentin, Bcl-2, and CD99 and by focal expression of cytokeratin, CAM5.2, or EMA (predominantly in the epithelial component of the biphasic type but also in the monophasic variant; see Fig. 11-7, B and C).130-132 CD34 is of value in the differential diagnosis of this tumor from its closest mimic, solitary fibrous tumor, being positive in the latter and negative in synovial sarcoma.130
A
C
Another interesting mesenchymal neoplasm that is rare in general and even rarer in the mediastinum is inflammatory pseudotumor (inflammatory myofibroblastic tumor).133,134 These tumors characteristically are composed of a mix of bland fibroblastic and myofibroblastic spindle cells admixed with inflammatory cells, in particular plasma cells. True to their myofibroblastic origin, the neoplastic spindle cells are generally positive for vimentin, SMA, muscle-specific actin (MSA), and calponin and can sometimes also stain with desmin and even cytokeratin (as many as 14% of cases).135-139 The most important feature, however, is the fact that inflammatory pseudotumors harbor ALK gene rearrangements in the majority of cases, and this is reflected by expression of ALK on a protein level in as many as 64% of cases.136,138,139 Because the plasma cell infiltrate may be so pronounced as to mimic a plasma cell neoplasm, clonality studies with kappa and lambda antibodies may be necessary to demonstrate a polyclonal cell population.137 More recently, inflammatory pseudotumor has been linked to IgG4-related sclerosing disease based on morphologic similarities and the fact that IgG4-positive plasma cells are often a component of inflammatory pseudotumors.139 Lastly, malignant fibrous histiocytoma (pleomorphic sarcoma) is a highly malignant spindle cell tumor that often occurs in the extremities of elderly
B
Figure 11-6 A, Mediastinal solitary fibrous tumor (SFT) characterized by a bland spindle cell proliferation and ropelike collagen (hematoxylin and eosin). B, CD34 is typically strongly and diffusely positive in SFT of the mediastinum. C, Bcl-2 is another marker that commonly shows a strong reaction in mediastinal SFT.
Mesenchymal Neoplasms of the Mediastinum
A
C patients. In the mediastinum, these tumors are often found to be metastatic, but if primary, they may arise from the thymus or the mediastinal soft tissue.140,141 Because of their fibrohistiocytic differentiation, the only positive markers often are vimentin and CD68, and tumor cells generally lack expression of cytokeratin, SMA, S-100 protein, CD34, or CD45.
Myogenic Neoplasms of the Mediastinum Primary mediastinal neoplasms with smooth muscle differentiation are rare tumors thought to arise either from small vessels of the mediastinal soft tissue, from displaced heterotopic smooth muscle, or as detached parasitic tumors that originate from the esophageal wall.142,143 These neoplasms can be found in either the anterior or posterior compartments and include both benign and malignant forms.142-146 These mediastinal spindle cell malignancies show the same IHC phenotype as their counterparts elsewhere and have been shown to be uniformly reactive for SMA, desmin, MSA, and vimentin,142,143,146 whereas more variable results have been reported for h-caldesmon (Fig. 11-8, A-B).145-147 Conversely, cytokeratin, CK5/6, calretinin, WT1, MyoD1, myoglobin, myogenin, and S-100 protein are
375
B
Figure 11-7 Monophasic synovial sarcoma arising in the mediastinum. A, Hematoxylin and eosin. B, Diffuse expression of Bcl-2. C, Focal and weak membranous expression of epithelial membrane antigen.
absent in smooth muscle tumors, thereby distinguishing them from other spindle cell neoplasms, namely, spindle cell thymoma, spindle cell thymic carcinoma, rhabdomyosarcoma, and sarcomatoid mesothelioma.143-146 Pure rhabdomyosarcomas of the mediastinum are extremely rare neoplasms that predominantly affect the young adult population (Fig. 11-9, A).148 Mediastinal rhabdomyosarcomas are more often identified as components of a GCT, teratoma, or carcinosarcoma and only rarely consist of pure rhabdomyosarcomatous elements.148 Histologically, they can show alveolar, embryonal, spindle cell, and pleomorphic architectural features.148 Mediastinal rhabdomyosarcomas express desmin and MSA with vimentin (see Fig. 11-9, B).148 Myoglobin staining is only focally observed and is usually present only in large maturing rhabdomyoblasts, therefore it is not a particularly useful marker for rhabdomyosarcomas composed predominantly of small cells. Focal, weak SMA and S-100 protein–positive cells can also be observed.148 The specificity of desmin and actin for the diagnosis of rhabdomyosarcoma can be challenged, because these determinants are also observed in smooth muscle– derived neoplasms. However, several nuclear proteins appear to be restricted to striated muscle—namely, MyoD1 and myogenin—that can be applied effectively to the differential diagnosis in question (see Fig. 11-9,
376
Immunohistology of the Mediastinum
A
B
Figure 11-8 A, Primary leiomyosarcoma of the mediastinum (hematoxylin and eosin). B, Smooth muscle actin positivity in mediastinal leiomyosarcoma.
C).149,150 In this context, a recent study to investigate the use of p63 in various myogenic neoplasms revealed that strong and diffuse cytoplasmic p63 reactivity is a good marker for skeletal muscle differentiation as opposed to very focal and weak cytoplasmic staining in smooth muscle tumors.151
A
C
More recently, aberrant expression of epithelial and neuroendocrine markers has been demonstrated in as many as 40% of cases of a large series of alveolar rhabdomyosarcomas, emphasizing the need to use a more extensive immunopanel in the diagnosis of small round cell neoplasms.152
B
Figure 11-9 Rhabdomyosarcoma of the mediastinum. A, This tumor shows numerous rhabdomyoblasts (hematoxylin and eosin). B, Desmin immunostain decorates the tumor cells. C, Diffuse reactivity of MyoD1 is shown.
Mesenchymal Neoplasms of the Mediastinum
Adipocytic Neoplasms of the Mediastinum Virtually all benign and malignant adipocytic neoplasms of the soft tissues can also arise from mediastinal structures. Although in most of these cases, the diagnosis is primarily based on the typical morphologic features of these tumors, advances in the discovery of the cytogenetic changes leading to the development of some of these tumors have also resulted in new immunomarkers that can be used to differentiate some of these lesions from other adipocytic or nonadipocytic mimics. For instance, the discovery that well-differentiated and dedifferentiated liposarcomas are characterized genetically by amplification of MDM2 and CDK4 genes on chromosome 12q13-15 has led to the observation that the products of these genes can also be detected on a protein level. Thus diffuse and strong nuclear expression of MDM2 and CDK4 can be used to distinguish well-differentiated and dedifferentiated liposarcomas from benign adipose tumors and other poorly differentiated sarcomas.153 In a similar manner, He and colleagues154 found that nuclear expression of p16 is seen in well-differentiated/dedifferentiated liposarcomas but not lipomas. The IHC phenotype of other benign and malignant lipomatous tumors is less specific and may vary quite considerably. Myxoid liposarcomas have been reported to be positive for vimentin and may stain with S-100 protein in as many as 40% of cases155; pleomorphic liposarcomas may show reactivity for SMA (45% of cases), cytokeratin 21%, S-100 protein (33%), and desmin (13%),156 whereas spindle cell lipomas are consistently positive for CD34 and negative for S-100 protein.157 Interestingly, normal and neoplastic adipose tissue can show significant expression of calretinin, which is therefore interpreted to be a useful, albeit nonspecific, marker of adipocytic differentiation.158
Neuroectodermal Tumors of the Mediastinum Although the majority of neuroectodermal-derived neoplasms occur in the posterior mediastinum, rarely these
A
377
tumors may arise in association with the thymus or in other compartments of the mediastinum. Neural tumors of the mediastinum derived from Schwann cells include neurofibroma, schwannoma (Fig. 11-10, A), and their malignant counterpart, malignant peripheral nerve sheath tumor (MPNST). Both neurofibromas and schwannomas express S-100 protein, although schwannomas do so more diffusely and consistently than neurofibromas (see Fig. 11-10, B).159 In addition, neurofibromas can show reactivity for neurofilament protein (NFP) and also CD34 in a characteristic “fingerprint” pattern, and a subset of schwannomas can also show immunoreactivity for glial fibrillary acidic protein (GFAP), podoplanin, calretinin, SOX10, and, very rarely, cytokeratin.159-162 MPNST shows a very similar IHC phenotype, however, expression of S-100 protein is usually more focal, and this is true also for NFP, podoplanin, and SOX10.159 A rare tumor commonly associated with neurofibromatosis type 1 (NF1) is an MPNST with heterologous differentiation in the form of skeletal muscle, the malignant Triton tumor. The rhabdomyosarcomatous elements in this tumor can be identified by using desmin, MyoD1, MSA, and myogenin in keeping with skeletal muscle differentiation.163 Ganglioneuroma, ganglioneuroblastoma, and neuroblastoma are another set of neoplasms that can arise in the mediastinum, primarily the posterior compartment. These tumors mainly affect children and young adults but have also been described in older patients.164-167 Whereas ganglioneuroma represents the benign, mature end of this spectrum of tumors, neuroblastoma forms the malignant, immature one. Ganglioneuromas are composed of two cell types, spindle cells and ganglion cells. The spindle cell component of these tumors usually stains with NFP and S-100 protein.168,169 NeuN, synaptophysin, and to a lesser extent chromogranin A are helpful determinants for the labeling of ganglion cells, which may be focal or widely scattered in ganglioneuromas.169,170 On the other hand, neuroblastomas are high-grade tumors composed of sheets of small round cells and varying amounts of neuropil that can easily mimic other small round cell neoplasms such as rhabdomyosarcoma, primitive neuroectodermal tumor
B
Figure 11-10 A, Mediastinal schwannoma with extensive degenerate changes (hematoxylin and eosin). B, S-100 protein outlines the Schwannian origin of mediastinal schwannoma.
378
Immunohistology of the Mediastinum
(PNET), or lymphoma. The small round cells in neuroblastoma show variable reactivity for NSE, NFP, chromogranin A, synaptophysin, CD57, and CD56.167,171-173 In addition, NB84 is a monoclonal antibody raised specifically against neuroblastoma; although it is not specific for that neoplasm, this marker is seen in the great majority of neuroblastic tumors.174,175 Neuroblastomas are universally devoid of markers of myogenous differentiation (desmin, MyoD1, and myogenin), hematolymphoid lineage (CD45), and epithelial differentiation (CK, EMA, or CD99). PNETs may rarely occur in the mediastinum, either in the anterior or in the posterior compartments (Fig. 11-11, A). The immunophenotype of PNETs is similar to that of neuroblastoma, in that PNET may be labeled for NFP, NSE, S-100 protein, CD57, and synaptophysin.176,177 Moreover, expression of CD99 is consistently seen in PNET but not in neuroblastoma (see Fig. 11-11, B).176,178 Of note, divergent differentiation can also demonstrate focal reactivity for CK.179 More recently, two new markers have emerged as potential sensitive tools in the differential diagnosis of small round cell soft tissue neoplasms: antibodies to FLI1 and CAV1 gene products.178,180 These markers show nuclear staining in as many as 96% of PNETs but have not been detected in rhabdomyosarcoma, desmoplastic small round cell tumors, or pleomorphic sarcomas. Another unusual tumor of neuroectodermal origin at this site is malignant melanoma. Primary malignant melanomas of the mediastinum are very infrequent but have been described sporadically in the pediatric and adult population.181-184 Similar to melanomas in other organ systems, mediastinal melanomas are characterized by IHC expression of vimentin, S-100 protein, melan-A, and HMB-45.184 In recent years, two neural crest transcription factors, SOX9 and SOX10, have been found to play a crucial role in melanogenesis and have been shown to be sensitive and specific markers for melanocytic tumors.185,186 Ependymomas commonly arise in the central nervous system but may also arise in a number of extraaxial sites
A
such as the mediastinum, where they originate primarily in a paravertebral location in the posterior mediastinum. Ependymomas are characteristically diffusely positive for GFAP, S-100 protein, and CD99 and show variable reactivity for a range of cytokeratins including AE1/AE3, CAM5.2, CK7, HMWCK, and CK18.187-190 Recent discovery of estrogen and progesterone receptor expression in these tumors has also been reported.188 The neuroendocrine markers chromogranin A and synaptophysin, however, are uniformly negative.187-189
Vascular and Perivascular Neoplasms of the Mediastinum The lesions in this category comprise hemangioma, lymphangioma, glomus tumor, epithelioid hemangioendothelioma, and angiosarcoma. Hemangioma and lymphangioma are benign neoplasms that can affect the mediastinum. They often occur in children but can also be diagnosed in the adult population. Diagnosis of these lesions is usually straightforward, but IHC stains can be used to confirm the diagnosis. Hemangiomas and lymphangiomas can show an overlapping IHC phenotype, because they both show a positive reaction with factor VIII (FVIII)–related antigen and CD31.191-193 Contrary to hemangiomas that often also show positivity for CD34,191 lymphangiomas tend to express D2-40 (podoplanin) and lymphatic endothelial receptor factor.191 Glomus tumor is a neoplasm thought to be of perivascular origin. These tumors can arise in many anatomic locations and can also occur in the mediastinum, most often in the posterior or superior compartments (Fig. 11-12, A). Glomus tumors are typically strongly and diffusely positive for smooth muscle markers (SMA, MSA, and calponin) and can show variable coexpression for CD34 and collagen type IV (see Fig. 11-12, B).194-197 Also, desmin may be focally positive in isolated cases.194-197 Markers that have been proved consistently negative include cytokeratin, synaptophysin, chromogranin A, CD31, and S-100 protein.195,196
B
Figure 11-11 A, Primitive neuroectodermal tumor of the mediastinum characterized by a proliferation of small round cells (hematoxylin and eosin). B, Consistent expression of CD99 is a common finding in primitive neuroectodermal tumors.
Mediastinal Germ Cell Tumors
A
379
B
Figure 11-12 A, Glomus tumor of the posterior mediastinum (hematoxylin and eosin). B, Mediastinal glomus tumor shows a diffuse and strong reaction with smooth muscle antigen.
Epithelioid hemangioendotheliomas and angiosarcomas are malignant vascular neoplasms that have been reported to arise in the anterior mediastinum (Fig. 11-13, A).191,198 They fundamentally express the same IHC markers and are best distinguished based on different morphologic features. FVIII-related antigen, CD31, CD34, von Willebrand factor (vWF), and Ulex europaeus lectin show consistent labeling of the cells in both tumors (see Fig. 11-13, B).191,198-200 Importantly, a subset of both tumors may show focal cytokeratin positivity that necessitates the use of a more extensive immunopanel in equivocal cases.200,201 Interesting results have also been observed with two novel markers in the context of benign and malignant vascular neoplasms. Recently, γ-synuclein has been investigated in a series of lymphoid proliferations, as well as vascular neoplasms, and was found to be strongly expressed by the neoplastic endothelial cells of hemangiomas, Kaposi sarcomas, and angiosarcomas.202 In addition, claudin-5 has been found to be expressed in epithelioid hemangioendothelioma, Kaposi sarcoma, and angiosarcoma.203 A caveat, however, is that both
A
markers also label aggressive carcinomas, thereby limiting their specificity for vascular neoplasms. The most important IHC characteristics of mediastinal vascular neoplasms are summarized in Table 11-4.
Mediastinal Germ Cell Tumors Pure or combined mediastinal GCTs with seminomatous, embryonal carcinomatous, yolk sac tumor, and choriocarcinomatous elements all have the potential to arise in the mediastinum or to involve it metastatically.204-209 Immunohistology is often indispensable for characterizing such neoplasms, especially in limited tissue samples. The IHC phenotype of germ cell neoplasms has been largely studied in the gonads, and only occasional studies have tried to investigate primary mediastinal GCTs in this context. In the last 5 years, a dramatic increase has been seen in the number of novel markers that have proven useful for GCTs. The most common nonteratomatous primary mediastinal GCT is seminoma (Fig. 11-14, A).205,210 Besides the traditional
B
Figure 11-13 A, High-grade angiosarcoma of the anterior mediastinum (hematoxylin and eosin). B, CD31 confirms the vascular nature of mediastinal angiosarcoma.
380
Immunohistology of the Mediastinum
TABLE 11-4 Immunophenotype of Mediastinal Vascular Neoplasms Hemangioma
Lymphangioma
Epithelioid Hemangioendothelioma
Angiosarcoma
Glomus Tumor
Factor VIII–related antigen
+
+
+
+
N
CD34
+
N
+
+
S
CD31
+
+
+
+
N
D2-40/podoplanin
N
+
S
S
N
CK
N
N
S
S
N
γ-Synuclein
+
NK
NK
+
N
Claudin-5
N
N
+
+
N
SMA
N
N
N
N
+
Antibody
+, Positive; S, sometimes positive; N, negative; NK, not known; CK, cytokeratin; SMA, smooth muscle actin.
markers used to detect seminomatous differentiation, such as vimentin and placental alkaline phosphatase (PLAP; see Fig. 11-14, B),205,211 more recent studies have shown that positive markers for this neoplasm include c-Kit, D2-40, Pouf5F1 (Oct-3/4), SALL4, TCL1A, SOX17, and melanoma-associated gene C2 (MAGEC2; see Fig. 11-14, C-E).208,212-218 Contrary to that, SOX2, glypican-3, and CD30 have consistently proved to be negative in this tumor.212,215,219,220 Interestingly, reactivity for cytokeratin has been described in a significant number of mediastinal seminomas (up to 80%)208,211 in contrast to only 20% of testicular seminomas.211 Embryonal carcinomas can show significant IHC overlap with seminoma. Markers such as cytokeratin, PLAP, c-Kit, Pouf5F1, and SALL4 may also show positive staining in embryonal carcinoma.211,213,214,216,218 However, CD30 and SOX2 appear to be exclusively expressed by embryonal carcinoma.212,220 In contrast to seminoma and yolk sac tumor, glypican-3, SOX17, MAGEC2, and TCL1A are consistently negative in embryonal carcinoma.212,217,219,220 Yolk sac tumors show coexpression of cytokeratin, AFP, glypican-3, and c-Kit but not Pouf5F1, unlike both seminoma and embryonal carcinoma, which do not express glypican-3 but do stain with Pouf5F1.213,214,219,221 As in all other GCT types, yolk sac tumors also show reactivity for SALL4,213 and Pax-2 and Pax-8 have recently been demonstrated to be positive in 50% and 25% of yolk sac tumors respectively.222 Choriocarcinoma is also globally CK reactive and shows staining of the syncytiotrophoblasts for β-human chorionic gonadotropin (β-hCG),211 but it is further typified by the immunohistologic presence of glypican-3, SALL4, and D2-40, 213,218,219 whereas CEA and TCL1A are uniformly negative.212,223 Finally, teratomas are highly variable tumors composed of cells from different germ cell layers. Hence, no consistent IHC phenotype is observed, and few studies have included these tumors in their series. A reported positive marker for teratomas is EMA, and variable reactivity has been described for SOX2 and SOX17.213,216,220 Negative results have been obtained with Pouf5F1, TCL1A, D2-40, c-Kit, CD30, and glypican-3.212,219,224 Table 11-5 summarizes the IHC characteristics of mediastinal GCTs.
KEY DIAGNOSTIC POINTS Seminoma vs. Embryonal Carcinoma • CD30 and SOX2 appear to be exclusively expressed by embryonal carcinoma. • In contrast to seminoma and yolk sac tumor, glypican-3, SOX17, MAGEC2, and TCL1A are consistently negative in embryonal carcinoma.
Histiocytic Tumors of the Mediastinum Very rarely, the mediastinum may be the site of origin of histiocytic proliferations. One of these, Langerhans cell histiocytosis (LCH), is a clonal disorder believed to be derived from the dendritic system. It often affects children but can also occur in adults and has occasionally been described as a primary mediastinal lesion.225-228 LCH is characterized by diffuse IHC expression of the neoplastic cells of S-100 protein and CD1a and patchy expression of CD68.229,230 Two newer markers that have been described in these lesions are langerin, which has been presented as a sensitive and relatively specific marker for LCH,231 and fascin, a highly selective marker for dendritic cell derivation.232 Another lesion that falls into this category is RosaiDorfman disease, also called sinus histiocytosis with massive lymphadenopathy. Contrary to LCH, this condition is primarily seen in adults and can sporadically involve the soft tissue of the mediastinum.233,234 Immunohistochemically, Rosai-Dorfman disease shows a similar staining pattern to LCH, with consistent positivity of lesional cells for S-100 protein and CD68.235-237 The most important discriminator between the two lesions in this context is CD1a, which is not expressed by the cells of Rosai-Dorfman disease.238
Lymphoproliferative Disorders of the Mediastinum Lymphoproliferative lesions can either arise in the mediastinum as primary disease or they can involve it
Lymphoproliferative Disorders of the Mediastinum
A
B
C
D
E
in a systemic manner. Included are lymphoproliferative disorders such as Castleman disease, Hodgkin and nonHodgkin lymphomas, and leukemias; among these the most common tumors are Hodgkin, mediastinal large B-cell, and lymphoblastic lymphoma. Lymphoproliferative disorders usually present as large, bulky mediastinal masses that cannot be differentiated radiographically from other mediastinal tumors. Table 11-6 summarizes the most pertinent IHC properties of mediastinal
381
Figure 11-14 A, Primary mediastinal seminoma showing a proliferation of neoplastic tumor cells with a lymphocytic component (hematoxylin and eosin). B, Placental alkaline phosphatase stains the tumor cells in a mediastinal seminoma. C, Strong membranous expression of c-Kit in a seminoma of mediastinal origin. D, Nuclear expression of Pouf5F1 (Oct-3/4) in mediastinal seminoma. E, SALL4 is another marker that stains mediastinal seminomas in a nuclear fashion.
lymphoproliferative disorders. Further detailed discussion of these entities is found in Chapter 6.
Castleman Disease Castleman disease is a heterogeneous group of diseases that was initially described as a benign localized mass of lymph nodes found in the mediastinum of asymptomatic patients.239 Subsequently, unicentric and
382
Immunohistology of the Mediastinum
TABLE 11-5 Immunophenotype of Mediastinal Germ Cell Tumors Antibody
Seminoma
Embryonal Carcinoma
Yolk Sac Tumor
Choriocarcinoma
CK
S
+
+
+
PLAP
+
N
N
N
Pouf5F1 (Oct-3/4)
+
+
N
NK
SALL4
+
+
+
+
c-Kit
+
+
+
NK
CD30
N
+
N
NK
Glypican-3
N
N
+
+
SOX2
N
+
NK
NK
SOX17
+
N
NK
NK
TCL1A
+
N
N
N
MAGEC2
+
N
NK
NK
AFP
N
N
+
N
D2-40/podoplanin
+
S
N
+
β-hCG
N
N
N
+
Pax-8
NK
NK
S
NK
Pax-2
NK
NK
S
NK
+, Positive; S, sometimes positive; N, negative; NK, not known; AFP, α-fetoprotein; β-hCG, beta–human chorionic gonadotropin; CK, cytokeratin; PLAP, placental alkaline phosphatase; MAGEC2, melanoma-associated gene C2.
multicentric disease has been identified; the two main subtypes are the hyaline vascular and plasma cell variants.240 The hyaline vascular variant shows a widened mantle zone of concentric rings of small lymphocytes that surround regressed germinal centers, and the plasma cell variant shows hyperplastic germinal centers and vascular interfollicular areas that contain polyclonal plasma cells. Immunohistochemically, a typical meshwork of follicular dendritic cells in the germinal centers can be highlighted by CD21 and CD35. The cells of the mantle zones express CD20 and CD79a, whereas interfollicular areas comprise primarily T cells that are positive for CD3, CD4, and CD43. Staining with CD138 will label the plasma cells in the interfollicular zones, and polytypic expression will be identified with immunoglobulin (Ig) light chains.241
Lymphoblastic Lymphoma Lymphoblastic lymphomas are neoplasms of immature B or T cells that often affect children or young adults and males in particular.242 Lymphoblastic lymphomas of T-cell lineage in particular can present as an anterior mediastinal mass and can cause diagnostic confusion with lymphocyte-rich thymomas, especially because the morphologic appearance and immunophenotype of the lymphocytes in both conditions can be very similar (CD1a, CD2, CD3, CD5, CD7, CD99, and TdT+).242-244 Consequently, IHC distinctions between these neoplasms must be made with extreme caution. The most helpful immunostain in this differential diagnosis is cytokeratin, which will highlight the distinct
network of interconnecting epithelial cells in thymoma. Although lymphoblastic lymphoma can occasionally contain entrapped keratin-positive epithelial cells, the distinct lacelike pattern of keratin-reactive cells typical for thymoma is not seen in hematopoietic neoplasms.
Mediastinal Large B-Cell Lymphoma and Mediastinal Gray-Zone Lymphoma Primary mediastinal large B-cell lymphoma is a subtype of diffuse large B-cell lymphoma that presents primarily in the mediastinum of young women as a bulky mass lesion (Fig. 11-15, A).245 The neoplastic cells are commonly positive for CD45 and B-cell antibodies, including CD20, CD79a, and Pax-5 (see Fig. 11-15, B-D).246,247 Other markers that are frequently positive include MUM1, Bcl-6, Bcl-2, and CD23.246-248 CD30 can be seen in 80% of cases but is usually weak and focal, whereas CD15 is generally negative.246,249 Primary mediastinal large B-cell lymphoma can show morphologic and IHC overlap with classic Hodgkin lymphoma of the nodular sclerosing type.245,250 In this setting, the most useful markers to differentiate these two lesions are CD45 and CD15. As mentioned above, CD45 will show diffuse positivity of the lymphoma cells in mediastinal large B-cell lymphoma, whereas CD15 will be negative in these cells. On the other hand, the classic Reed-Sternberg cells in Hodgkin lymphoma will be CD45 negative and CD15 positive. Markers such as CD30 and pan–B-cell markers may be expressed by both neoplasms. However, cases remain that show morphologic or immunophenotypic overlap between the two entities and thus evade
N N N N N
N N N + + + +
+ + + N N N N N N N N N N N N N N N N
CD3
CD5
TdT
CD20
CD79a
Pax-5
MUM1
CD10
Bcl-2
CD15
CD30
ALK
CD138
CD21
CD35
EMA
CD34
Myeloperoxidase
S-100 protein
N
N
N
N
N
N
N
N
N
N
N
N
N
N
+ N
N
N
N N
N
N
N
+
N
+
N
N
+ N
+
N
+
N
N
N
+
+
N
+ N
N
N
N
N
N
N
N
N
N
N
N
N
S
Dendritic Cell Tumors
N
N
N
N
N
S
S
+
N
N
N
N
+
Extraosseous Plasmacytoma
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
+
Granulocytic Sarcoma
+, Positive S, sometimes positive; N, negative; ALK, anaplastic lymphoma kinase; EMA, epithelial membrane antigen; MALT, mucosa-associated lymphoid tissue; TdT, terminal deoxynucleotidyl transferase.
N
N
N
N
N
N
N
N
+
+
N N
N
+
N
N
N
N
+
+
+
N
N
N
+
MALT Lymphoma
+
N
N
+
N
N
N
N
N
N
N
+
+
+
Anaplastic Lymphoma
S
N
N
N
+
+
CD45
Hodgkin Lymphoma
Mediastinal Large B-Cell Lymphoma
Antibody
T-Cell Lymphoblastic Lymphoma
TABLE 11-6 Immunophenotype of Mediastinal Hematopoietic Tumors
Lymphoproliferative Disorders of the Mediastinum
383
384
Immunohistology of the Mediastinum
A
B
C
D
Figure 11-15 A, Diffuse proliferation of neoplastic lymphoid cells characterizes mediastinal large B-cell lymphoma (hematoxylin and eosin). B, CD45 reactivity confirms lymphoid differentiation in a mediastinal large B-cell lymphoma. C, The B cell marker CD20 shows strong and diffuse positivity in mediastinal large B-cell lymphoma. D, Mediastinal large B-cell lymphoma shows nuclear reactivity for Pax-5.
definitive classification. These tumors have recently been termed B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classic Hodgkin lymphoma or mediastinal gray-zone lymphoma.251
Hodgkin Lymphoma Hodgkin lymphoma is thought to arise in the thymus or perithymic mediastinal lymph nodes and is in fact the most common malignant mediastinal neoplasm. Most cases of mediastinal Hodgkin lymphoma are recognizable without the need for immunohistology; however, one of its variants, syncytial Hodgkin lymphoma, in which mononuclear Reed-Sternberg cells are arranged in sheets, may closely simulate the appearance of a large cell lymphoma.252 The typical immunophenotype of the Reed-Sternberg cells in Hodgkin disease is CD45 negative, in contrast to almost all cases of nonHodgkin lymphoma, which are CD45 positive. On the other hand, Reed-Sternberg cells coexpress CD15 and CD30, whereas CD15 is not expressed in most non-Hodgkin lymphoma.249,250 Nonetheless, a potential
overlap exists between Hodgkin lymphoma and selected examples of CD30-positive non-Hodgkin lymphoma, which makes cytogenetic studies an important adjunct in this context.
Other Mediastinal Lymphoproliferative Disorders ANAPLASTIC LARGE CELL LYMPHOMA
Rare examples of anaplastic large cell lymphoma have been reported in the mediastinum.253,254 These lymphomas are characterized by large pleomorphic tumor cells with consistent CD30 reactivity and negative staining for CD15.255 Most of these tumors express a T-cell phenotype (CD3+, CD4+, CD8−, CD43+), however, some cases do not exhibit a specific phenotype and are classified as null type.256 In addition, the neoplastic cells show EMA positivity in the majority of cases and positive staining for ALK in 80% of tumors,257,258 which makes the latter marker an important diagnostic tool in the assessment of mediastinal large cell lymphomas.
Summary
MUCOSA-ASSOCIATED LYMPHOID TISSUE LYMPHOMA
Mucosa-associated lymphoid tissue (MALT) lymphomas are indolent lymphomas of B-cell lineage that are rarely identified as primary thymic neoplasms.259,260 These tumors often, but not exclusively, affect middleaged women with a history of autoimmune disease.260 MALT lymphoma has a nonspecific B-cell immunophenotype and is positive for pan–B-cell markers, such as CD20 and CD79a, and shows monotypic expression of Ig light chains. Although CD10 is usually negative, it is important to note that CD5 can show variable results that can complicate its distinction from other CD5positive B-cell lymphomas, such as chronic lymphocytic leukemia/small lymphocytic lymphoma or mantle cell lymphoma.260,261 When in doubt, the classic histologic features, coupled with cytogenetic studies for trisomy 3, can lead to the correct diagnosis.261 GRANULOCYTIC SARCOMA (EXTRAMEDULLARY MYELOGENOUS LEUKEMIA)
Solitary mass lesions composed of immature myeloid cells are extremely rare but are known to occur in the mediastinum.262 Histologically, these may mimic lymphoma, and IHC studies are often required for the correct diagnosis. Immunoreactivity for CD45, CD43, CD34, and myeloperoxidase is expected in granulocytic sarcoma, but contrary to the main lymphoma types, the tumor cells are negative for B-cell markers (CD20, CD79a), T-cell markers (CD3, CD5), natural killer (NK) cell markers (CD56), plasmacytic markers (CD138), CD30, and ALK.263,264 EXTRAOSSEOUS PLASMACYTOMA
Extraosseous plasmacytoma can potentially imitate the microscopic appearance of neuroendocrine neoplasms and large cell lymphomas.265,266 Plasmacytomas are reactive for CD45, CD79a, CD38, CD138, and EMA with associated surface light-chain restriction for kappa or lambda, and they show variable expression of MUM1, Pax-5, and cyclin D1.267,268 In contrast to neuroendocrine carcinoma, these tumors do not stain with cytokeratin, synaptophysin, or chromogranin.269 Light-chain restriction normally excludes reactive conditions,270 although it is important to highlight that CD138 is not specific for plasmacellular proliferations. As shown by Kambham and colleagues,271 it may be observed in a variety of mesenchymal and epithelial neoplasms as well. MEDIASTINAL DENDRITIC CELL TUMORS
Neoplasms that show differentiation toward lymphoreticular dendritic cells are increasingly recognized and may arise in the mediastinal soft tissue, lymph nodes or the thymus. Based on their histologic appearance of bland, spindle-shaped cells with interspersed inflammatory cells, predominantly lymphocytes, these tumors can be easily confused with spindle cell thymomas. The
385
two main categories of mediastinal dendritic cell tumors are currently classified as follicular and interdigitating dendritic cell sarcomas.272 In contrast to thymoma, dendritic cell tumors consistently lack cytokeratin and p63. The constituent spindle cells are variably reactive for CD45, and tumors in the follicular dendritic cell sarcoma group label for combinations of CD21, CD23, CD35, podoplanin, and clusterin,272-274 whereas interdigitating dendritic cell sarcomas lack these markers but are frequently positive for S-100 protein.272
Prognostic Markers for Mediastinal Neoplasms In recent years, more and more attempts to use IHC stains as a prognostic tool have been explored. This is particularly true for thymic epithelial neoplasms, given that there is still much debate about the importance of histologic type versus staging as the most important predictive factor for these tumors. However, despite best efforts, most of the markers investigated are used more for experimental, rather than diagnostic or prognostic, reasons and are not routinely applied in the assessment of thymic epithelial neoplasms. Nevertheless, markers that have been associated with more aggressive disease, increased metastatic potential, or reduced survival include podoplanin,29 HMWCK 34βE12,275 c-Kit,276 p53,277 insulin-like growth factor 1 receptor (IGF1R), phosphorylated v-act murine thymoma viral homolog 1 oncogene (p-AKT),278 LAT1,70 GLUT-1,67 and MUC1.71 On the other hand, expression of estrogen receptors is inversely correlated with tumor size, clinical stage, WHO type, and Ki-67 labeling and is associated with better clinical outcome.279 In addition, EGFR and HER2 overexpression has been demonstrated in thymomas and thymic carcinomas respectively.280-286 Unlike other tumors, in which the expression of these markers is correlated with a poor prognosis,287-290 in a recent study, Weissferdt and colleagues291 have shown no such association in thymic carcinomas, which casts some doubt on the use of these markers for prognostic purposes in these tumors.
Summary Mediastinal lesions, and thymic epithelial neoplasms in particular, can pose diagnostic challenges because of their relative rarity and morphologic variability. A complicating factor is the fact that a diagnosis is often required to be made on material derived from small mediastinoscopic biopsies, therefore familiarity with the IHC features is of utmost importance in the assessment of mediastinal tumors and tumorlike conditions. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Fabien N, Auger C, Monier JC: Immunolocalization of thymosin alpha 1, thymopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: a light and electron microscopic study. Immunology. 63:721–727, 1988. 2. Goldstein G: The isolation of thymopoietin (thymin). Ann N Y Acad Sci. 249:177–185, 1975. 3. Savino W, Dardenne M: Thymic hormone-containing cells VI. Immunohistologic evidence for the simultaneous presence of thymulin, thymopoietin and thymosin alpha 1 in normal and pathological human thymuses. Eur J Immunol. 11:987–991, 1984. 4. Hirokawa K, Utsuyama M, Moriizumi E, et al: Immunohistochemical studies in human thymomas. Localization of thymosin and various cell marker. Virchows Arch B Cell Pathol Incl Mol Pathol. 55:371–380, 1988. 5. Suster S, Rosai J: Histology of the normal thymus. Am J Surg Pathol. 14:284–303, 1990. 6. Weissferdt A, Moran CA: Pax8 expression in thymic epithelial neoplasms: an immunohistochemical analysis. Am J Surg Pathol. 35:1305–1310, 2011. 7. Kodama T, Watanabe S, Sato Y, et al: An immunohistochemical study of thymic epithelial tumors. I. Epithelial component. Am J Surg Pathol. 10:26–33, 1986. 8. Fukai I, Masaoka A, Hashimoto T, et al: An immunohistologic study of the epithelial components of 81 cases of thymoma. Cancer. 69:2463–2468, 1992. 9. Raica M, Encică S, Motoc A, et al: Structural heterogeneity and immunohistochemical profile of Hassall corpuscles in normal human thymus. Ann Anat. 188:345–352, 2006. 10. Dotto J, Pelosi G, Rosai J: Expression of p63 in thymomas and normal thymus. Am J Clin Pathol. 127:415–420, 2007. 11. Wu M, Sun K, Gil J, et al: Immunohistochemical detection of p63 and XIAP in thymic hyperplasia and thymomas. Am J Clin Pathol. 131:689–693, 2009. 12. Pan CC, Chen PC, Chiang H: KIT (CD117) is frequently overexpressed in thymic carcinomas but is absent in thymomas. J Pathol. 202:375–381, 2004. 13. Raica M, Kondylis A, Mogoantă L, et al: Diagnostic and clinical significance of D2-40 expression in the normal human thymus and thymoma. Rom J Morphol Embryol. 51:229–234, 2010. 14. Engel P, Francis D, Graem N: Expression of bcl-2 in fetal thymus, thymomas and thymic carcinomas. Association with p53 expression and review of the literature. APMIS. 106:449–455, 1998. 15. Chan JK, Tsang WY, Seneviratne S, et al: The MIC2 antibody 013. Practical application for the study of thymic epithelial tumors. Am J Surg Pathol. 19:1115–1123, 1995. 16. Marchevsky A: The thymus gland. In Marchevsky A, Kaneko M, editors: Surgical pathology of the mediastinum, New York, 1992, Raven Press, pp 27–42. 17. Isaacson PG, Norton AJ, Addis BJ: The human thymus contains a novel population of B lymphocytes. Lancet. 2:1488–1491, 1987. 18. Taubenberger JK, Jaffe ES, Medeiros LJ: Thymoma with abundant L26-positive “asteroid” cells. A case report with an analysis of normal thymus and thymoma specimens. Arch Pathol Lab Med. 115:1254–1257, 1991. 19. Hofmann WJ, Momburg F, Möller P, et al: Intra- and extrathymic B cells in physiologic and pathologic conditions. Immunohistochemical study on normal thymus and lymphofollicular hyperplasia of the thymus. Virchows Arch A Pathol Anat Histopathol. 412:431–442, 1988. 20. Ordóñez NG: Value of PAX 8 immunostaining in tumor diagnosis: a review and update. Adv Anat Pathol. 19:140–151, 2012. 21. Bernatz PE, Harrison EG, Clagett OT: Thymoma: a clinicopathologic study. J Thorac Cardiovasc Surg. 42:424–444, 1961. 22. Levine GD, Rosai J: Thymic hyperplasia and neoplasia: a review of current concepts. Hum Pathol. 9:495–515, 1978. 23. Marino M, Müller-Hermelink HK: Thymoma and thymic carcinoma: relation of thymoma epithelial cells to the cortical and medullary differentiation of thymus. Virch Arch A Pathol Anat Histopathol. 407:119–149, 1985. 24. Suster S, Moran CA: Thymoma, atypical thymoma, and thymic carcinoma. A novel conceptual approach to the classification of
385.e1
thymic epithelial neoplasms. Am J Clin Pathol. 111:826–833, 1999. 25. Travis WD, Brambilla E, Müller-Hermellink HK, et al: Pathology & Genetics. Tumours of the Lung, Pleura, Thymus and heart, Lyon, 2004, World Health Organization, IARC Press. 26. Ring NP, Addis BJ: Thymoma: an integrated clinicopathological and immunohistochemical study. J Pathol. 149:327–337, 1986. 27. Pan CC, Chen PC, Chou TY, Chiang H: Expression of calretinin and other mesothelioma-related markers in thymic carcinoma and thymoma. Hum Pathol. 34:1155–1162, 2003. 28. Nonaka D, Henley JD, Chiriboga L, et al: Diagnostic utility of thymic epithelial markers CD205 (DEC205) and Foxn1 in thymic epithelial neoplasms. Am J Surg Pathol. 31:1038–1044, 2007. 29. Tateyama H, Sugiura H, Yamatani C, et al: Expression of podoplanin in thymoma: its correlation with tumor invasion, nodal metastasis, and poor clinical outcome. Hum Pathol. 42:533–540, 2011. 30. Tateyama H, Eimoto T, Tada T, et al: Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma. Am J Clin Pathol. 111:235–240, 1999. 31. Kornstein MJ, Rosai J: CD5 labeling of thymic carcinomas and other nonlymphoid neoplasms. Am J Clin Pathol. 109:722–726, 1998. 32. Petrini I, Zucali PA, Lee HS, et al: Expression and mutational status of c-kit in thymic epithelial tumors. J Thorac Oncol. 5:1447–1453, 2010. 33. Pan CC, Chen WY, Chiang H: Spindle cell and mixed spindle/ lymphocytic thymomas: an integrated clinicopathologic and immunohistochemical study of 81 cases. Am J Surg Pathol. 25:111–120, 2001. 34. Kornstein MJ, Kay S: B cells in thymomas. Mod Pathol. 3:61–63, 1990. 35. Weissferdt A, Hernandez JC, Kalhor N, et al: Spindle cell thymomas: an immunohistochemical study of 30 cases. Appl Immunohistochem Mol Morphol. 19:329–335, 2011. 36. Yan B, Seng SC, Petersson F: Thymoma with nuclear expression of thyroid transcription factor-1: a potential diagnostic pitfall on core biopsy. Appl Immunohistochem Mol Morphol. 19:76–81, 2011. 37. Wick MR: Assessing the prognosis of thymomas. Ann Thorac Surg. 50:521–522, 1990. 38. Battifora H, Sun TT, Bahu RM, et al: The use of antikeratin antiserum as a diagnostic tool: thymoma versus lymphoma. Hum Pathol. 11:635–641, 1980. 39. Wick MR, Rosai J: Epithelial tumors. In Givel JC, editor: Surgery of the Thymus, Berlin, 1990, Springer-Verlag, pp 79–107. 40. Yu GH, Salhany KE, Gokaslan ST, et al: Thymic epithelial cells as a diagnostic pitfall in the fine-needle aspiration diagnosis of primary mediastinal lymphoma. Diagn Cytopathol. 16:460–465, 1997. 41. Suster S, Moran CA: Micronodular thymoma with lymphoid B-cell hyperplasia: clinicopathologic and immunohistochemical study of eighteen cases of a distinctive morphologic variant of thymic epithelial neoplasm. Am J Surg Pathol. 23:955–962, 1999. 42. Tateyama H, Saito Y, Fujii Y, et al: The spectrum of micronodular thymic epithelial tumours with lymphoid B-cell hyperplasia. Histopathology. 38:519–527, 2001. 43. Weissferdt A, Kalhor N, Suster S, et al: Adenomatoid spindle cell thymomas: a clinicopathological and immunohistochemical study of 20 cases. Am J Surg Pathol. 34:1544–1549, 2010. 44. Kalhor N, Suster S, Moran CA: Spindle cell thymomas (WHO Type A) with prominent papillary and pseudopapillary features: a clinicopathologic and immunohistochemical study of 10 cases. Am J Surg Pathol. 35:372–377, 2011. 45. Suster S, Moran CA, Chan JK: Thymoma with pseudosarcomatous stroma: report of an unusual histologic variant of thymic epithelial neoplasm that may simulate carcinosarcoma. Am J Surg Pathol. 21:1316–1323, 1997. 46. Yoneda S, Marx A, Heimann S, et al: Low-grade metaplastic carcinoma of the thymus. Histopathology. 35:19–30, 1999. 47. Noh TW, Kim SH, Lim BJ, et al: Thymoma with pseudosarcomatous stroma. Yonsei Med J. 42:571–575, 2001.
385.e2
Immunohistology of the Mediastinum
48. Moritani S, Ichihara S, Mukai K, et al: Sarcomatoid carcinoma of the thymus arising in metaplastic thymoma. Histopathology. 52:409–411, 2008. 49. Fukuda T, Ohnishi Y, Emura I, et al: Microcytic variant of thymoma: histological and immunohistochemical findings in two cases. Virchows Arch A Pathol Anat Histopathol. 420:185– 189, 1992. 50. Weissferdt A, Moran CA: Thymomas with prominent signet ring cell-like features: a clinicopathologic and immunohistochemical study of 10 cases. Hum Pathol. 2012 [Epub ahead of print]. 51. Moran CA, Suster S: Ancient” (sclerosing) thymomas: a clinicopathologic study of 10 cases. Am J Clin Pathol. 121:867–871, 2004. 52. Kuo T: Sclerosing thymoma–a possible phenomenon of regression. Histopathology. 25:289–291, 1994. 53. Moran CA, Koss MN: Rhabdomyomatous thymoma. Am J Surg Pathol. 17:633–636, 1993. 54. Salih Deveci M, Ceyhan K, Deveci G, et al: Pericardial rhabdomyomatous spindle cell thymoma with mucinous cystic degeneration. Histopathology. 38:479–481, 2001. 55. Moran CA, Suster S, Koss MN: Plasma cell-rich thymoma. Am J Clin Pathol. 102:199–201, 1994. 56. Anderson ET, Vye MV: Dysproteinemia of the myeloma type associated with a thymoma. Ann Intern Med. 66:141–149, 1967. 57. Gilbert EF, Harley JB, Anido V, et al: Thymoma, plasma cell myeloma, red cell aplasia and malabsorption syndrome. Am J Med. 44:820–829, 1968. 58. Weissferdt A, Moran CA: Desmoplastic spindle cell thymoma: a cliniciopathological and immunohistochemical study of 14 cases. Human Pathol. 44:623–627, 2013. 59. Weissferdt A, Moran CA: Thymic carcinoma, part 1: a clinicopathologic and immunohistochemical study of 65 cases. Am J Clin Pathol. 138:103–114, 2012. 60. Chalabreysse L, Etienne-Mastroianni B, Adeleine P, et al: Thymic carcinoma: a clinicopathological and immunohistological study of 19 cases. Histopathology. 44:367–374, 2004. 61. Truong LD, Mody DR, Cagle PT, et al: Thymic carcinoma. A clinicopathologic study of 13 cases. Am J Surg Pathol. 14:151– 166, 1990. 62. Suster S, Moran CA: Primary thymic epithelial neoplasms showing combined features of thymoma and thymic carcinoma. A clinicopathologic study of 22 cases. Am J Surg Pathol. 20:1469– 1480, 1996. 63. Suster S, Moran CA: Spindle cell thymic carcinoma: clinicopathologic and immunohistochemical study of a distinctive variant of primary thymic epithelial neoplasm. Am J Surg Pathol. 23:691–700, 1999. 64. Brown JG, Familiari U, Papotti M, et al: Thymic basaloid carcinoma: a clinicopathologic study of 12 cases, with a general discussion of basaloid carcinoma and its relationship with adenoid cystic carcinoma. Am J Surg Pathol. 33:1113–1124, 2009. 65. Saad RS, Landreneau RJ, Liu Y, et al: Utility of immunohistochemistry in separating thymic neoplasms from germ cell tumors and metastatic lung cancer involving the anterior mediastinum. Appl Immunohistochem Mol Morphol. 11:107–112, 2003. 66. Nakagawa K, Matsuno Y, Kunitoh H, et al: Immunohistochemical KIT (CD117) expression in thymic epithelial tumors. Chest. 128:140–144, 2005. 67. Kojika M, Ishii G, Yoshida J, et al: Immunohistochemical differential diagnosis between thymic carcinoma and type B3 thymoma: diagnostic utility of hypoxic marker, GLUT-1, in thymic epithelial neoplasms. Mod Pathol. 22:1341–1350, 2009. 68. Pomplun S, Wotherspoon AC, Shah G, et al: Immunohistochemical markers in the differentiation of thymic and pulmonary neoplasms. Histopathology. 40:152–158, 2002. 69. Dorfman DM, Shahsafaei A, Chan JK: Thymic carcinomas, but not thymomas and carcinomas of other sites, show CD5 immunoreactivity. Am J Surg Pathol. 21:936–940, 1997. 70. Kaira K, Oriuchi N, Imai H, et al: L-type amino acid transporter 1 (LAT1) is frequently expressed in thymic carcinomas but is absent in thymomas. J Surg Oncol. 99:433–438, 2009. 71. Kaira K, Murakami H, Serizawa M, et al: MUC1 expression in thymic epithelial tumors: MUC1 may be useful marker as
differential diagnosis between type B3 thymoma and thymic carcinoma. Virchows Arch. 458:615–620, 2011. 72. Toprani TH, Tamboli P, Amin MB, et al: Thymic carcinoma with rhabdoid features. Ann Diagn Pathol. 7:106–111, 2003. 73. Franke A, Ströbel P, Fackeldey V, et al: Hepatoid thymic carcinoma: report of a case. Am J Surg Pathol. 28:250–256, 2004. 74. Weissferdt A, Moran CA: Micronodular thymic carcinoma with lymphoid hyperplasia: a clinicopathological and immunohistochemical study of five cases. Mod Pathol. 25:993–999, 2012. 75. Choi WW, Lui YH, Lau WH, et al: Adenocarcinoma of the thymus: report of two cases, including a previously undescribed mucinous subtype. Am J Surg Pathol. 27:124–130, 2003. 76. Matsuno Y, Morozumi N, Hirohashi S, et al: Papillary carcinoma of the thymus: report of four cases of a new microscopic type of thymic carcinoma. Am J Surg Pathol. 22:873–880, 1998. 77. Takahashi F, Tsuta K, Matsuno Y, et al: Adenocarcinoma of the thymus: mucinous subtype. Hum Pathol. 36:219–223, 2005. 78. Maeda D, Ota S, Ikeda S, et al: Mucinous adenocarcinoma of the thymus: a distinct variant of thymic carcinoma. Lung Cancer. 64:22–27, 2009. 79. Ra SH, Fishbein MC, Baruch-Oren T, et al: Mucinous adenocarcinomas of the thymus: report of 2 cases and review of the literature. Am J Surg Pathol. 31:1330–1336, 2007. 80. Kapur P, Rakheja D, Bastasch M, et al: Primary mucinous adenocarcinoma of the thymus: a case report and review of the literature. Arch Pathol Lab Med. 130:201–204, 2006. 81. Hasserjian RP, Klimstra DS, Rosai J: Carcinoma of the thymus with clear-cell features. Report of eight cases and review of the literature. Am J Surg Pathol. 19:835–841, 1995. 82. Nakano T, Endo S, Tsubochi H, et al: Thymic clear cell carcinoma. Gen Thorac Cardiovasc Surg. 58:98–100, 2010. 83. Nishimura M, Kodama T, Nishiyama H, et al: A case of sarcomatoid carcinoma of the thymus. Pathol Int. 47:260–263, 1997. 84. Kapila K, Pathan SK, Amir T, et al: Mucoepidermoid thymic carcinoma: a challenging mediastinal aspirate. Diagn Cytopathol. 37:433–436, 2009. 85. Banki F, Khalil K, Kott MM, et al: Adenoid cystic carcinoma of the thymus gland: a rare tumor. Ann Thorac Surg. 90:e56–e58, 2010. 86. Di Tommaso L, Kuhn E, Kurrer M, et al: Thymic tumor with adenoid cystic carcinomalike features: a clinicopathologic study of 4 cases. Am J Surg Pathol. 31:1161–1167, 2007. 87. Viebahn R, Hiddemann W, Klinke F, et al: Thymus carcinoid. Pathol Res Pract. 180:445–451, 1985. 88. Duh QY, Hybarger CP, Geist R, et al: Carcinoids associated with multiple endocrine neoplasia syndromes. Am J Surg. 154:142– 148, 1987. 89. Moran CA, Suster S: Neuroendocrine carcinomas (carcinoid tumor) of the thymus. A clinicopathologic analysis of 80 cases. Am J Clin Pathol. 114:100–110, 2000. 90. Moran CA, Suster S: Spindle-cell neuroendocrine carcinomas of the thymus (spindle-cell thymic carcinoid): a clinicopathologic and immunohistochemical study of seven cases. Mod Pathol. 12:587–591, 1999. 91. Moran CA, Suster S: Primary neuroendocrine carcinoma (thymic carcinoid) of the thymus with prominent oncocytic features: a clinicopathologic study of 22 cases. Mod Pathol. 13:489–494, 2000. 92. Moran CA, Suster S: Angiomatoid neuroendocrine carcinoma of the thymus: report of a distinctive morphological variant of neuroendocrine tumor of the thymus resembling a vascular neoplasm. Hum Pathol. 30:635–639, 1999. 93. Suster S, Moran CA: Thymic carcinoid with prominent mucinous stroma. Report of a distinctive morphologic variant of thymic neuroendocrine neoplasm. Am J Surg Pathol. 19:1277– 1285, 1995. 94. Wick MR, Rosai J: Neuroendocrine neoplasms of the thymus. Pathol Res Pract. 183:188–199, 1988. 95. de Montpréville VT, Macchiarini P, Dulmet E: Thymic neuroendocrine carcinoma (carcinoid): a clinicopathologic study of fourteen cases. J Thorac Cardiovasc Surg. 111:134–141, 1996. 96. Tiffet O, Nicholson AG, Ladas G, et al: A clinicopathologic study of 12 neuroendocrine tumors arising in the thymus. Chest. 124:141–146, 2003.
References 97. Herbst WM, Kummer W, Hofmann W, et al: Carcinoid tumors of the thymus. An immunohistochemical study. Cancer. 60:2465–2470, 1987. 98. Wick MR, Scheithauer BW: Thymic carcinoid. A histologic, immunohistochemical, and ultrastructural study of 12 cases. Cancer. 53:475–484, 1984. 99. Ordóñez NG: Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol. 24:1217–1223, 2000. 100. Sturm N, Rossi G, Lantuejoul S, et al: Expression of thyroid transcription factor-1 in the spectrum of neuroendocrine cell lung proliferations with special interest in carcinoids. Hum Pathol. 33:175–182, 2002. 101. Moran CA, Suster S, Fishback N, et al: Mediastinal paragangliomas. A clinicopathologic and immunohistochemical study of 16 cases. Cancer. 72:2358–2364, 1993. 102. Suster S, Moran CA: Neuroendocrine neoplasms of the mediastinum. Am J Clin Pathol. 115(Suppl):S17–S27, 2001. 103. Ayadi-Kaddour A, Braham E, Ismail O, et al: Posterior mediastinal paragangliomas: a report of three patients with peculiar tumours. Respirology. 14:459–461, 2009. 104. Ortega PF, Sosa LA, Patel M, et al: Cystic paraganglioma of the anterior mediastinum. Ann Diagn Pathol. 14:341–346, 2010. 105. Nathaniels EK, Nathaniels AM, Wang CA: Mediastinal parathyroid tumors: a clinical and pathological study of 84 cases. Ann Surg. 171:165–170, 1970. 106. Moran CA, Suster S: Primary parathyroid tumors of the mediastinum: a clinicopathologic and immunohistochemical study of 17 cases. Am J Clin Pathol. 124:749–754, 2005. 107. Ordoñez NG, Ibañez ML, Samaan NA, et al: Immunoperoxidase study of uncommon parathyroid tumors. Report of two cases of nonfunctioning parathyroid carcinoma and one intrathyroid parathyroid tumor-producing amyloid. Am J Surg Pathol. 7:535– 542, 1983. 108. Plaza JA, Dominguez F, Suster S: Cystic adenomatoid tumor of the mediastinum. Am J Surg Pathol. 28:132–138, 2004. 109. Bendek M, Ferenc M, Freudenberg N: Post-irradiation pericardial malignant mesothelioma: an autopsy case and review of the literature. Cardiovasc Pathol. 19:377–379, 2010. 110. Padgett DM, Cathro HP, Wick MR, et al: Podoplanin is a better immunohistochemical marker for sarcomatoid mesothelioma than calretinin. Am J Surg Pathol. 32:123–127, 2008. 111. Marchevsky AM: Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Arch Pathol Lab Med. 132:397–401, 2008. 112. Betta PG, Magnani C, Bensi T, et al: Immunohistochemistry and molecular diagnostics of pleural malignant mesothelioma. Arch Pathol Lab Med. 136:253–261, 2012. 113. Wachter DL, Wünsch PH, Hartmann A, et al: Adenomatoid tumors of the female and male genital tract. A comparative clinicopathologic and immunohistochemical analysis of 47 cases emphasizing their site-specific morphologic diversity. Virchows Arch. 458:593–602, 2011. 114. Minato H, Nojima T, Kurose N, et al: Adenomatoid tumor of the pleura. Pathol Int. 59:567–571, 2009. 115. Sangoi AR, McKenney JK, Schwartz EJ, et al: Adenomatoid tumors of the female and male genital tracts: a clinicopathological and immunohistochemical study of 44 cases. Mod Pathol. 22:1228–1235, 2009. 116. Kennedy AD, King G, Kerr KM: HBME-1 and antithrombomodulin in the differential diagnosis of malignant mesothelioma of pleura. J Clin Pathol. 50:859–862, 1997. 117. Ordóñez NG: Value of thrombomodulin immunostaining in the diagnosis of mesothelioma. Histopathology. 31:25–30, 1997. 118. Hinterberger M, Reineke T, Storz M, et al: D2-40 and calretinin—a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation. Mod Pathol. 20:248–255, 2007. 119. Laury AR, Hornick JL, Perets R, et al: PAX8 reliably distinguishes ovarian serous tumors from malignant mesothelioma. Am J Surg Pathol. 34:627–635, 2010. 120. Wick MR, Loy T, Mills SE, et al: Malignant epithelioid pleural mesothelioma versus peripheral pulmonary adenocarcinoma: a histochemical, ultrastructural, and immunohistologic study of 103 cases. Hum Pathol. 21:759–766, 1990.
385.e3
121. Owens CL, Sharma R, Ali SZ: Deep fibromatosis (desmoid tumor): cytopathologic characteristics, clinicoradiologic features, and immunohistochemical findings on fine-needle aspiration. Cancer. 111:166–172, 2007. 122. Bhattacharya B, Dilworth HP, Iacobuzio-Donahue C, et al: Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions. Am J Surg Pathol. 29:653–659, 2005. 123. Ferenc T, Wroński JW, Kopczyński J, et al: Analysis of APC, alpha-, beta-catenins, and N-cadherin protein expression in aggressive fibromatosis (desmoid tumor). Pathol Res Pract. 205:311–324, 2009. 124. Takeshima Y, Nakayori F, Nakano T, et al: Extra-abdominal desmoid tumor presenting as an intrathoracic tumor: case report and literature review. Pathol Int. 51:824–828, 2001. 125. Flieder DB, Suster S, Moran CA: Idiopathic fibroinflammatory (fibrosing/sclerosing) lesions of the mediastinum: a study of 30 cases with emphasis on morphologic heterogeneity. Mod Pathol. 12:257–264, 1999. 126. Miyata T, Takahama M, Yamamoto R, et al: Sclerosing mediastinitis mimicking anterior mediastinal tumor. Ann Thorac Surg. 88:293–295, 2009. 127. Hanau CA, Miettinen M: Solitary fibrous tumor: histological and immunohistochemical spectrum of benign and malignant variants presenting at different sites. Hum Pathol. 26:440–449, 1995. 128. van de Rijn M, Lombard CM, Rouse RV: Expression of CD34 by solitary fibrous tumors of the pleura, mediastinum, and lung. Am J Surg Pathol. 18:814–820, 1994. 129. De Raet J, Sacré R, Hoorens A, et al: Malignant giant solitary fibrous tumor of the mediastinum. J Thorac Oncol. 3:1068– 1070, 2008. 130. Witkin GB, Miettinen M, Rosai J: A biphasic tumor of the mediastinum with features of synovial sarcoma. A report of four cases. Am J Surg Pathol. 13:490–499, 1989. 131. Suster S, Moran CA: Primary synovial sarcomas of the mediastinum: a clinicopathologic, immunohistochemical, and ultrastructural study of 15 cases. Am J Surg Pathol. 29:569–578, 2005. 132. Trupiano JK, Rice TW, Herzog K, et al: Mediastinal synovial sarcoma: report of two cases with molecular genetic analysis. Ann Thorac Surg. 73:628–630, 2002. 133. Chen CH, Lin RL, Liu HC, et al: Inflammatory myofibroblastic tumor mimicking anterior mediastinal malignancy. Ann Thorac Surg. 86:1362–1364, 2008. 134. Makimoto Y, Nabeshima K, Iwasaki H, et al: Inflammatory myofibroblastic tumor of the posterior mediastinum: an older adult case with anaplastic lymphoma kinase abnormalities determined using immunohistochemistry and fluorescence in situ hybridization. Virchows Arch. 446:451–455, 2005. 135. Coffin CM, Watterson J, Priest JR, et al: Extrapulmonary inflammatory myofibroblastic tumor (inflammatory pseudotumor). A clinicopathologic and immunohistochemical study of 84 cases. Am J Surg Pathol. 19:859–872, 1995. 136. Coffin CM, Hornick JL, Fletcher CD: Inflammatory myofibroblastic tumor: comparison of clinicopathologic, histologic, and immunohistochemical features including ALK expression in atypical and aggressive cases. Am J Surg Pathol. 31:509–520, 2007. 137. Pettinato G, Manivel JC, De Rosa N, et al: Inflammatory myofibroblastic tumor (plasma cell granuloma). Clinicopathologic study of 20 cases with immunohistochemical and ultrastructural observations. Am J Clin Pathol. 94:538–546, 1990. 138. Cessna MH, Zhou H, Sanger WG, et al: Expression of ALK1 and p80 in inflammatory myofibroblastic tumor and its mesenchymal mimics: a study of 135 cases. Mod Pathol. 15:931–938, 2002. 139. Saab ST, Hornick JL, Fletcher CD, et al: IgG4 plasma cells in inflammatory myofibroblastic tumor: inflammatory marker or pathogenic link? Mod Pathol. 24:606–612, 2011. 140. Imai K, Saito H, Minamiya Y, et al: Malignant fibrous histiocytoma originating from the thymus. Gen Thorac Cardiovasc Surg. 56:606–609, 2008. 141. Nishida T, Nishiyama N, Kawata Y, et al: Mediastinal malignant fibrous histiocytoma developing from a foreign body granuloma. Jpn J Thorac Cardiovasc Surg. 53:583–586, 2005. 142. Ouadnouni Y, Achir A, Bekarsabein S, et al: Primary mediastinal leiomyoma: a case report. Cases J. 2:8555, 2009.
385.e4
Immunohistology of the Mediastinum
143. Moran CA, Suster S, Perino G, et al: Malignant smooth muscle tumors presenting as mediastinal soft tissue masses. A clinicopathologic study of 10 cases. Cancer. 74:2251–2260, 1994. 144. Uno A, Sakurai M, Onuma K, et al: A case of giant mediastinal leiomyoma with long-term survival. Tohoku J Exp Med. 156:1–6, 1988. 145. Iwata T, Miura T, Inoue K, et al: Primary leiomyosarcoma of the anterior mediastinum encasing the aortic arch, left common carotid and left subclavian arteries. Ann Thorac Cardiovasc Surg. 18:140–143, 2012. 146. Conner WC, Fink GW, McGinnis KM, et al: Surgical management of leiomyosarcoma of the mediastinum. Ann Thorac Surg. 77:334–336, 2004. 147. Hisaoka M, Wei-Qi S, Jian W, et al: Specific but variable expression of h-caldesmon in leiomyosarcomas: an immunohistochemical reassessment of a novel myogenic marker. Appl Immunohistochem Mol Morphol. 9:302–308, 2001. 148. Suster S, Moran CA, Koss MN: Rhabdomyosarcomas of the anterior mediastinum: report of four cases unassociated with germ cell, teratomatous, or thymic carcinomatous components. Hum Pathol. 25:349–356, 1994. 149. Cessna MH, Zhou H, Perkins SL, et al: Are myogenin and myoD1 expression specific for rhabdomyosarcoma? A study of 150 cases, with emphasis on spindle cell mimics. Am J Surg Pathol. 25:1150–1157, 2001. 150. Stock N, Chibon F, Binh MB, et al: Adult-type rhabdomyosarcoma: analysis of 57 cases with clinicopathologic description, identification of 3 morphologic patterns and prognosis. Am J Surg Pathol. 33:1850–1859, 2009. 151. Martin SE, Temm CJ, Goheen MP, et al: Cytoplasmic p63 immunohistochemistry is a useful marker for muscle differentiation: an immunohistochemical and immunoelectron microscopic study. Mod Pathol. 24:1320–1326, 2011. 152. Bahrami A, Gown AM, Baird GS, et al: Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol. 21:795–806, 2008. 153. Binh MB, Sastre-Garau X, Guillou L, et al: MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing welldifferentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol. 29:1340–1347, 2005. 154. He M, Aisner S, Benevenia J, et al: p16 immunohistochemistry as an alternative marker to distinguish atypical lipomatous tumor from deep-seated lipoma. Appl Immunohistochem Mol Morphol. 17:51–56, 2009. 155. Graadt van Roggen JF, Hogendoorn PC, Fletcher CD: Myxoid tumours of soft tissue. Histopathology. 35:291–312, 1999. 156. Hornick JL, Bosenberg MW, Mentzel T, et al: Pleomorphic liposarcoma: clinicopathologic analysis of 57 cases. Am J Surg Pathol. 28:1257–1267, 2004. 157. Templeton SF, Solomon AR, Jr: Spindle cell lipoma is strongly CD34 positive. An immunohistochemical study. J Cutan Pathol. 23:546–550, 1996. 158. Cates JM, Coffing BN, Harris BT, et al: Calretinin expression in tumors of adipose tissue. Hum Pathol. 37:312–321, 2006. 159. Rodriguez FJ, Folpe AL, Giannini C, et al: Pathology of peripheral nerve sheath tumors: diagnostic overview and update on selected diagnostic problems. Acta Neuropathol. 123:295–319, 2012. 160. Fine SW, McClain SA, Li M: Immunohistochemical staining for calretinin is useful for differentiating schwannomas from neurofibromas. Am J Clin Pathol. 122:552–559, 2004. 161. Jokinen CH, Dadras SS, Goldblum JR, et al: Diagnostic implications of podoplanin expression in peripheral nerve sheath neoplasms. Am J Clin Pathol. 129:886–893, 2008. 162. Nonaka D, Chiriboga L, Rubin BP: Differential expression of S100 protein subtypes in malignant melanoma, and benign and malignant peripheral nerve sheath tumors. J Cutan Pathol. 35:1014–1019, 2008. 163. Stasik CJ, Tawfik O: Malignant peripheral nerve sheath tumor with rhabdomyosarcomatous differentiation (malignant triton tumor). Arch Pathol Lab Med. 130:1878–1881, 2006. 164. Chavez Espinosa JI, Chavez Fernández JA, Hoyer OH, et al: Endothoracic neurogenic neoplasm (analysis of 30 cases). Rev Interam Radiol. 5:49–54, 1980.
165. Argani P, Erlandson RA, Rosai J: Thymic neuroblastoma in adults: report of three cases with special emphasis on its association with the syndrome of inappropriate secretion of antidiuretic hormone. Am J Clin Pathol. 108:537–543, 1997. 166. Asada Y, Marutsuka K, Mitsukawa T, et al: Ganglioneuroblastoma of the thymus: an adult case with the syndrome of inappropriate secretion of antidiuretic hormone. Hum Pathol. 27:506–509, 1996. 167. Ohtaki Y, Ishii G, Hasegawa T, et al: Adult neuroblastoma arising in the superior mediastinum. Interact Cardiovasc Thorac Surg. 13:220–222, 2011. 168. Shimada H, Chatten J, Newton WA, Jr, et al: Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. J Natl Cancer Inst. 73:405–416, 1984. 169. Hironaka M, Fukayama M, Takayashiki N, et al: Pulmonary gangliocytic paraganglioma: case report and comparative immunohistochemical study of related neuroendocrine neoplasms. Am J Surg Pathol. 25:688–693, 2001. 170. Wolf HK, Buslei R, Schmidt-Kastner R, et al: NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem. 44:1167–1171, 1996. 171. Mukai M, Torikata C, Iri H, et al: Expression of neurofilament triplet proteins in human neural tumors. An immunohistochemical study of paraganglioma, ganglioneuroma, ganglioneuroblastoma, and neuroblastoma. Am J Pathol. 122:28–35, 1986. 172. Osborn M, Dirk T, Käser H, et al: Immunohistochemical localization of neurofilaments and neuron-specific enolase in 29 cases of neuroblastoma. Am J Pathol. 122:433–442, 1986. 173. Hachitanda Y, Tsuneyoshi M, Enjoji M: Expression of pan-neuroendocrine proteins in 53 neuroblastic tumors. An immunohistochemical study with neuron-specific enolase, chromogranin, and synaptophysin. Arch Pathol Lab Med. 113:381– 384, 1989. 174. Miettinen M, Chatten J, Paetau A, et al: Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors. Am J Surg Pathol. 22:327–332, 1998. 175. Thomas JO, Nijjar J, Turley H, et al: NB84: a new monoclonal antibody for the recognition of neuroblastoma in routinely processed material. J Pathol. 163:69–75, 1991. 176. Ambros IM, Ambros PF, Strehl S, et al: MIC2 is a specific marker for Ewing’s sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of Ewing’s sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer. 67:1886–1893, 1991. 177. Carter RL, al-Sams SZ, Corbett RP, et al: Comparative study of immunohistochemical staining for neuron-specific enolase, protein gene product 9.5 and S-100 protein in neuroblastoma, Ewing’s sarcoma and other round cell tumours in children. Histopathology. 16:461–467, 1990. 178. Llombart-Bosch A, Machado I, Navarro S, et al: Histological heterogeneity of Ewing’s sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support. Virchows Arch. 455:397–411, 2009. 179. Gu M, Antonescu CR, Guiter G, et al: Cytokeratin immunoreactivity in Ewing’s sarcoma: prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol. 24:410–416, 2000. 180. Rossi S, Orvieto E, Furlanetto A, et al: Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody. Mod Pathol. 17:547–552, 2004. 181. Takao H, Shimizu S, Doi I, et al: Primary malignant melanoma of the anterior mediastinum: CT and MR findings. Clin Imaging. 32:58–60, 2008. 182. Karuppiah SV, Buchan KG: Primary malignant melanoma: a rare cause of mediastinal mass. Jpn J Thorac Cardiovasc Surg. 54:396– 398, 2006. 183. Pandya B, Ramraje S, Darade A: Primary malignant melanoma of the mediastinum. J Assoc Physicians India. 52:924–925, 2004. 184. Vlodavsky E, Ben-Izhak O, Best LA, et al: Primary malignant melanoma of the anterior mediastinum in a child. Am J Surg Pathol. 24:747–749, 2000. 185. Nonaka D, Chiriboga L, Rubin BP: Sox10: a pan-schwannian and melanocytic marker. Am J Surg Pathol. 32:1291–1298, 2008.
References 186. Rao P, Fuller GN, Prieto VG: Expression of Sox-9 in metastatic melanoma–a potential diagnostic pitfall. Am J Dermatopathol. 32:262–266, 2010. 187. Wilson RW, Moran CA: Primary ependymoma of the mediastinum: a clinicopathologic study of three cases. Ann Diagn Pathol. 2:293–300, 1998. 188. Idowu MO, Rosenblum MK, Wei XJ, et al: Ependymomas of the central nervous system and adult extra-axial ependymomas are morphologically and immunohistochemically distinct–a comparative study with assessment of ovarian carcinomas for expression of glial fibrillary acidic protein. Am J Surg Pathol. 32:710–718, 2008. 189. Estrozi B, Queiroga E, Bacchi CE, et al: Myxopapillary ependymoma of the posterior mediastinum. Ann Diagn Pathol. 1:283– 287, 2006. 190. Maeda S, Takahashi S, Koike K, et al: Primary ependymoma in the posterior mediastinum. Ann Thorac Cardiovasc Surg. 17:494–497, 2011. 191. Weissferdt A, Moran CA: Primary vascular tumors of the lungs: a review. Ann Diagn Pathol. 14:296–308, 2010. 192. Yoshino N, Okada D, Ujiie H, et al: Venous Hemangioma of the Posterior Mediastinum. Ann Thorac Cardiovasc Surg. 2011 Oct 28. [Epub ahead of print]. 193. Kanzaki M, Kikkawa T, Obara T, et al: Successful excision of an isolated mediastinal cystic lymphangioma with bilateral thoracoscopic surgery. Ann Thorac Cardiovasc Surg. 17:570–572, 2011. 194. Koss MN, Hochholzer L, Moran CA: Primary pulmonary glomus tumor: a clinicopathologic and immunohistochemical study of two cases. Mod Pathol. 11:253–258, 1998. 195. Mentzel T, Hügel H, Kutzner H: CD34-positive glomus tumor: clinicopathologic and immunohistochemical analysis of six cases with myxoid stromal changes. J Cutan Pathol. 29:421–425, 2002. 196. Gaertner EM, Steinberg DM, Huber M, et al: Pulmonary and mediastinal glomus tumors–report of five cases including a pulmonary glomangiosarcoma: a clinicopathologic study with literature review. Am J Surg Pathol. 24:1105–1114, 2000. 197. Khoury T, Balos L, McGrath B, et al: Malignant glomus tumor: a case report and review of literature, focusing on its clinicopathologic features and immunohistochemical profile. Am J Dermatopathol. 27:428–431, 2005. 198. Suster S, Moran CA, Koss MN: Epithelioid hemangioendothelioma of the anterior mediastinum. Clinicopathologic, immunohistochemical, and ultrastructural analysis of 12 cases. Am J Surg Pathol. 18:871–881, 1994. 199. Deyrup AT, Miettinen M, North PE, et al: Angiosarcomas arising in the viscera and soft tissue of children and young adults: a clinicopathologic study of 15 cases. Am J Surg Pathol. 33:264– 269, 2009. 200. Mentzel T, Beham A, Calonje E, et al: Epithelioid hemangioendothelioma of skin and soft tissues: clinicopathologic and immunohistochemical study of 30 cases. Am J Surg Pathol. 21:363–374, 1997. 201. Meis-Kindblom JM, Kindblom LG: Angiosarcoma of soft tissue: a study of 80 cases. Am J Surg Pathol. 22:683–697, 1998. 202. Zhang H, Maitta RW, Bhattacharyya PK, et al: γ-Synuclein is a promising new marker for staining reactive follicular dendritic cells, follicular dendritic cell sarcoma, Kaposi sarcoma, and benign and malignant vascular tumors. Am J Surg Pathol. 35:1857–1865, 2011. 203. Miettinen M, Sarlomo-Rikala M, Wang ZF: Claudin-5 as an immunohistochemical marker for angiosarcoma and hemangioendotheliomas. Am J Surg Pathol. 35:1848–1856, 2011. 204. Moran CA, Suster S: Primary germ cell tumors of the mediastinum: I. Analysis of 322 cases with special emphasis on teratomatous lesions and a proposal for histopathologic classification and clinical staging. Cancer. 80:681–690, 1997. 205. Moran CA, Suster S, Przygodzki RM, et al: Primary germ cell tumors of the mediastinum: II. Mediastinal seminomas–a clinicopathologic and immunohistochemical study of 120 cases. Cancer. 80:691–698, 1997. 206. Moran CA, Suster S, Koss MN: Primary germ cell tumors of the mediastinum: III. Yolk sac tumor, embryonal carcinoma, choriocarcinoma, and combined nonteratomatous germ cell tumors of
385.e5
the mediastinum–a clinicopathologic and immunohistochemical study of 64 cases. Cancer. 80:699–707, 1997. 207. Przygodzki RM, Hubbs AE, Zhao FQ, et al: Primary mediastinal seminomas: evidence of single and multiple KIT mutations. Lab Invest. 82:1369–1375, 2002. 208. Sung MT, Maclennan GT, Lopez-Beltran A, et al: Primary mediastinal seminoma: a comprehensive assessment integrated with histology, immunohistochemistry, and fluorescence in situ hybridization for chromosome 12p abnormalities in 23 cases. Am J Surg Pathol. 32:146–155, 2008. 209. Weidner N: Germ-cell tumors of the mediastinum. Semin Diagn Pathol. 16:42–50, 1999. 210. Weissferdt A, Suster S, Moran CA: Primary mediastinal “thymic” seminomas. Adv Anat Pathol. 19:75–80, 2012. 211. Suster S, Moran CA, Dominguez-Malagon H, et al: Germ cell tumors of the mediastinum and testis: a comparative immunohistochemical study of 120 cases. Hum Pathol. 29:737–742, 1998. 212. Lau SK, Weiss LM, Chu PG: TCL1 protein expression in testicular germ cell tumors. Am J Clin Pathol. 133:762–766, 2010. 213. Cao D, Li J, Guo CC, et al: SALL4 is a novel diagnostic marker for testicular germ cell tumors. Am J Surg Pathol. 33:1065–1077, 2009. 214. Jung SM, Chu PH, Shiu TF, et al: Expression of OCT4 in the primary germ cell tumors and thymoma in the mediastinum. Appl Immunohistochem Mol Morphol. 14:273–275, 2006. 215. Liu A, Cheng L, Du J, et al: Diagnostic utility of novel stem cell markers SALL4, OCT4, NANOG, SOX2, UTF1, and TCL1 in primary mediastinal germ cell tumors. Am J Surg Pathol. 34:697– 706, 2010. 216. Iczkowski KA, Butler SL, Shanks JH, et al: Trials of new germ cell immunohistochemical stains in 93 extragonadal and metastatic germ cell tumors. Hum Pathol. 39:275–281, 2008. 217. Bode PK, Barghorn A, Fritzsche FR, et al: MAGEC2 is a sensitive and novel marker for seminoma: a tissue microarray analysis of 325 testicular germ cell tumors. Mod Pathol. 24:829–835, 2011. 218. Idrees M, Saxena R, Cheng L, et al: Podoplanin, a novel marker for seminoma: A comparison study evaluating immunohistochemical expression of podoplanin and OCT3/4. Ann Diagn Pathol. 14:331–336, 2010. 219. Zynger DL, Dimov ND, Luan C, et al: Glypican 3: a novel marker in testicular germ cell tumors. Am J Surg Pathol. 30: 1570–1575, 2006. 220. Nonaka D: Differential expression of SOX2 and SOX17 in testicular germ cell tumors. Am J Clin Pathol. 131:731–736, 2009. 221. Kao CS, Idrees MT, Young RH, et al: Solid pattern yolk sac tumor: a morphologic and immunohistochemical study of 52 cases. Am J Surg Pathol. 36:360–367, 2012. 222. Sangoi AR, McKenney JK, Brooks JD, et al: Evaluation of Putative Renal Cell Carcinoma Markers PAX-2, PAX-8, and hKIM-1 in Germ Cell Tumors: A Tissue Microarray Study of 100 Cases. Appl Immunohistochem Mol Morphol. 2012 Apr 10. [Epub ahead of print]. 223. Moran CA, Suster S: Primary mediastinal choriocarcinomas: a clinicopathologic and immunohistochemical study of eight cases. Am J Surg Pathol. 21:1007–1012, 1997. 224. Lau SK, Weiss LM, Chu PG: D2-40 immunohistochemistry in the differential diagnosis of seminoma and embryonal carcinoma: a comparative immunohistochemical study with KIT (CD117) and CD30. Mod Pathol. 20:320–325, 2007. 225. al-Ali F, Gooding CA, Jacques CJ: Calcified mass in anterior part of mediastinum caused by Langerhans’ cell histiocytosis. AJR Am J Roentgenol. 162:467–468, 1994. 226. Mogul M, Hartman G, Donaldson S, et al: Langerhans’ cell histiocytosis presenting with the superior vena cava syndrome: a case report. Med Pediatr Oncol. 21:456–459, 1993. 227. Abramson SJ, Berdon WE, Reilly BJ, et al: Cavitation of anterior mediastinal masses in children with histiocytosis-X. Report of four cases with radiographic, pathologic findings and clinical follow up. Pediatr Radiol. 17:10–14, 1987. 228. Nakata H, Suzuki H, Sato Y, et al: Histiocytosis X with anterior mediastinal mass as its initial manifestation. Pediatr Radiol. 12:84–85, 1982.
385.e6
Immunohistology of the Mediastinum
229. Singhi AD, Montgomery EA: Gastrointestinal tract langerhans cell histiocytosis: A clinicopathologic study of 12 patients. Am J Surg Pathol. 35:305–310, 2011. 230. Edelweiss M, Medeiros LJ, Suster S, et al: Lymph node involvement by Langerhans cell histiocytosis: a clinicopathologic and immunohistochemical study of 20 cases. Hum Pathol. 38:1463– 1469, 2007. 231. Lau SK, Chu PG, Weiss LM: Immunohistochemical expression of Langerin in Langerhans cell histiocytosis and non-Langerhans cell histiocytic disorders. Am J Surg Pathol. 32:615–619, 2008. 232. Pinkus GS, Lones MA, Matsumura F, et al: Langerhans cell histiocytosis immunohistochemical expression of fascin, a dendritic cell marker. Am J Clin Pathol. 118:335–343, 2002. 233. Costa AL, Silva NO, Motta MP, et al: Soft tissue Rosai-Dorfman disease of the posterior mediastinum. J Bras Pneumol. 35:717– 720, 2009. 234. Cunha BA, Durie N, Selbs E, et al: Fever of unknown origin (FUO) due to Rosai-Dorfman disease with mediastinal adenopathy mimicking lymphoma: diagnostic importance of elevated serum ferritin levels and polyclonal gammopathy. Heart Lung. 38:83–88, 2009. 235. Montgomery EA, Meis JM, Frizzera G: Rosai-Dorfman disease of soft tissue. Am J Surg Pathol. 16:122–129, 1992. 236. Wenig BM, Abbondanzo SL, Childers EL, et al: Extranodal sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) of the head and neck. Hum Pathol. 24:483–492, 1993. 237. Paulli M, Rosso R, Kindl S, et al: Immunophenotypic characterization of the cell infiltrate in five cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). Hum Pathol. 23:647–654, 1992. 238. Kong YY, Kong JC, Shi DR, et al: Cutaneous rosai-dorfman disease: a clinical and histopathologic study of 25 cases in China. Am J Surg Pathol. 31:341–350, 2007. 239. Castleman B, Iverson L, Menendez VP: Localized mediastinal lymphnode hyperplasia resembling thymoma. Cancer. 9:822– 830, 1956. 240. Dham A, Peterson BA: Castleman disease. Curr Opin Hematol. 14:354–359, 2007. 241. Kazakov DV, Fanburg-Smith JC, Suster S, et al: Castleman disease of the subcutis and underlying skeletal muscle: report of 6 cases. Am J Surg Pathol. 28:569–577, 2004. 242. Cortelazzo S, Ponzoni M, Ferreri AJ, et al: Lymphoblastic lymphoma. Crit Rev Oncol Hematol. 79:330–343, 2011. 243. Robertson PB, Neiman RS, Worapongpaiboon S, et al: 013 (CD99) positivity in hematologic proliferations correlates with TdT positivity. Mod Pathol. 10:277–282, 1997. 244. Soslow RA, Bhargava V, Warnke RA: MIC2, TdT, bcl-2, and CD34 expression in paraffin-embedded high-grade lymphoma/ acute lymphoblastic leukemia distinguishes between distinct clinicopathologic entities. Hum Pathol. 28:1158–1165, 1997. 245. Hutchinson CB, Wang E: Primary mediastinal (thymic) large B-cell lymphoma: a short review with brief discussion of mediastinal gray zone lymphoma. Arch Pathol Lab Med. 135:394– 398, 2011. 246. Pileri SA, Zinzani PL, Gaidano G, et al: Pathobiology of primary mediastinal B-cell lymphoma. Leuk Lymphoma. 44(Suppl 3):S21–S26, 2003. 247. Pileri SA, Gaidano G, Zinzani PL, et al: Primary mediastinal B-cell lymphoma: high frequency of BCL-6 mutations and consistent expression of the transcription factors OCT-2, BOB.1, and PU.1 in the absence of immunoglobulins. Am J Pathol. 162:243–253, 2003. 248. Calaminici M, Piper K, Lee AM, et al: CD23 expression in mediastinal large B-cell lymphomas. Histopathology. 45:619– 624, 2004. 249. Higgins JP, Warnke RA: CD30 expression is common in mediastinal large B-cell lymphoma. Am J Clin Pathol. 112:241–247, 1999. 250. Pervez S, Khawaja RD: Mediastinal lymphomas: primary mediastinal (thymic) large B-cell lymphoma versus classical Hodgkin lymphoma, histopathologic dilemma solved? Pathol Res Pract. 206:365–367, 2010. 251. Jaffe ES, Stein H, Swerdlow SH, et al: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma. In Swerdlow SH,
Campo E, Harris NL, et al, editors: Tumours of Haematopoetic and Lymphoid Tissues, Lyon, France, 2008, IARC Press, pp 267– 268. World Health Organization Classification of Tumours; vol. 3. 252. Strickler JG, Michie SA, Warnke RA, et al: The “syncytial variant” of nodular sclerosing Hodgkin’s disease. Am J Surg Pathol. 10:470–477, 1986. 253. Kayao J, Tamaru J, Wakita H, et al: Anaplastic large cell lymphoma of the mediastinum diagnosed by transbronchial scratch cytology. A case report. Acta Cytol. 46:405–411, 2002. 254. Nakamura S, Takagi N, Kojima M, et al: Clinicopathologic study of large cell anaplastic lymphoma (Ki-1-positive large cell lymphoma) among the Japanese. Cancer. 68:118–129, 1991. 255. Stein H, Mason DY, Gerdes J, et al: The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood. 66:848–858, 1985. 256. Jaffe ES: Anaplastic large cell lymphoma: the shifting sands of diagnostic hematopathology. Mod Pathol. 14:219–228, 2001. 257. Wellmann A, Otsuki T, Vogelbruch M, et al: Analysis of the t(2;5)(p23;q35) translocation by reverse transcriptionpolymerase chain reaction in CD30+ anaplastic large-cell lymphomas, in other non-Hodgkin’s lymphomas of T-cell phenotype, and in Hodgkin’s disease. Blood. 86:2321–2328, 1995. 258. Benharroch D, Meguerian-Bedoyan Z, Lamant L, et al: ALKpositive lymphoma: a single disease with a broad spectrum of morphology. Blood. 91:2076–2084, 1998. 259. Isaacson PG, Chan JK, Tang C, et al: Low-grade B-cell lymphoma of mucosa-associated lymphoid tissue arising in the thymus. A thymic lymphoma mimicking myoepithelial sialadenitis. Am J Surg Pathol. 14:342–351, 1990. 260. Weissferdt A, Moran CA: Primary MALT-type lymphoma of the thymus: a clinicopathological and immunohistochemical study of six cases. Lung. 189:461–466, 2011. 261. Jaso J, Chen L, Li S, et al: CD5-positive mucosa-associated lymphoid tissue (MALT) lymphoma: a clinicopathologic study of 14 cases. Hum Pathol. 43:1436–1443, 2012. 262. Byrd JC, Edenfield WJ, Shields DJ, et al: Extramedullary myeloid cell tumors in acute nonlymphocytic leukemia: a clinical review. J Clin Oncol. 13:1800–1816, 1995. 263. Wong WS, Loong F, Ooi GC, et al: Primary granulocytic sarcoma of the mediastinum. Leuk Lymphoma. 45:1931–1933, 2004. 264. Ravandi-Kashani F, Cortes J, Giles FJ: Myelodysplasia presenting as granulocytic sarcoma of mediastinum causing superior vena cava syndrome. Leuk Lymphoma. 36:631–637, 2000. 265. Niwa K, Tanaka T, Mori H, et al: Extramedullary plasmacytoma of the mediastinum. Jpn J Clin Oncol. 17:95–100, 1987. 266. Miyazaki T, Kohno S, Sakamoto A, et al: A rare case of extramedullary plasmacytoma in the mediastinum. Intern Med. 31:1363–1365, 1992. 267. Lorsbach RB, Hsi ED, Dogan A, et al: Plasma cell myeloma and related neoplasms. Am J Clin Pathol. 136:168–182, 2011. 268. Aref S, Goda T, El-Sherbiny M: Syndecan-1 in multiple myeloma: relationship to conventional prognostic factors. Hematology. 8:221–228, 2003. 269. Tong AW, Lee JC, Stone MJ: Characterization of a monoclonal antibody having selective reactivity with normal and neoplastic plasma cells. Blood. 69:238–245, 1987. 270. Lorsbach RB, Hsi ED, Dogan A, et al: Plasma cell myeloma and related neoplasms. Am J Clin Pathol. 136:168–182, 2011. 271. Kambham N, Kong C, Longacre TA, et al: Utility of syndecan-1 (CD138) expression in the diagnosis of undifferentiated malignant neoplasms: a tissue microarray study of 1,754 cases. Appl Immunohistochem Mol Morphol. 13:304–310, 2005. 272. Kairouz S, Hashash J, Kabbara W, et al: Dendritic cell neoplasms: an overview. Am J Hematol. 82:924–928, 2007. 273. Chan JK, Fletcher CD, Nayler SJ, et al: Follicular dendritic cell sarcoma. Clinicopathologic analysis of 17 cases suggesting a malignant potential higher than currently recognized. Cancer. 79:294–313, 1997. 274. Xie Q, Chen L, Fu K, et al: Podoplanin (d2-40): a new immunohistochemical marker for reactive follicular dendritic cells and follicular dendritic cell sarcomas. Int J Clin Exp Pathol. 1:276– 284, 2008.
References 275. Cîmpean AM, Raica M, Encica S: Overexpression of cytokeratin 34beta E12 in thymoma: could it be a poor prognosis factor? Rom J Morphol Embryol. 45:153–157, 1999-2004. 276. Petrini I, Zucali PA, Lee HS, et al: Expression and mutational status of c-kit in thymic epithelial tumors. J Thorac Oncol. 5:1447–1453, 2010. 277. Weirich G, Schneider P, Fellbaum C, et al: p53 alterations in thymic epithelial tumours. Virchows Arch. 431:17–23, 1997. 278. Zucali PA, Petrini I, Lorenzi E, et al: Insulin-like growth factor-1 receptor and phosphorylated AKT-serine 473 expression in 132 resected thymomas and thymic carcinomas. Cancer. 116:4686– 4695, 2010. 279. Ishibashi H, Suzuki T, Suzuki S: Sex steroid hormone receptors in human thymoma. J Clin Endocrinol Metab. 88:2309–2317, 2003. 280. Ionescu DN, Sasatomi E, Cieply K, et al: Protein expression and gene amplification of epidermal growth factor receptor in thymomas. Cancer. 103:630–636, 2005. 281. Girard N, Shen R, Guo T, et al: Comprehensive genomic analysis reveals clinically relevant molecular distinctions between thymic carcinomas and thymomas. Clin Cancer Res. 15:6790–6799, 2009. 282. Suzuki E, Sasaki H, Kawano O, et al: Expression and mutation statuses of epidermal growth factor receptor in thymic epithelial tumors. Jpn J Clin Oncol. 36:351–356, 2006. 283. Pan CC, Chen PC, Wang LS, et al: Expression of apoptosisrelated markers and HER-2/neu in thymic epithelial tumours. Histopathology. 43:165–172, 2003. 284. Meister M, Schirmacher P, Dienemann H, et al: Mutational status of the epidermal growth factor receptor (EGFR) gene in
385.e7
thymomas and thymic carcinomas. Cancer Lett. 248:186–191, 2007. 285. Aisner SC, Dahlberg S, Hameed MR, et al: Epidermal growth factor receptor, C-kit, and Her2/neu immunostaining in advanced or recurrent TENs staged according to the 2004 World Health Organization in patients treated with octreotide and prednisone: an Eastern Cooperative Oncology Group study. J Thorac Oncol. 5:885–892, 2010. 286. Yoh K, Nishiwaki Y, Ishii G, et al: Mutational status of EGFR and KIT in thymoma and thymic carcinoma. Lung Cancer. 62:316–320, 2008. 287. Meert AP, Martin B, Delmotte P, et al: The role of EGF-R expression on patient survival in lung cancer: a systematic review with meta-analysis. Eur Respir J. 20:975–981, 2002. 288. Liu L, Shao X, Gao W, et al: The role of human epidermal growth factor receptor 2 as a prognostic factor in lung cancer: a meta-analysis of published data. J Thorac Oncol. 5:1922–1932, 2010. 289. Hirsch FR, Varella-Garcia M, Franklin WA, et al: Evaluation of HER-2/neu gene amplification and protein expression in non-small cell lung carcinomas. Br J Cancer. 86:1449–1456, 2002. 290. Begnami MD, Fukuda E, Fregnani JH, et al: Prognostic implications of altered human epidermal growth factor receptors (HERs) in gastric carcinomas: HER2 and HER3 are predictors of poor outcome. J Clin Oncol. 29:3030–3036, 2011. 291. Weissferdt A, Lin H, Woods D, et al: HER family receptor and ligand status in thymic carcinoma. Lung Cancer. 77:515–521, 2012.
C H A P T E R 1 2
IMMUNOHISTOLOGY OF LUNG AND PLEURAL NEOPLASMS SAMUEL P. HAMMAR, SANJA DACIC
Overview 386 Primary Lung Neoplasms 387 Differential Diagnosis and Pitfalls of Lung Neoplasms 418 Theranostic Applications in Lung Neoplasms 431 Pleural Neoplasms 437 Diagnostic Pitfalls of Pleural Neoplasms 468 Molecular Biology and Theranostic Features in Diffuse Malignant Mesothelioma 477 Prognosis of Mesothelioma Based on Morphology and Immunohistochemical Analysis 478 Summary 478
Overview Immunohistochemistry (IHC) is an effective adjuvant technique commonly used to diagnose primary and metastatic neoplasms of the lung and pleura. Because of its relative ease of use and specificity, IHC has largely replaced mucin histochemistry and electron microscopy in diagnosing pulmonary and pleural neoplasms. In some instances, electron microscopy is diagnostically superior to IHC. In other cases, neither IHC nor electron microscopy is specific for a given neoplasm. As described in this chapter, IHC is now being used for therapeutic/prognostic reasons. Significant new information has accumulated concerning the IHC features of lung and pleural neoplasms. As might be expected, specificities and sensitivities of some markers have changed, new and highly sensitive and specific markers have been developed, and markers thought to initially be restricted to certain neoplasms have been observed in neoplasms not thought to express such markers. Some antibodies are now being used to 386
determine the degree of differentiation of a neoplasm and predictors of prognosis.1-7 For example, Pelosi and colleagues6 evaluated fascin, an actin-bundling protein that induces cell membrane protrusions and increases the motility of normal and transformed epithelial cells, to predict lymph node metastases in typical and atypical carcinoids. The authors found that fascin immunoreactivity was closely correlated with occurrence of lymph node metastases in typical and atypical carcinoid tumors. However, the authors found no correlation in high-grade neuroendocrine (NE) tumors of the lung. Fascin expression was also associated with an increased proliferative activity (Ki-67). Pelosi and others7 evaluated fascin immunoreactivity in stage I non–small cell lung carcinoma (NSCLC) and concluded that fascin was upregulated and that invasive and more aggressive NSCLCs were an independent, not a prognostic, predictor of unfavorable clinical course. The authors also suggested that targeting the fascin pathway could be used in a therapeutic sense. The majority of IHC reports have dealt with diagnostic and therapeutic issues. These issues are discussed later in the chapter. An overview of IHC titled Immunohistochemistry: Then and Now, by Jaishree Jagirdar,8 is an excellent review of people who have been involved in diagnostic IHC from the beginning and those who are entering the discipline. The role of the pathologist is to provide not only accurate subtyping of lung cancers, but also increasingly to provide prognostic and predictive information critical to patient management. The 2012 review article by Dubinski and colleagues9 that concerns ancillary testing in lung cancer diagnosis provides a good overview of various ancillary tests such as IHC and molecular diagnostics used in the diagnosis and treatment of lung cancer. Most primary lung cancers can be diagnosed by histologic criteria alone, although when lung neoplasms are more poorly differentiated, and when clinical situations are more complicated, IHC techniques are often used to confirm or eliminate a pathologic diagnosis. In addition, many neoplasms of different primary origins are morphologically similar to primary lung and pleural neoplasms, and IHC is an effective way to distinguish them from each other.
Primary Lung Neoplasms
Primary Lung Neoplasms A wide variety of primary neoplasms occur in the lung. Four major types comprise 85% to 90% of primary lung neoplasms: adenocarcinoma, squamous cell carcinoma (SCC), small cell carcinoma, and large cell undifferentiated carcinoma. The new classification by the International Association of Lung Cancer (IALC), American Thoracic Society (ATS), and European Respiratory Society (ERS) International Multidisciplinary Classification of Lung Adenocarcinoma10 is shown in Table 12-1. Adenocarcinoma is currently the most frequently diagnosed primary lung cancer in the United States and usually occurs in a subpleural location, although occasionally it is central or intrabronchial. SCC is the second most common primary lung cancer and occurs predominantly in a central distribution, arising from mainstem and lobar bronchi. Approximately 10% of primary pulmonary SCC occurs in the periphery of the lung. Small cell lung cancers (SCLCs) occur in the central region of the lung and arise from NE cells in the mainstem bronchi and lobar bronchi, although as many as 10% of SCLCs occur in the periphery of the lung. Large cell undifferentiated carcinomas may occur in any location in the lung and comprise approximately 8% to 10% of primary lung cancers. Using the proposed IALC/ATS/ERS revised classification of lung adenocarcinoma, Yoshizawa and
TABLE 12-1 Adenocarcinoma of the Lung Preinvasive Lesions Atypical adenomatous hyperplasia Adenocarcinoma In Situ (≤3 cm, formerly bronchioloalveolar carcinoma) Nonmucinous Mucinous Mixed mucinous/nonmucinous Minimally Invasive Adenocarcinoma (≤3 cm lepidic predominant tumor with ≤5 mm invasion) Nonmucinous Mucinous Mixed mucinous/nonmucinous Invasive Adenocarcinoma Lepidic predominant (formerly nonmucinous bronchioloalveolar carcinoma pattern, with >5 mm invasion) Acinar predominant Papillary predominant Micropapillary predominant Solid predominant with mucin production Variants of Invasive Adenocarcinoma Invasive mucinous adenocarcinoma (formerly mucinous bronchioloalveolar carcinoma) Colloid Fetal (low and high grade) Enteric
387
colleagues11 sought to investigate the usefulness of this classification to identify prognostically significant lung adenocarcinoma subtypes. The authors evaluated 514 patients with neoplasms classified as adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), lepidic predominant nonmucinous (LPNM), acinar predominant (AP), papillary predominant (PP), micropapillary predominant (MPP), solid predominant (SP), colloid predominant (CP), and mucinous adenocarcinoma (MA). Statistical analysis was performed by using SPSS version 17 with crosstable and Chi-square statistics. Survival analysis was performed by using KaplanMeier analysis for disease-free survival and Cox regression. Of the 514 patients, 323 (63%) were females and 191 (37%) were males; 376 (73%) were stage 1A, and 138 (27%) were stage 1B; mean age was 68 years (33 to 89 yrs). Five-year disease-free survival for males was significantly worse (77%) than for females (88%, P = .011). It was also worse for stage 1B (75%) than for stage 1A (86%, P = .001). Three overall prognostic groups for 5-year disease-free survival were identified as follows: 1. 100% for AIS (n = 1), MIA (n = 7), and LPNM adenocarcinoma (n = 28) 2. 85% for PP (n = 143); 86% for acinar predominant (n = 232); and 86% for MA (n = 15) 3. 69% for SP (n = 66); 62% for MPP (n = 12); and 69% for CP (n = 9). In addition, the authors11 stated that survival was significantly worse for mucinous adenocarcinoma (76%) compared with LPNM (100%, P = .014) and that in multivariate analysis stratified for stage, proposed IALC classification, lymphatic invasion, and sex were independent prognostic factors of survival. The authors concluded that the proposed IALC/ATS/ERS classification identified prognostically significant categories of stage I lung adenocarcinoma; that AIS and MIA were rare tumors at Memorial Sloan–Kettering, comprising less than 2% of all cases; and that LPNM adenocarcinoma accounted for only 5.4% of all tumors. The authors opined that the data supported the proposal to use the predominant subtype for classifying the remaining 93% of lung adenocarcinomas, which were invasive.
Biology of Antigens and Antibodies Several antibodies are useful in confirming or eliminating primary lung cancers. Those used are dependent on the type of neoplasm suspected and the clinical situation encountered. A list of the antibodies commonly used in detecting, confirming, or eliminating primary lung carcinomas (excluding NE carcinomas [NECs], which is discussed later in the chapter) is shown in Table 12-2. A list of tumor-specific markers and their staining patterns is shown in Table 12-3. ANTIBODIES USED TO DETECT NONNEUROENDOCRINE LUNG NEOPLASMS
Keratins are a family of polypeptides that have been separated according to molecular weight and isoelectric
388
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-2 Commonly Used Antibodies to Evaluate Potential Lung Neoplasms (Excluding Neuroendocrine Lung Neoplasms) Antibody Directed Against
Clone
Characteristics of Antigen
Immunogen
Manufacturer
Dilution
Keratin
AE1/AE3
AE1: acidic subfamily 40, 48, 50, 56.5 kD AE3: basic subfamily 52, 56, 58, 59, 64, 65-67 kD
Human epidermal keratin
Dako
1 : 200
Keratin
5D3
CK8, 18
Colorectal carcinoma cell line
BioGenex
1 : 100
Keratin
MAK6
CK8, 14/16, 18, and 19
Extracellular antigen from MCF-tissue culture and from human sole epidermis
Zymed
1 : 100
Keratin
35βH11
CK8: 54 kD
Hep3B hepatocellular carcinoma line
Dako
1 : 50
Keratin
34βE12
Keratins: Moll numbers 1, 5, 10, and 14
Human stratum corneum keratin
Dako
1 : 100
CK5/6
D5/16B4
Intermediate filament CK5/CK6 and to a slight degree CK4
Purified CK5
Biocare Medical
1 : 100
CK7
OV-TL 12/30
Moll CK7: 54 kD
OTN 11 ovarian cell carcinoma line
Cell Marque
NA
CK20
Ks20.8
Moll CK20
Cytoskeletal protein from human duodenal mucosa
Cell Marque
NA
Vimentin
Vim 3B4
Intermediate filament: 57 kD
Vimentin from bovine eye lens
Dako
1 : 100
EMA
E29
Glycoprotein: 250-400 kD
Delapidated extract of human milk fat
Ventana
NA
HMFG-2
115D8
MAM-6 mucus glycoprotein >400 kD
Purified HMFG protein
BioGenex
1 : 25
pCEA
—
Antibody recognizes CEA and CEA-like proteins, including nonspecific cross-reacting substance and biliary glycoprotein
Human CEA isolated from metastatic colonic adenocarcinoma
Ventana
NA
CD15 (LeuM1)
C3D-1
3-Fucosyl-N-acetyllactosamine
Purified neutrophils from normal human peripheral blood
Ventana
NA
TAG
B72.3
Glycoprotein in a variety of adenocarcinomas
Membrane-enriched fraction of metastatic breast carcinoma
Cell Marque
NA
Human epithelial antigen
VU-1D9
Glycoproteins of 34 and 49 kD on surface and in cytoplasm of all epithelial cells except squamous epithelium, hepatocytes, and parietal cells
MCF-7 cell line
Ventana
NA
TTF-1
8G7G3/1
40-kD member of NKx2 family of homeodomain transcription factors
Mouse ascites
Cell Marque
NA
S-100 protein
—
S-100 proteins A and B
S-100 protein isolated from cow brain
Dako
1 : 3,000
SP-A
PE-10
Surfactant A
Surfactant apoproteins isolated from lung lavages of patients with alveolar proteinosis
Dako
1 : 100
Primary Lung Neoplasms
389
TABLE 12-2 Commonly Used Antibodies to Evaluate Potential Lung Neoplasms (Excluding Neuroendocrine Lung Neoplasms)—cont’d Antibody Directed Against
Clone
Characteristics of Antigen
Immunogen
Manufacturer
Dilution
CDX-2
CDX2-88
Homeobox family of intestinespecific transcription factor regulates proliferation and differentiation of intestinal epithelial cells
Full-length CDX-2
BioGenex
NA
p63
4A4
Human p63 protein, a member of the p53 family
Mouse monoclonal antibody
Cell Marque
1 : 100
Fascin
55K-2
55 kD actin-bundling protein
Fascin purified from HeLa cells
Dako
1 : 1000
Antigens were obtained by heat-induced epitope retrieval. CK, Cytokeratin; EMA, epithelial membrane antigen; HMFG-2, human milk fat globule protein 2; pCEA, polyclonal carcinoembryonic antigen; SP-A, surfactant apoprotein A; TAG, tumor-associated glycoprotein; TTF-1, thyroid transcription factor-1.
point (acidic or basic). Twenty molecular species exist and have been catalogued by Moll and colleagues.12,13 Cytokeratin 7 (CK7) is expressed in many pulmonary epithelial cells, although it is also found in a variety of other epithelial cells and in a variety of nonpulmonary carcinomas,14 and it is the most commonly found molecular species of keratin in primary pulmonary adenocarcinoma; CK5 is found predominantly in SCC. When used diagnostically, CK7 is often used with CK20 and nonkeratin antibodies in diagnosing and classifying
glandular neoplasms. Most primary pulmonary carcinomas contain several molecular species of keratins, with the exception of small cell carcinoma, which typically contains low-molecular-weight (LMW) keratins, including CK7. Chu and colleagues15 evaluated 435 epithelial neoplasms from various organs by using IHC with CK7 and CK20 antibodies. CK7 was seen in the majority of carcinoma cases, with the exception of carcinomas that arise from the colon, prostate, kidney, and thymus;
TABLE 12-3 Tumor-Specific Markers and Their Immunostaining Pattern Marker
Tumor
Staining Pattern
Calretinin
Mesothelioma, sex cord–stromal, adrenocortical
Nuclear/cytoplasmic
CDX-2
Colorectal/duodenal
Nuclear
D2-40
Mesothelioma, lymphatic endothelial cell marker
Membranous
Estrogen/progesterone receptors
Breast, ovary, endometrium
Nuclear
Gross cystic disease fluid protein 15
Breast
Cytoplasmic
HepPar1 (hepatocyte paraffin)
Hepatocellular
Cytoplasmic
Inhibin
Sex cord–stromal, adrenocortical
Cytoplasmic
Mammaglobin
Breast
Cytoplasmic
Melan-A
Adrenocortical, melanoma
Cytoplasmic
Mesothelin
Mesothelioma
Cytoplasmic/membranous
Prostate acid phosphatase
Prostate
Cytoplasmic
Prostate-specific antigen
Prostate
Cytoplasmic
Renal cell carcinoma marker
Renal
Membranous
Thyroglobulin
Thyroid
Cytoplasmic
Thyroid transcription factor 1
Lung, thyroid
Nuclear
Uroplakin III
Urothelial
Membranous
Villin
Gastrointestinal (epithelia with brush border)
Apical
Wilms tumor 1
Ovarian serous, mesothelioma, Wilms, desmoplastic, small round cell
Nuclear
390
Immunohistology of Lung and Pleural Neoplasms
carcinoid tumors of the lung and gastrointestinal (GI) tract; and Merkel cell carcinomas of the skin. The majority of SCCs from various organs were CK7 negative, with the exception of cervical SCC. CK20 was seen in almost all colorectal carcinomas and in Merkel cell carcinomas and was also observed in 62% of pancreatic carcinomas, 50% of gastric carcinomas, 43% of cholangiocarcinomas, and 29% of transitional cell carcinomas. As shown in Table 12-4, CK7 expression was seen in 10 of 10 lung adenocarcinomas, 3 of 7 (43%) SCLCs, 2 of 9 (22%) lung carcinoid tumors, and 0 of 15 squamous cell lung carcinomas. As shown in Table 12-5, CK20 expression was seen in 1 of 10 adenocarcinomas, 0 of 7 SCLCs, 0 of 9 carcinoid tumors, and 0 of 15 squamous cell lung carcinomas. An update of CK7 and CK20 expression in various neoplasms is shown in Table 12-6.
Sano and colleagues16 evaluated 50 cases of squamous cell carcinoma resected by lobectomy between 2005 and 2010. Centrally located SCCs were defined as a tumor from trachea to segmental bronchi, and peripheral SCCs were defined as being in a more peripheral location. IHC staining for CK7, TTF-1, p63, CK14, napsin A (Nap-A), 34βE12 (high-molecular-weight [HMW] keratin), CK5/6, and p53 was performed. The authors also studied the levels of entrapped pneumocytes inside the core by observing CK7. The IHC patterns were identical, and none showed statistically significant differences between centrally located SCC and peripherally located SCC. The 5-year survival rate showed no difference in the prognosis between these two entities. Vimentin is a 58-kD intermediate filament found predominantly in mesenchymal cells, although it is also
TABLE 12-4 Cytokeratin 7 in Carcinomas Organ
Tumor Type
Total Cases (n)
Positive Cases (n)
% Positive
Lung
Adenocarcinoma
10
10
100
Ovary
Adenocarcinoma
24
24
100
Pancreas
Adenocarcinoma
13
12
92
Stomach
Adenocarcinoma
8
3
38
Colon
Adenocarcinoma
20
1
5
Prostate
Adenocarcinoma
18
0
0
Lung
Carcinoid tumor
9
2
22
Gastrointestinal tract
Carcinoid tumor
15
2
13
Kidney
Carcinoma
19
2
11
Liver
Cholangiocarcinoma
14
13
93
Adrenal
Cortical carcinoma
10
0
0
Breast
Ductal and lobular carcinoma
26
25
96
Uterus
Endometrial carcinoma
10
10
100
Soft tissue
Epithelioid sarcoma
12
0
0
Germ cell
Germ cell tumor
14
1
7
Liver
Hepatoma
11
1
9
Skin
Merkel cell tumor
9
0
0
Mesothelium
Mesothelioma, malignant
17
11
65
Lung, liver, small bowel
Neuroendocrine carcinoma
9
5
56
Lung
Small cell carcinoma
7
3
43
Cervix
Squamous cell carcinoma
15
13
87
Head and neck
Squamous cell carcinoma
30
8
27
Esophagus
Squamous cell carcinoma
14
3
21
Lung
Squamous cell carcinoma
15
0
0
Thymus
Thymoma
8
0
0
Bladder
Transitional cell carcinoma
24
21
88
Salivary gland
Tumors, all
9
9
100
Thyroid
Tumors, all
55
54
98
Primary Lung Neoplasms
391
TABLE 12-5 Cytokeratin 20 in Carcinomas Organ
Tumor Type
Total Cases (n)
Positive Cases (n)
% Positive
Colon
Adenocarcinoma
20
20
100
Pancreas
Adenocarcinoma
13
8
62
Stomach
Adenocarcinoma
8
4
50
Lung
Adenocarcinoma
10
1
10
Ovary
Adenocarcinoma
24
1
4
Prostate
Adenocarcinoma
18
0
0
Gastrointestinal tract
Carcinoid tumor
15
1
7
Lung
Carcinoid tumor
9
0
0
Kidney
Carcinoma
19
0
0
Liver
Cholangiocarcinoma
14
6
43
Adrenal
Cortical carcinoma
10
0
0
Uterus
Endometrial carcinoma
10
0
0
Soft tissue
Epithelioid sarcoma
12
0
0
Germ cell
Germ cell tumor
14
0
0
Liver
Hepatocellular carcinoma
11
1
9
Breast
Lobular and ductal carcinoma
26
0
0
Skin
Merkel cell tumor
9
7
78
Mesothelium
Mesothelioma, malignant
17
0
0
Lung, liver, small bowel
Neuroendocrine carcinoma
9
0
0
Lung
Small cell carcinoma
7
0
0
Head and neck
Squamous cell carcinoma
30
2
7
Cervix
Squamous cell carcinoma
15
0
0
Esophagus
Squamous cell carcinoma
14
0
0
Lung
Squamous cell carcinoma
15
0
0
Thymus
Thymoma
Bladder
Transitional cell carcinoma
Salivary gland Thyroid
8
0
0
24
7
29
Tumors, all
9
0
0
Tumors, all
55
0
0
expressed in most spindle cell carcinomas17 and is reported by some to be expressed in a relatively high percentage of pulmonary adenocarcinomas.18 Thyroid transcription factor 1 (TTF-1), a 38- to 40-kD transcription factor member of the NKx2 family of homeodomain transcription factors, is expressed in thyroid and pulmonary epithelial cells.19,20 TTF-1 binds to and activates the promoters for Clara cell secretory protein and surfactant proteins A, B, and C.21,22 TTF-1 is expressed in the nuclei of 60% to 75% of pulmonary adenocarcinomas23-26 and in most SCLCs, atypical carcinoids, large cell NECs, and approximately 35% of typical carcinoids.27 Data that concern TTF-1 expression in various tumors are shown in Table 12-7. Another member of the p53 family is p63, which is significant in the development of epithelial tissue and SCCs. Expression of p63 was evaluated in 408 cases of
lung cancer by tissue microarray in two different laboratories by Au and associates.28 Table 12-8 shows the results for p63 expression in lung carcinoma for both laboratories. As expected, the majority of SCCs expressed p63, as did a sizable number of large cell NECs and small cell carcinomas. Expression of p63 was stated to be of prognostic significance in NECs, and high-grade tumors were found to be more likely to express p63 than low-grade tumors. From a practical point of view, we use p63 expression as a marker of squamous cell differentiation (Fig. 12-1). A relatively unknown antibody that recognizes ΔNp63, a p63 isoform suggested to be highly specific for squamous/basal cells, is p40; it might be a more valuable marker for cases in which p63 has traditionally been used—to identify SCCs, and it is a good marker; however, there is now evidence that p63 can also be
392
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-6 Coordinate CK7/20: Expression in Carcinomas CK7+/CK20+
CK7+/CK20−
CK7−/CK20+
CK7−/CK20−
Urothelial carcinoma Uroplakin+ Thrombomodulin+ p63+ CK5/6 (~1/2+)
Breast carcinoma ER/PR+ GCDFP+ Mammaglobin+ CEA+
Lung small cell carcinoma (majority) TTF-1+ NE† markers+ p63− NE* markers, including synaptophysin, chromogranin, and CD56
Colorectal adenocarcinoma CDX-2+ CEA+ MUC2+ MUC5AC−
Prostate adenocarcinoma PSA+ PAP+ CEA− Uroplakin− Thrombomodulin− p63− CK5/6−
Nonseminoma GCTs* PLAP+ EMA− Yolk sac tumor: AFP+ Embryonal carcinoma: OCT3/4+ CD30+
Pancreatic adenocarcinoma (~2/3) CEA+ CA 19-9+ MUC5AC+ MUC2− CDX-2 (variable)
Endometrial adenocarcinoma Vimentin+ ER/PR+ CEA−
Mesothelioma (~2/3) Calretinin+ WT1+ CK5/6+ Thrombomodulin+ D2-40+ Mesothelin+ p63− CEA− MOC-31− BerEP4− TTF-1−
Merkel cell carcinoma NE† markers+
Squamous cell carcinoma p63+ CK5/6+ Thrombomodulin+
Mesothelioma (~1/3)
Ovarian mucinous carcinoma MUC5AC+ MUC2− CDX-2 (variable)
Endocervical adenocarcinoma CEA+ Vimentin− ER/PR−
Thyroid carcinoma TTF-1+‡ thyroglobulin+‡ CEA− (except medullary CA)
Gastric adenocarcinoma (subset)
Renal cell carcinoma vimentin+ RCC marker+ CD10+ CEA−
Lung small cell carcinoma (minor subset)
Bladder adenocarcinoma Thrombomodulin+ CDX-2 (variable)
Ovarian serous carcinoma WT1+ ER/PR+ Mesothelin+ CEA−
Squamous cell carcinoma of cervix
Hepatocellular carcinoma HepPar1+ pCEA+§ CD10+§ MOC-31− CK19−
Gastric adenocarcinoma (subset)
Gastric adenocarcinoma (subset)
Lung adenocarcinoma TTF-1+ CEA+ CK5/6− p63−
Salivary gland tumor
Cholangiocarcinoma (minor subset)
Cholangiocarcinoma CEA+ CK19+ MOC-31+ CA19-9+ CDX-2 (variable) HepPar1−
Urothelial carcinoma (subset) Pancreatic and gastric adenocarcinoma (subset)
Adrenocortical carcinoma Inhibin+ Calretinin+ melan-A+ Vimentin+ CEA−
*Seminoma is keratin negative, OCT3/4 positive. † Including synaptophysin, chromogranin, and CD56. ‡ Undifferentiated anaplastic thyroid carcinoma is often negative for TTF-1 and thyroglobulin. § Characteristic canalicular pattern. AFP, Alpha-fetoprotein; CA 19-9, carbohydrate antigen; 19-9 CEA, carcinoembryonic antigen; CK, cytokeratin; EMA, epithelial membrane antigen; ER/PR, estrogen receptor/progesterone receptor; GCDFP, gross cystic disease fluid protein; GCT, germ cell tumor; NE, neuroendocrine; PAP, peroxidase-antiperoxidase; pCEA, polyclonal carcinoembryonic antigen; PLAP, placental alkaline phosphatase; PSA, prostate-specific antigen; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor 1.
Primary Lung Neoplasms
393
TABLE 12-7 Thyroid Transcription Factor 1 Expression in Tumors Diagnosis
% Positive
Adenocarcinoma, bronchioloalveolar, mixed
91
Adenocarcinoma, bronchioloalveolar, mucinous
21
Adenocarcinoma, bronchioloalveolar, nonmucinous
87
Adenocarcinoma, colorectal
2
Adenocarcinoma, colorectal, metastatic
4
Adenocarcinoma, endometrial
6
Adenocarcinoma, enteric differentiation, lung
70
Adenocarcinoma, lung and metastases
77
Adenocarcinoma, mucinous, lung
73
Adenocarcinoma, peritoneal, primary
8
Adenocarcinoma, thyroid (note: anaplastic carcinomas of thyroid are less frequently positive)
100
Alveolar adenoma
100
Carcinoid tumor, atypical, lung
100
Carcinoid tumor, intestinal
2
Carcinoid tumor, lung
57
Carcinoid tumor, metastatic, lung primary
80
Carcinoid, atypical
26
Carcinoid, not otherwise specified
24
Carcinoma, adenosquamous, lung
75
Carcinoma, large cell, metastatic
63
Carcinoma, large cell, neuroendocrine
46
Carcinoma, large cell, not otherwise specified
20
Carcinoma, oat cell, pulmonary
88
Carcinoma, rhabdoid features, lung, nonrhabdoid component
56
Carcinoma, small cell, bladder
34
Carcinoma, small cell, cervix
20
Carcinoma, small cell, esophageal
34
Carcinoma, small cell, gastrointestinal
17
Carcinoma, small cell, metastatic to lung
50
Carcinoma, small cell, prostate
58
Carcinoma, small cell, vaginal
34
Carcinoma, squamous cell, lung Carcinoma, undifferentiated type, metastatic
7 34
Cystadenocarcinoma, ovarian
2
Mesothelioma, all types
0
Sarcoma, not otherwise specified
6
Sclerosing hemangioma of lung, cuboidal cells and polygonal cells
100
Signet-ring cell carcinoma, lung
85
Synovial sarcoma
20
Figure 12-1 Nuclear p63 expression in a poorly differentiated squamous cell carcinoma of lung (×100).
found in a subset of lung adenocarcinomas. In a 2012 study by Bishop and colleagues,29 p40 staining was equivalent to p63 in sensitivity for SCC, but p40 was markedly superior to p63 with respect to specificity, which eliminated a potential pitfall for misinterpreting a p63-positive adenocarcinoma or suspected lymphoma as SCC. The authors compared the standard p63 antibody (4A4) with p40 in a series of 470 cases (81 squamous cell carcinomas; 237 adenocarcinomas; and 152 large cell lymphomas) and found that p63 was positive in 100% of SCCs, 31% of adenocarcinomas, and 54% of large cell lymphomas (sensitivity 100%, specificity 60%). In contrast, although p40 was also positive in 100% of SCCs, only 3% of adenocarcinomas and no large cell lymphomas showed p40 labeling (sensitivity 100%, specificity 98%). The mean percentage of p63 versus p40 immunoreactive cells in SCCs was equivalent: 97% versus 96%, respectively. The findings were stated to strongly support the routine use of p40 in place of p63 for the diagnosis of pulmonary SCC. Desmoglein 3 (DSG3) is a calcium-binding transmembrane glycoprotein component of desmosomes in vertebrate epithelial cells. Desmosomes are intercellular junctions that connect cells to one another and are typically fairly common in squamous cells, both neoplastic and nonneoplastic. A study by Savci-Heijink and colleagues30 showed that DSG3 had a sensitivity of 98% and a specificity of 99% for lung cancer. The authors found that 64 of 65 SCCs of the lung (98.5%) expressed DSG3 immunostaining, whereas only 1 of 47 pulmonary adenocarcinomas (2.1%) expressed DSG3 immunostaining. There was stated to be no immunostaining for DSG3 in 0 of 36 large cell carcinomas of the lung and 0 of 9 small cell carcinomas of the lung. The authors stated that the limitations to their study were that DSG3 expression by IHC was assessed only on large surgical specimens, and the tumors included in their study were at least moderately differentiated to guarantee diagnostic accuracy and to provide a gold standard to assess specificity and sensitivity of DSG3. However, the authors concluded DSG3 was a promising
394
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-8 p63 Expression in Lung Carcinoma Tumor Type
Negative (n)
Weak (n)
Strong (n)
Uninterpretable (n)
Positive (%)
PhenoPath Laboratory Result Adenocarcinoma
56
14
10
13
30.0
Atypical carcinoid
18
4
4
5
30.8
Classic carcinoid
51
1
0
16
1.9
Large cell carcinoma
34
5
15
14
37.0
Large cell neuroendocrine carcinoma
4
3
1
3
50.0
Small cell carcinoma
3
5
5
1
76.9
Squamous cell carcinoma
3
1
93
26
96.9
Adenocarcinoma
74
2
5
12
8.6
Atypical carcinoid
23
1
3
4
14.8
Classic carcinoid
57
0
1
10
1.7
Large cell carcinoma
42
3
10
13
23.6
Large cell neuroendocrine carcinoma
9
0
0
2
0
Small cell carcinoma
9
2
2
1
30.8
Squamous cell carcinoma
4
11
89
19
96.2
GPEC Laboratory Result
GPEC, Genetic Pathology Evaluation Centre.
diagnostic marker for distinguishing SCCs of the lung from other subtypes of lung cancers. In a study by Fukuoka and colleagues,31 the authors suggested that DSG3 was a prognostic marker in that positive DSG3 staining significantly correlated with a favorable prognosis in non-SCLC and carcinoid tumors. However, DSG3 did not appear to be as sensitive and specific in that study, which showed that 79.5% of SCCs and 54.8% of adenocarcinomas express DSG3 staining. In addition, de Paralta-Venturina and colleagues32 studied DSG3 to evaluate its use in the distinction between SCCs of the lung and nonsquamous lung carcinomas in small biopsy specimens. A total of 47 patients with resected lung SCCs who had previous bronchial, core, or fine needle aspiration (FNA) biopsy were identified from the files, and 97% of the biopsy cases and 72% of the FNA cases were diagnosed as SCCs based on histomorphology. The use of IHC identified all SCCs in all types of biopsy material based on positivity with at least one squamous marker, with p63 the most sensitive antibody (98%) followed by DSG3 (94%) and CK5/6 (85%). DSG3 was found to show membranous staining and tended to be variable and patchy compared with p63 and CK5/6. Four cases (8.5%) of SCC expressed TTF-1 staining, and none expressed Nap-A. However, Ordonez33 recently evaluated this issue and concluded that TTF-1 was not expressed in pulmonary or extrapulmonary SCCs, and TTF-1 staining may have been caused by a variety of other factors. In the study by de Paralta-Venturina and others,32 of the 20 adenocarcinoma biopsies, 8 (40%) showed rare faint p63 staining, and none showed staining for CK5/6 and
DSG3. Sensitivity and specificity for the diagnosis of SCC with DSG3 was 94% and 100%, CK5/6 was 85% and 100%, and p63 was 98% and 60% respectively. The authors32 concluded that IHC in small biopsy material, such as FNA biopsy with an adequate cell block, could provide definitive diagnosis of SCC; and although p63 was the most sensitive, DSG3 and CK5/6 had high specificities for diagnosing SCC. The authors32 recommended a panel of two squamous markers, DSG3 and CK5/6, and two adenocarcinoma markers, TTF-1 and Nap-A, for distinguishing SCC from non-SCC in small biopsy specimens. Findeis-Hosey and colleagues34 studied 97 resected SCCs of the lung for squamous precursor lesions that included basal cell hyperplasia, squamous metaplasia, low-grade dysplasia, high-grade dysplasia, and SCC in situ. The authors stated that insulin-like growth factor 2 (IGF-2) messenger RNA (mRNA)–binding protein 3 (IMP3) was reported to be expressed in multiple malignant neoplasms, but only a few studies had looked at IMP3 immunostaining with respect to SCC of the lung, and none had examined IMP3 expression in squamous precursor lesions. In addition, 86 of 97 SCCs (88.7%) showed predominantly strong, diffuse positive staining for IMP3, which was also expressed in the majority of cases of high-grade squamous dysplasia, squamous carcinoma in situ, and invasive SCC of the lung. These findings indicated that IMP3 might play an important role in the initiation of progression of pulmonary SCC. Sheikh and coworkers35 evaluated 33 cases of adenocarcinoma, 43 cases of benign lungs with fibrosis and metaplasia, 5 cases of atypical adenomatous
Primary Lung Neoplasms
and convoluted tubules of the kidney.45 It is present in lysosomes of type II pneumocytes and in alveolar macrophages, presumably secondary to phagocytosis, and to a lesser degree in pancreatic acini and ducts. Nap-A is thought to be involved in the maturation of biologically active surfactant protein B (SP-B) peptide, and Nap-A is strongly expressed in the cytoplasm of as many as 80% of primary lung adenocarcinomas studied by using IHC. Poorly differentiated cancers do not stain as well as well-differentiated cancers. SCCs and small cell carcinomas of the lung are reported to be negative for Nap-A expression.45 Sainz and colleagues46 studied the presence of Nap-A in 967 lung neoplasms and found that less than 5% of carcinomas of the bladder, pancreas, breast, liver, biliary tract, colon, ovary, and uterus; lung SCC; and lung small cell carcinoma were positive for Nap-A and that 74% of lung adenocarcinomas were positive for Nap-A versus 63% that were positive for TTF-1. Also, 11% of lung adenocarcinomas detected by Nap-A were missed by TTF-1 staining. The authors46 concluded that Nap-A was a valuable marker for detecting lung adenocarcinomas versus other adenocarcinomas, such as those from the breast, colon, biliary tract, pancreas, urinary bladder, and ovary. It was less useful in distinguishing primary pulmonary adenocarcinomas from thyroid carcinomas and renal carcinomas. In 2012, two articles were published that concerned Nap-A and the use of this marker to diagnose adenocarcinomas of the lung and other types of cancer. Turner and colleagues47 evaluated 1674 cases of carcinoma: 303/1674, or 18.1%, were primary lung adenocarcinomas; 200/1674, or 11.9%, were primary squamous cell lung carcinomas; and 52/1674, or 3.1%, were primary small cell carcinomas of the lung; also investigated were carcinomas of the kidney, thyroid, biliary tract, bladder, breast, colon, liver, ovaries, pancreas, prostate, stomach, and uterus. This study was done to compare Nap-A with TTF-1 in the typing of primary lung carcinoma and the differentiation of primary lung adenocarcinomas from
hyperplasia, 5 cases of adenosquamous carcinoma, and 3 cases of squamous carcinomas for nuclear p63 expression by IHC. The benign lung conditions included usual interstitial pneumonia, parenchymal scar, cryptogenic organizing pneumonia, and diffuse alveolar damage. In a normal lung, p63 was expressed in the reserve cells of large and small airways and in occasional cells of the distal lobular unit. In the fibrotic reactive processes, nuclear staining was observed in the basal cells of the airways and the bronchiolar and squamous metaplastic epithelium, and p63 immunoreactivity was stated to be less uniformly expressed in acute lung injury. Strong reactivity was shown in 1 of 33 pulmonary adenocarcinomas and in most epithelial cells of 2 of 5 atypical adenomatous hyperplasias. Three pulmonary adenocarcinomas were stated to highlight only rare tumor cells. The authors concluded that their results highlighted the differential p63 expression across various bronchioloalveolar lesions and that p63 could be helpful in distinguishing reactive from neoplastic glandular proliferations in the lung. A variety of epithelial cell markers—carcinoembryonic antigen (CEA), human milk fat globule protein 2 (HMFG-2), epithelial membrane antigen (EMA), Leu-M1 (CD15), B72.3, BerEP4, and surfactant apoprotein—are expressed in primary lung carcinomas, predominantly pulmonary adenocarcinomas. Most are nonspecific and are frequently used in distinguishing epithelial mesothelioma from pulmonary and nonpulmonary adenocarcinoma (see the paragraphs that follow) and in evaluating metastatic adenocarcinoma of unknown primary origin (see Chapter 8).36 The immunohistogram of common non-NE lung neoplasms is shown in Figure 12-2. Additional IHC findings in lung neoplasms are listed in Table 12-9,15,37-44 and Table 12-10 shows an update on the differential expression of TTF-1, CK7, and CK20 in common primary and metastatic tumors of the lung. Nap-A is an aspartic proteinase expressed in normal lung parenchymal type II pneumocytes and in proximal
1 FTT
rE Be
3 B7 2.
1 M Le u
EA pC
EM
A
an d
Vi
m
HM
en t
FG
2
in
7 CK
/6 CK 5
E1 2 34 β
11 35 βH
AE
P4
Adenocarcinoma Squamous carcinoma Large cell carcinoma
100 90 80 70 60 50 40 30 20 10 0 1/ AE 3
Percent positive
395
Figure 12-2 Immunohistogram of common primary pulmonary carcinomas, excluding small cell carcinoma. CK, Cytokeratin; EMA, epithelial membrane antigen; HMFG2, human milk fat globule protein 2; pCEA, polyclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor 1β.
37
38
40
Johansson et al41
Simsir et al
Lau et al
39
Nakamura et al
Amin et al
Chu et al
15
Reference
Moderately differentiated
29
Mucinous
12
Large cell pleomorphic
Adenocarcinoma
11
9
Small cell carcinoma
13
Mixed
6 Squamous cell carcinoma
Nonmucinous
4
12
Mucinous
Bronchioloalveolar:
6
16
Mixed
Nonmucinous
48
7
Bronchioloalveolar:
67
Large cell undifferentiated
Small cell carcinoma
18
8
Squamous cell carcinoma
26
Poorly differentiated
Well differentiated
19
4
Adenocarcinoma
52
Micropapillary adenocarcinoma
Typical carcinoid
9
15
Small cell carcinoma
Squamous cell carcinoma
15
7
Adenocarcinoma
Histologic Lung Cancer Type
10
No. Cases Studied
0 (0%)
0 (0%)
0 (0%)
12 (100%)
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
CK5
5 (55%)
11 (100%)
13 (100%)
3 (25%)
6 (100%)
4 (100%)
4 (67%)
7 (100%)
10 (83%)
46 (96%)
Not done
Not done
Not done
Not done
Not done
Not done
Not done
14 (93%)
2 (22%)
3 (43%)
0 (0%)
10 (100%)
CK7
TABLE 12-9 Expression of Keratins, TTF-1, and SP-A in Primary Pulmonary Neoplasms
9 (100%)
11 (100%)
13 (100%)
12 (100%)
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Cam5.2 CK8
0 (0%)
0 (0%)
0 (0%)
5 (55%)
11 (100%)
13 (100%)
0 (0%)
5 (83%)
6 (100%) Focally + 0 (0%)
2 (50%)
0 (0%)
6 (86%)
0 (0%)
36 (75%)
0 (0%)
16 (88.9%)
0 (0%)
4 (100%)
27 (93%)
19 (100%)
50 (96.2%)
12 (80%)
Not done
Not done
Not done
Not done
TTF-1
0 (0%)
4 (67%)
0 (0%)
3 (25%)
0 (0%)
Not done
Not done
Not done
Not done
Not done
Not done
Not done
2 (13%)
0 (0%)
0 (0%)
0 (0%)
1 (10%)
CK20
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
0 (0%)
0 (0%)
0 (0%)
2 (50%)
18 (62%)
17 (89.5%)
38 (73.1%)
Not done
Not done
Not done
Not done
Not done
SP-A
396 Immunohistology of Lung and Pleural Neoplasms
Nonmucinous Mucinous
18
Bronchioloalveolar:
“Conventional” adenocarcinomas
Nonlung metastatic neoplasm
32
50
125
Metastatic* lung neoplasm
Pseudomesothelioma
1
83
Mesothelioma
1
Sclerosing hemangioendothelioma
Atypical carcinoid
3
44
Typical carcinoid
Lymphoepithelial-like
25
8
Pleomorphic
Large cell undifferentiated
23
0
Small cell carcinoma
Squamous cell carcinoma
0
36
Adenosquamous carcinoma
12
Adenocarcinoma
Squamous carcinoma
Adenocarcinoma
*Positive for all thyroid cancers. CK, Cytokeratin; SP-A, surfactant protein A; TTF-1, thyroid transcription factor 1.
Saad et al
44
99
Chang et al43
176
64
Yatabe et al42
Not done Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
4 (22.2%)
20 (62.5%)
30 (60%)
0 (0%)
5 (6%)
0 (0%)
0 (0%)
39 (89%)
0 (0%)
0 (0%)
0 (0%)
19 (53%)
12 (100%)
169 (96%)
4 (4%)
54 (84.4%)
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
41 (64.1%)
Primary Lung Neoplasms
397
398
Immunohistology of Lung and Pleural Neoplasms
carcinomas of other sites. The authors found Nap-A to be superior to TTF-1 in distinguishing primary lung adenocarcinomas from other carcinomas (except kidney), particularly primary SCLC and primary thyroid carcinoma. A combination of Nap-A and TTF-1 was found to be useful in distinguishing primary lung adenocarcinoma (Nap-A+, TTF-1+) from primary lung SCC (Nap-A−, TTF-1−) and primary SCLC (Nap-A−, TTF1+). Whithaus and colleagues48 evaluated 197 adenocarcinomas and 66 SCCs for Nap-A, CK5/6, p63, and TTF-1 to find the most cost-effective panel to use to distinguish lung adenocarcinoma from SCC of the lung. For adenocarcinomas, the authors found that Nap-A had an 83% sensitivity and 98% specificity, whereas TTF-1 had a 60% sensitivity and 98% specificity. For SCCs, CK5/6 had a 53% sensitivity and 96% specificity, whereas p63 had a 95% sensitivity and 86% specificity for SCC. The authors concluded that the best panel to distinguish adenocarcinoma from SCC was Nap-A and p63, which showed a specificity of 94% and a sensitivity of 96%. The authors also stated that it was important to note the source of the antibody to avoid false-negative results. TTF-1 plays a critical role in the normal development of embryonic epithelial cells of the thyroid and lung and is the most commonly used IHC marker in differentiating carcinomas of the lung and thyroid. In a 2012 review article, Ordonez49 pointed out that TTF-1 was not as specific for lung and thyroid cancers as previously thought and presented a comprehensive, critical review of the literature on the expression of TTF-1 in tumors such as thyroid tumors, lung tumors, extrapulmonary small cell carcinomas, extrapulmonary non–small cell NECs, non-NECs of the female genital system and breast, adenocarcinomas of the digestive system, nonNECs of the urinary tract and prostate, tumors of the central nervous system (CNS) and pituitary gland, hepatocellular carcinomas, and rare tumors such as adenoid cystic carcinomas of the salivary gland and nasopharyngeal adenocarcinomas. The results showed that TTF-1 can be expressed in a percentage of tumors of the CNS such as glioblastomas, pituicytomas, granular cell tumors of the neurohypophysis, spindle cell oncocytoma of the adenohypophysis, and, rarely, in choroid plexus tumors and pituitary adenomas. TTF-1 has also been reported in a relatively small percentage of carcinomas of the female genital system and breast and in adenocarcinomas of the digestive tract and colon, although this is uncommon. TTF-1 expression has been consistently absent in parathyroid adenomas, thymic epithelial tumors, adrenal cortical carcinomas, and mesotheliomas. Also, because SCLCs, but not Merkel cell carcinomas, frequently express TTF-1 immunostaining, TTF-1 can be a useful marker in distinguishing between these two malignancies when used in conjunction with other markers such as CK20. TTF-1 is often expressed in lung adenocarcinomas but not in SCCs, and for this reason, TTF-1 has proved to be a useful marker in assisting with this differential diagnosis. Ordonez49 pointed out that although TTF-1 does not appear to be as specific for carcinomas of the lung and thyroid as previously thought, it remains a very useful
IHC marker in diagnostic pathology. Recently, commercially available SPT24 anti–TTF-1 monoclonal antibody was used in a large majority of cases in which TTF-1 positivity was demonstrated; however, reactivity was also reported with the more commonly used 8G7G3/1 clone, albeit in a lower percentage of cases. This information is of significant use in lung neoplasms, especially when separating certain types of lung neoplasms from one another and in expression of TTF-1 in certain thyroid tumors. TTF-1 expression correlates with predominantly histologic subtypes and recurrence in stage 1 lung adenocarcinomas. In a study of 506 stage I lung adenocarcinoma patients, Kadota and colleagues50 investigated whether TTF-1 expression correlated with the 2011 IASLC/ ATS/ERS lung adenocarcinoma classification and the prognosis based on histologic subtypes and stated that the majority of lung adenocarcinomas express TTF-1, but lack of TTF-1 expression correlated with worse prognosis. Of the 506 cases, 459 had adequate cores available for TTF-1 expression analysis. Intensity of staining was scored as follows: 0, no expression; 1, mild expression; 2, intermediate expression; and 3, strong expression. According to the intensity score, TTF-1 expression was divided into two groups: the low group represented scores from 0 to 1, and the high group represented scores of 2 to 3. A log-rank test was used to analyze the association between histologic variables and recurrence-free probability. The authors found that high TTF-1 expression was identified in 8/8 of AIS and MIA; 25/26 (96%) of LPNM; 113/129 (88%) of PP; 188/215 (87%) of acinar; 9/12 (75%) of MPP; 44/57 (77%) of solid; 4/9 (44%) of invasive mucinous; and 1/3 (33%) of colloid adenocarcinoma. High TTF-1 expression was more frequent in tumors with low architectural grade (P < .001), and the authors concluded that TTF-1 expression significantly correlated with architectural grade based on histologic subtype. In addition, TTF-1 expression was stated to correlate with recurrence and further stratified the acinar subtype. The distinction of lung adenocarcinoma from SCC of the lung has gained increased importance because of the emergence of histology-based therapies. Ang and colleagues51 state that IHC for squamous markers (p63 and HMW keratin) in combination with TTF-1 is suggested to aid in this distinction. The authors studied p63 (4A4 clone), HMW keratin (34βE12), and TTF-1 in 185 unselected adenocarcinomas that included 88 consecutive resections and 97 tumors and concluded that reactivity for both p63 and 34βE12 was frequent in lung adenocarcinoma, therefore neither p63 nor 34βE12 alone could be used to distinguish adenocarcinoma from SCC. However, no adenocarcinoma in this study had a three-marker coexpression profile that overlapped with typical SCC (TTF-1−/p63+/34βE12+), suggesting the utility of a panel approach. Zenali and colleagues52 found mucinous (colloid) carcinoma of the lung to be an uncommon subtype of pulmonary adenocarcinomas and discovered that differentiating between primary pulmonary and metastatic mucinous adenocarcinoma of extrapulmonary origin— namely, of lower GI tract origin—can be challenging
399
Primary Lung Neoplasms
TABLE 12-10 Differential Expression of TTF-1 and CK7/20 in the Most Common Primary and Metastatic Lung Neoplasms Diagnosis
TTF-1 (%)
CK7 (%)
CK20 (%)
Adenocarcinoma, bronchioloalveolar, mixed
91
100
64
Adenocarcinoma, mucinous, lung
21
87
67
Adenocarcinoma, bronchioloalveolar, nonmucinous
87
99
4
Adenocarcinoma, enteric differentiation, lung
70
100
24
Adenocarcinoma, follicular, papillary thyroid
94
100
0
Adenocarcinoma, lung
77
97
9
Adenocarcinoma, lung, metastatic
77
98
9
Sclerosing hemangioma of lung
99
100
0
Signet-ring cell carcinoma, lung
85
100
0
0
83
58
21
98
83
Carcinoma, signet ring, stomach
0
69
35
Carcinoma, transitional cell, not otherwise specified
0
91
403
Cystadenocarcinoma, mucinous, ovarian, not otherwise specified
0
93
70
88
13
3
Adenocarcinoma, endometrial
6
95
5
Adenocarcinoma, gallbladder
0
94
28
Adenocarcinoma, gastric
1
70
45
Adenocarcinoma, metastatic
0
100
0
Adenocarcinoma, pancreas
0
94
43
Adenocarcinoma, peritoneal, primary
8
100
0
Carcinoma, breast
0
88
0
Carcinoma, breast, metastatic
0
88
2
Carcinoma, embryonal, not otherwise specified
0
79
0
20
80
12
Carcinoma, signet ring, breast
0
100
4
Carcinoma, squamous cell, cervical
0
87
0
Cholangiocarcinoma
0
95
44
Cystadenocarcinoma, ovarian
2
97
16
Mesothelioma, malignant, localized
0
100
0
Mesothelioma, not otherwise specified
0
77
4
Mucoepidermoid carcinoma, lung
0
77
4
Neuroendocrine carcinoma, high grade, ampulla of Vater
0
88
38
Papillary cystadenocarcinoma, metastatic
0
100
0
TTF-1+ CK7+ CK20+ Bronchioloalveolar Carcinoma
TTF-1+ CK7+ CK20− Tumors
TTF-1− CK7+ CK20+ Tumors Adenocarcinoma, ampullary Adenocarcinoma, bronchioloalveolar, mucinous
TTF-1+ CK7− CK20− Tumors Carcinoma, oat cell, pulmonary TTF-1− CK7+ CK20−
Carcinoma, large cell, not otherwise specified
CK, Cytokeratin; TTF-1, thyroid transcription factor 1.
400
Immunohistology of Lung and Pleural Neoplasms
because of the considerable histologic and immunophenotypic overlap between the two. The authors evaluated hematoxylin and eosin (H&E)–stained sections from 17 surgical resection cases of pulmonary mucinous carcinoma from MD Anderson Cancer Center, however, 5 cases were excluded on the basis that they were mucinous adenocarcinomas of extrapulmonary origin. IHC analysis with a panel of immunostains (CK7, CK20, TTF-1, surfactant protein A [SP-A], and CDX-2) was performed, and extent of expression was assessed by light microscopy on a scale of 0 to 4+, 0 meaning none and 4+ meaning more than 75% staining. CK7 showed 4+ staining in 10 of 12 cases, 3+ staining in 1 case, and 2+ staining in 1 case. CK20 showed 4+ staining in 5 of 12 cases, 3+ staining in 2 cases, 2+ staining in 3 cases; 1+ staining in 1 case; and no staining in 1 case. TTF-1 showed 1+ staining in 4 of 12 cases, 7 showed no staining, and one case showed results that were not available. SP-A showed 1+ staining in 1 of 12 cases, and the remainder showed no staining. CDX-2 showed 4+ positive staining in 5 of 12 cases, 3+ staining in 2 cases, 2+ staining in 4 cases, and one case in which the results were not available. The authors concluded that their results indicate the use of IHC and clinical/radiologic correlation remains the gold standard for the site of origin in mucinous carcinomas that occur in the lung. Strong and diffuse expression of CK7 in colloid carcinoma of the lung can help differentiate this type of carcinoma from metastatic mucinous adenocarcinoma of the lower GI tract.
Neuroendocrine Lung Neoplasms Small cell lung carcinoma (SCLC) is the most common NE neoplasm of the lung.53,54 The previously described subtypes, lymphocyte-like and intermediate-polygonal/ fusiform, are currently lumped together as SCLC.55 The entity referred to as large cell–small cell carcinoma has been eliminated. The World Health Organization (WHO) International Histologic Classification of Tumors defines small cell carcinoma as “a malignant epithelial tumor consisting of small cells with scant cytoplasm, ill-defined cell borders, finely granular nuclear chromatin, and absent or inconspicuous nucleoli. The cells are round, oval and spindle shaped, and nuclear molding is prominent. The mitotic count is high.” A variant of small cell carcinoma is referred to as combined small cell carcinoma and is defined as “a small cell carcinoma combined with an additional component that consists of any of the histologic types of non–small cell carcinoma, usually adenocarcinoma, squamous cell carcinoma, or large cell carcinoma, but less commonly, spindle cell or giant cell carcinoma.” Other primary NE neoplasms of the lung include typical carcinoid, atypical carcinoid, and large cell NEC. The WHO definition of typical carcinoid is “a tumor with carcinoid morphology and less than 2 mitoses/10 hpf (2 mm2) lacking necrosis and 0.5 cm or larger.” Significant confusion has surrounded the entity known as atypical carcinoid, and several different names have been applied. The designation atypical carcinoid was used by Arrigoni and associates56 to describe a NE
lung neoplasm that differed from typical carcinoid. Atypical carcinoid has also been referred to as malignant carcinoid,57 well-differentiated NEC,58 peripheral small cell carcinoma of lung resembling carcinoid tumor,59 and Kulchitzky cell carcinoma II.60 The current WHO International Histological Typing of Lung, along with Travis and colleagues,61 describe atypical carcinoid as a tumor with NE morphology with between 2 and 10 mitoses/ 10 hpf (2 mm2) and/or with foci of punctate necrosis or both. Additional reports have been published that compare typical and atypical pulmonary carcinoids.62,63 A report by Thomas and colleagues62 provides evidence that atypical pulmonary carcinoid tumors with regional lymph node metastasis have a high likelihood of developing recurrent disease if treated with surgical resection only and that patients have a significantly worse outcome than those who have typical carcinoids with thoracic lymph node involvement. The existence of large cell NEC of the lung was suggested by Gould and Chejfec64 in 1978 and was further described by Hammond and Sause65 in 1985, Neal and coworkers66 in 1986, and Barbareschi and associates67 in 1989. It is uncertain whether the neoplasms described by McDowell68 in 1981 were large cell NECs or NSCLCs that showed NE differentiation. Travis and colleagues69 reported on 35 NE lung neoplasms in 1991 that included five large cell NE lung neoplasms. They included the following criteria for diagnosing a neoplasm as a large cell NEC: • A neuroendocrine appearance by light microscopy that includes an organoid, trabecular, palisading, or rosette pattern • Large cells with most cells greater than the nuclear diameter of three small resting lymphocytes • A low nuclear/cytoplasmic ratio, polygonal-shaped cells, finely granular eosinophilic cytoplasm with an eosinophilic hue, coarse nuclear chromatin, and frequent nucleoli • A mitotic rate greater than 10 mitoses per 10 hpf • Necrosis • Neuroendocrine features distinguished by IHC, electron microscopy, or both The WHO International Histological Classification of Tumors describes a large cell NEC as a “large cell carcinoma showing histologic features such as organoid, nesting, trabecular, rosette-like, and palisading patterns that suggest neuroendocrine differentiation in which the latter can be confirmed by immunohistochemistry or electron microscopy.” As Hammar et al70 published in 1989, a wide spectrum of differentiation for NE lung neoplasms exists, and some neoplasms do not fit into a well-defined category. Additional data have been published on large cell NEC.71-77 These articles continue to show that large cell NEC of the lung is an aggressive neoplasm. The publication by Peng and colleagues77 showed that the IHC features of large cell NEC have a similar biologic marker profile to SCLC and a different biologic profile than large cell carcinoma with NE features. However, a loss of heterozygosity (LOH) was noted at chromosome 3p in large cell NEC, large cell carcinoma with NE features,
Primary Lung Neoplasms
and SCLC. The authors suggested that morphologic NE differentiation might not be identical to biologic NE differentiation in large cell carcinomas of the lung. In a review article by Lim and collegues78 published in 2008, 1% to 2% of lung tumors are typical and atypical carcinoids; typical carcinoids account for the majority, whereas atypical carcinoids make up 11% to 24% of all pulmonary carcinoids. Typical carcinoids usually show organoid and/or trabecular architecture with mildly pleomorphic cells that comprise nuclei with granular chromatin and indistinct nucleoli with moderate amounts of eosinophilic cytoplasm. Mitoses are fewer than 2 per 2 mm2 and show no necrosis. Typical carcinoids sometimes arise on a background of diffuse idiopathic NE cell hyperplasia (DIPNECH). Atypical carcinoids are distinguished from typical carcinoids by the presence of necrosis and/or 2 to 10 mitoses per 2 mm2. Atypical carcinoids show greater architectural disorganization and increased pleomorphism, but these are not discriminatory criteria. According to one of the authors,78 large cell NECs and SCLCs are the two high-grade tumors in the NE spectrum. Large cell NECs and SCLCs comprise 3% and 15% to 20%, respectively, of all invasive lung cancers. No preinvasive lesions are recognized. Approximately 80% of large cell NECs are pure, and 20% are combined with other histologies such as adenocarcinoma or squamous carcinoma. Travis78 stated that Rossi and colleagues79 found the following percentage of IHC expression in large cell NEC: chromogranin A, 65%; synaptophysin, 53%; and CD56, 93%. NE differentiation can be seen in 10% to 20% of non–SCLCs, most likely adenocarcinoma. As many as two thirds of surgically resected SCLCs are “pure,” and the remaining are combined with other histologies such as adenocarcinoma or squamous carcinoma. In addition to the three standard IHC markers, other highlighted markers include AE1/AE3, Ki-67, neuron-specific enolase (NSE), bombesin-like peptides, protein gene product 9.5 (PGP9.5), and NE-specific proteins A and C. ANTIBODIES USED TO DETECT NEUROENDOCRINE LUNG NEOPLASMS
NE cells occur in many organs and tissues in the body and are part of the diffuse NE system, as described by Pearse,80 and the dispersed NE system, described by Gould and DeLellis.81 Not surprisingly, NE cells and neoplasms formed by cells that exhibit NE differentiation show similar IHC features. They contain a variety of biogenic amines, peptide hormones, and neurotransmitters that can be identified biochemically or immunohistochemically.82 IHC markers are useful in showing NE differentiation by a neoplasm but are not usually specific. The antibodies commonly used in demonstrating NE differentiation are shown in Table 12-11. Synaptophysin is a 38-kD glycoprotein component of presynaptic vesicles isolated from bovine neurons,83,84 and it is the most sensitive antibody for identifying NE neoplasms but is the least specific. Synaptophysin has occasionally been observed in non-NE NSCLCs.
401
Chromogranins are a family of acidic proteins that contain high concentrations of glutamic acid located in the matrix of NE granules in normal and neoplastic NE cells.85,86 In 1965, chromogranin A was discovered in adrenal medullary cells by Banks and Helle.87 Antibodies against chromogranin A are the most specific marker of normal and neoplastic NE cells. Expression in a given neoplasm generally correlates with the number of cytoplasmic NE granules visualized ultrastructurally. NSE catalyzes the interconversion of 2phosphoglycerate and phosphoenolpyruvate in the glycolytic pathway. Enolases are dimers composed of three subunits: alpha (α), beta (β), and gamma (γ). NSE contains a high concentration of γ-enolase and is usually present in high concentrations in neurons and NE cells. Unfortunately, NSE is not neuron or NE cell specific, rather it has been identified in a wide variety of nonneuron, non-NE cells that include smooth muscle, myoepithelial, renal epithelial, and plasma cells and megakaryocytes.88,89 NSE is not uncommonly referred to as nonspecific enolase. Despite its low specificity, it is a highly sensitive marker for neoplastic NE cells. Other NE markers occasionally used to identify normal NE lung cells and neoplastic NE cells include neurofilaments,90 neural cell adhesion molecules (NCAMs),91,92 and Leu-7.93 The most frequent neuropeptides, neuroamines, and hormones found in NE lung neoplasms are listed in Box 12-1. TTF-1 is found in a high percentage of small cell carcinomas, atypical carcinoids, and large cell NECs but in less than 50% of typical carcinoids. The current discriminative value of NE markers is shown in Table 12-12.78 Matsuki and colleagues94 evaluated histidine decarboxylase, an enzyme of the amine precursor uptake and decarboxylation system known to be distributed in mast cells and enterochromaffin-like cells, with the hypothesis that this enzyme was a marker for NE differentiation. They found that the antihistidine decarboxylase antibody stained most SCLCs (18/23; sensitivity 0.78) and was rarely reactive with non-NE lung tumors (2/44; specificity 0.95). They also found the reaction to be similar to that obtained with CD56. Histidine decarboxylase was expressed in 6 of 12 large cell NECs and in 4 of 7 GI small cell carcinomas, therefore Matsuki and colleagues concluded that histidine decarboxylase
Box 12-1 NEUROPEPTIDES, NEUROAMINES, AND HORMONES FREQUENTLY FOUND IN NEUROENDOCRINE LUNG NEOPLASMS Adrenocorticotropic hormone Arginine vasopressin Bombesin Calcitonin Gastrin Leu-enkephalin Neurotensin Serotonin Somatostatin Vasoactive intestinal polypeptide
402
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-11 Antibodies Commonly Used in Identifying Neuroendocrine Lung Neoplasms Antibody Directed Against
Clone
Characteristics of Antigen
Immunogen
Manufacturer
Dilution
Synaptophysin
—
38-kD membrane component of synaptic vesicles
Synthetic human synaptophysin coupled to ovalbumin
Dako
1 : 100
Chromogranin A
DAK-A3
439–amino acid protein encoded on chromosome 14 residing in NE granules
C-terminal 20-kD fragment of chromogranin A
Dako
1 : 100
NSE
—
46-kD gamma-gamma isoenzyme of enolase
NSE isolated from human brain
Dako
1 : 400
Leu-7, CD57
NK-1
110-kD human myeloid cellassociated surface glycoprotein
Antigen from human natural killer cells
BioGenex
1 : 20
NCAM
UJ13A
125-kD sialo glycoprotein
16-week-old fetal human brain homogenates
Dako
1 : 20
TTF-1
8G7G3/1
40-kD member of NKx2 family of homeodomain transcription factors in lung and thyroid
Mouse ascites
Cell Marque
NA
CD117
—
145-kD band of transmembrane receptor
Rabbit
Dako
1 : 400
Ki-67
_
Ki-67 nuclear antigen in proliferating cells
Mouse monoclonal antibody
Ventana
NA
Antigen retrieval was achieved with heat-induced epitope retrieval (HIER). NCAM, Neural cell-adhesion molecule; NE, neuroendocrine; NSE, neuron-specific enolase; TTF-1, thyroid transcription factor 1.
was useful in distinguishing SCLC from non-NE lung carcinoma and in demonstrating NE differentiation. Antibodies used in evaluating lung neoplasms for NE differentiation include LMW keratin, HMW keratin, synaptophysin, chromogranin A, TTF-1, and leukocyte common antigen (LCA). CEA is occasionally expressed in NE lung neoplasms,95 and NSE is expressed in nearly all NE lung neoplasms. An immunohistogram that shows the characteristic IHC staining reactions in NE lung neoplasms is shown in Figure 12-3. Small cell carcinoma, which comprises approximately 20% to 25% of all primary lung cancers, shows immunostaining for LMW keratin (CAM5.2, 35βH11); no immunostaining for HMW keratin; immunostaining for synaptophysin, NSE, and CEA (Fig. 12-4); variable staining for chromogranin A; and frequent nuclear
TABLE 12-12 Discriminative Value of Neuroendocrine Markers Marker
Sensitivity (%)
Specificity (%)
CD56 (NCAM)
95
97
34βE12
95
95
Chromogranin
85
97
NSE
82
68
Synaptophysin
80
97
Leu-7
39
97
NCAM, Neural cell-adhesion molecule; NSE, neuron-specific enolase.
immunostaining for TTF-1 (Fig. 12-5). The pattern of staining for LMW keratin and chromogranin is usually punctate (Figs. 12-6 and 12-7). In our experience, many SCLCs do not express chromogranin A, although the expression is dependent upon how many NE granules are in the cytoplasm of the neoplastic cells. When studied by electron microscopy, occasional small cell carcinomas are found in which tumor cells contain a moderate number of NE granules (Fig. 12-8). Typical carcinoids, atypical carcinoids, and large cell NECs characteristically express both LMW and HMW keratins, synaptophysin, and chromogranin A. Atypical carcinoids and large cell NECs frequently express TTF-1. IHC tests are helpful in identifying these neoplasms as neuroendocrine but are often not especially helpful in separating specific neoplasms from one another. Chromogranin A shows the greatest staining intensity in typical carcinoids (Fig. 12-9), which correlates with relatively frequent cytoplasmic NE granules. According to a study by Findeis-Hosey and colleagues,96 Nap-A is frequently expressed in pulmonary adenocarcinomas but is rarely detected in NE tumors of the lung based on a study of 14 typical carcinoids, 12 atypical carcinoids, 11 large cell NECs, and 9 SCLCs. These findings were stated to support the concept that the cells of origin in adenocarcinomas and NE tumors of the lung are different. The authors96 stated that IHC detection of Nap-A expression may serve as a useful diagnostic tool in the distinction between NE tumors of the lung and poorly differentiated pulmonary adenocarcinoma. In a study to evaluate the usefulness of the Ki-67 index and mitoses in tumor size for predicting
Primary Lung Neoplasms
403
100 90
Percent positive
80 70 60 50 40 30 20 10
IB -1 /M
7 11
F1 TT
u7 Le
CD
Ki -6 7
og ro m Ch
Sy
na
pt op
ra n
hy
in
A
sin
E NS
tin en
CK
7 Vi m
34
E
12
11 H 35
AE
1/ AE
3
0
Carcinoid Atypical carcinoid Large cell neuroendocrine carcinoma Small cell carcinoma Figure 12-3 Immunohistogram of neuroendocrine lung carcinomas. CK, Cytokeratin; NSE, neuron-specific enolase; TTF-1, thyroid transcription factor 1.
metastasis of carcinoid tumors of the lung, Shibani and colleagues97 studied 48 cases of carcinoid tumors of the lung and reported that finding more than two mitoses was a powerful predictor of metastases and that analysis of Ki-67 index along with metastases might be a useful adjunct for predicting metastases in lung carcinoid tumors, for which adjuvant therapy might be considered. Kirby and colleagues98 studied Pax-2, Pax-5, and Pax-8 expression in pulmonary NE tumors and stated that PAX genes 1 through 9 are transcription factors with DNA-binding capabilities that play key roles in fetal development, tissue maintenance, and repair. Pax-8 expression was identified in 16% (23/144) of pulmonary NE tumors and included 2.4% (1/42) of typical
carcinoids, 11.1% (3/27) of atypical carcinoids, 33.3% (8/24) of large cell NECs, and 21.6% (17/51) of SCLCs. Pax-8 expression correlated with NE tumor differentiation/grade and was observed in a significant percentage of large cell NECs and SCLCs. The authors stated that because Pax-8 expression was also seen in rare pulmonary carcinoids, its suggested site specificity for pancreatic/duodenal NE tumors should be used with caution. The vast majority of pulmonary NECs were negative for Pax-2 and Pax-5. Ko and colleagues99 evaluated 58 cases of pulmonary NE tumors that included carcinoid tumorlet, typical carcinoids, atypical carcinoids, SCLC, and large cell NEC for histone 1.5 (H1.5) immunostaining based on intensity and proportion of positive tumor cells. H1.5
Figure 12-4 Many small cell lung carcinomas express carcinoembryonic antigen (×200).
Figure 12-5 This small cell carcinoma of lung shows intense nuclear immunostaining for thyroid transcription factor 1 (×200).
404
Immunohistology of Lung and Pleural Neoplasms
Figure 12-6 Most small cell carcinomas of lung show punctate immunostaining for low-molecular-weight keratin (×400).
was universally found to show nuclear staining with no cytoplasmic staining. High nuclear expression of H1.5 in pulmonary NE tumors was directly correlated with high tumor grade, whereas low- and intermediate-grade typical carcinoids and atypical carcinoids had a low incidence of positive staining for H1.5 (weak and/or involving a small percentage of cells). In contrast, all high-grade large cell NECs and SCLCs showed a high percentage of H1.5-positive cells. These results were stated to be compatible with the theory that high expression of H1.5 reflects a constant state of DNA replication in high-grade pulmonary NE tumors. The authors stated that H1.5 staining might be useful in differentiating carcinoid tumors from high-grade NECs.
Rare Primary Lung Neoplasms
Figure 12-8 In this small cell carcinoma, the neoplastic cells contain a moderate number of cytoplasmic, dense, core neuroendocrine granules (×16,000).
Kaposi sarcoma, clear cell neoplasm (sugar tumor, perivascular epithelioid cell tumor [PEComa]), rhabdoid tumor, sclerosing hemangioma, and inflammatory pseudotumor.100 SARCOMATOID CARCINOMA
A variety of rare primary neoplasms occur in the lung that are occasionally encountered by pathologists and may cause diagnostic confusion. Examples include sarcomatoid carcinoma (carcinosarcoma, spindle cell carcinoma), pulmonary blastoma, malignant hemangioendothelioma (intravascular bronchioloalveolar tumor [IVBAT]), sarcoma, lymphoproliferative disorder/ lymphoma, pulmonary Langerhans cell histiocytosis,
Sarcomatoid carcinoma—also referred to as carcinosarcoma, spindle cell carcinoma, blastoma and teratocarcinoma—is a neoplasm of epithelial derivation that shows variable differentiation. Sarcomatoid carcinoma has been reviewed conceptually by Wick and Swanson.101 Several studies have evaluated sarcomatoid carcinoma by IHC by using keratin antibodies and/or electron microscopy and have concluded that the spindle cell component of the neoplasm was of
Figure 12-7 As shown in this photograph, the immunostaining pattern for chromogranin A in this small cell lung carcinoma is punctate (×400).
Figure 12-9 This typical carcinoid shows intense cytoplasmic immunostaining for chromogranin A (×400).
Primary Lung Neoplasms
Figure 12-10 This region of sarcomatoid carcinoma showed immunostaining of the spindle cells for low-molecular-weight keratin (×400).
epithelial derivation.102-106 In most instances, the spindle cells coexpress keratin and vimentin (Figs. 12-10 and 12-11) or occasionally keratin and other intermediate filaments, such as desmin or actin. PLEOMORPHIC CARCINOMA
Pleomorphic carcinoma, as defined by Fishback and colleagues,107 is a neoplasm that occurs predominantly in older individuals and is composed predominantly of spindle cells and large pleomorphic giant cells. In a review of 78 cases, the authors found foci of squamous differentiation in 8%, large cell undifferentiated carcinoma in 25%, and adenocarcinoma in 45%. The remaining 22% of the neoplasms were composed of neoplastic spindle cells and giant cells; neoplastic spindle cells usually express only vimentin, whereas the immunostaining pattern of the epithelial component of the neoplasm depends on what type of differentiation it shows. Pelosi and colleagues108 evaluated 31 cases of pleomorphic carcinoma of the lung that showed neoplastic epithelial cells and a spindle and/or giant cell component for cytokeratins, EMA, CEA, vimentin, S-100 protein, smooth muscle actin (SMA), desmin, cell-cycle
405
control and apoptosis factors (p53, p21Waf1, p27Kip1, FHIT), tumor growth (proliferative fraction assessed by Ki-67 antigen, microvascular density assessed by CD34 immunostaining), and tumor cell motility (fascin). The epithelial component of these tumors was immunoreactive for cytokeratins, EMA, CEA, cell-cycle inhibitors, and tumor suppressor gene expression, whereas the sarcomatoid component, independent of tumor stage and size, was more immunoreactive for vimentin, fascin, and microvascular density. The authors suggested a model of tumorigenesis whereby the mesenchymal phenotype of the pleomorphic cells was likely induced by selective activation and segregation of several molecules involved in cell differentiation, cell-cycle control, and tumor cell growth and motility. PULMONARY BLASTOMA
Pulmonary blastomas comprise an epithelial component that forms glandular structures, often resembling endometrial glands or fetal glands, and a spindle cell component. The epithelial tumor cells occasionally form squamous morulae. As reported by Koss and colleagues109 and Yousem others,110 these neoplasms differentiate in divergent ways and occasionally show NE differentiation. The epithelial cells form glands and immunostain for keratin, CEA, and EMA. The spindle cells express vimentin and, depending on the type of differentiation of the sarcomatoid component, desmin, actin, and S-100 protein. Those that contain NE elements show markers of NE cells. The epithelial component of pulmonary blastoma typically expresses β-catenin. PRIMARY SARCOMA
Primary sarcomas of the lung are rare, and a variety of them occur. The immunophenotype of the neoplastic cells that form such neoplasms is essentially identical to sarcomas that occur in soft tissue and in organs (see Chapter 4). Etienne-Mastroianni and colleagues111 performed a clinicopathologic study of 12 cases of primary sarcoma of the lung. The histologic diagnosis of these 12 neoplasms was confirmed by detailed IHC, and they reported 7 leiomyosarcomas, 2 monophasic synovial sarcomas, 1 peripheral nerve sheath tumor, 1 epithelioid sarcoma, and 1 malignant fibrous histiocytoma. In all, 9 of the 12 patients had surgery: 3 pneumonectomies and 6 lobectomies were performed, with further resection in 2 cases; 4 patients received chemotherapy, and 2 had radiation therapy. Follow-up was available on all 12 patients: survival ranged from 3 to 144 months, with a mean of 42 months; long-term survival up to 3 years was observed in 5 patients; overall 5-year survival rate was 38%. The authors concluded that primary sarcomas of the lung were rare and aggressive neoplasms, and treatment and prognosis did not differ from those of other soft tissue sarcomas. KAPOSI SARCOMA
Figure 12-11 Same neoplasm shown in Figure 12-10. Neoplastic spindle cells show intense immunostaining for vimentin (×400).
Kaposi sarcoma has become more common since the advent of acquired immunodeficiency syndrome
406
Immunohistology of Lung and Pleural Neoplasms
Figure 12-12 The neoplastic cells in Kaposi sarcoma are spindle shaped and are in a lymphatic distribution (×400).
(AIDS), and in most cases, it is a metastatic neoplasm in the lung.112-121 Kaposi sarcoma follows lymphatic pathways in the lung and often involves lymph nodes. The neoplastic cells are spindle shaped (Fig. 12-12) and immunostain for vimentin and endothelial cell markers such as CD31 (Fig. 12-13).
Figure 12-14 In this malignant epithelioid hemangioendothelioma, the neoplastic cells have an epithelial appearance and can be confused with carcinoma (×400).
ANGIOSARCOMA
Malignant hemangioendothelioma, also referred to as intravascular bronchioloalveolar tumor, usually occurs in relatively young women, is bilateral, and takes the form of multiple small nodules that fill alveolar spaces and undergo degeneration, necrosis, and calcification.100 This neoplasm was initially thought to be of alveolar cell origin. Histologically, the tumor cells are round or polygonal and have an epithelial appearance (Fig. 12-14). They characteristically express endothelial markers such as CD31, CD34, and factor VIII antigen (FVIII), and they immunostain for vimentin. Rarely, epithelioid hemangioendotheliomas express keratin (Fig. 12-15). Ultrastructurally, the neoplastic cells often contain Weibel-Palade bodies, which are pathognomonic markers of endothelial cells (Fig. 12-16).
Primary angiocarcinomas are rare, and they can be dedifferentiated without forming obvious vascular channels. The neoplastic cells usually show the same IHC profile as malignant hemangioendotheliomas. Adem and coworkers122 reported on seven patients with metastatic angiosarcoma that masqueraded as diffuse pulmonary hemorrhage. Of the seven patients, who ranged in age from 31 to 73 years, six were male. Six patients presented with hemoptysis, and all had diffuse abnormalities on radiographic studies. Clinical diagnoses included pulmonary hemorrhage syndrome (two cases), acute respiratory failure (one case) and infection (one case). Metastatic disease was included in the differential diagnosis in one patient. No patients had a previous diagnosis of malignancy, and all biopsies showed hemorrhage associated with atypical epithelioid and spindle cells that formed anastomosing vascular channels distributed along and within lymphatics and arteries. The neoplastic cells were immunoreactive for FVIII-related protein and CD31. Three patients with complete follow-up died of their disease, and three underwent autopsy examination in which primary sites
Figure 12-13 Kaposi sarcoma tumor cells express vascular markers such as CD31 (×400).
Figure 12-15 Occasional neoplastic epithelioid hemangioendothelioma cells show cytoplasmic immunostaining for keratin (×400).
HEMANGIOENDOTHELIOMA
Primary Lung Neoplasms
407
classification of lymphoproliferative lung lesions as sophisticated molecular biology and genetic studies have become available. Conditions thought to be nonneoplastic several years ago have now been shown to be low-grade lymphomas. BRONCHIAL-ASSOCIATED LYMPHOID TISSUE
Figure 12-16 In this case, the neoplastic epithelioid hemangioendothelioma cells contained cytoplasmic Weibel-Palade bodies (×20,000).
outside of the lung were identified. Two angiosarcomas arose in the heart, and one arose in the pelvic soft tissue. One patient likely had a primary site in the right atrium identified by cardiac ultrasound. The authors122 concluded that angiosarcoma should be included in the differential diagnosis of diffuse hemorrhage, especially in young adults. We observed a similar case in which the cells had a much more epithelioid morphology and presented as multiple nodules in the lung, although no primary tumor was identified (Fig. 12-17).
Lymphoproliferative Disorders of the Lung A variety of nonneoplastic and neoplastic lymphoproliferative disorders involve the lung (see Chapters 5 and 6). Significant evolution has ensued with respect to
A
Bronchial-associated lymphoid tissue (BALT) is inconspicuous or absent in most normal adult human lung tissue. There is debate as to whether BALT is seen in the normal human adult lungs.123 Exogenous or endogenous antigenic stimulation of a variety of types causes this lymphoid tissue to appear in human lung tissue, and it occurs in a fairly distinct anatomic location in most instances; namely, in association with bronchi and bronchioles. In nonsmoking adults, lymphoid tissue is seldom seen in lung tissue and, when present, it occurs as small aggregates usually found at bronchial divisions and adjacent to respiratory bronchioles. In smokers, BALT is significantly increased, with occasional large lymphoid follicles, some of which have germinal centers.124 As recently reviewed by Swigris and associates,125 BALT is part of a larger system referred to as mucosal associated lymphoid tissue (MALT) and is uncommonly seen in the normal adult lung and is also commonly seen in other mammals such as rats, rabbits, and sheep. The lymphoid tissue is usually poorly organized and consists of aggregates of lymphocytes around small bronchi and at bronchial divisions. Some lymphocytes reside in the bronchial epithelium between the epithelial cells. Lymphoid cells are thought to populate these areas by the contact of cell-surface homing receptors, such as integrins, on specific intrapulmonary vascular adhesion molecules, such as those seen in postcapillary vascular endothelial cells. Approximately 60% of BALT is composed of B cells; the remainder are T cells. BALT is thought to play an essential role in the prevention of infection of inhaled microorganisms and is a site for lymphoid differentiation, where lymphocytes come in contact with inhaled antigens to become antigen-specific memory or immune effector cells. When primed by an antigen, these cells are thought to circulate throughout
B Figure 12-17 Angiosarcoma of the lung. A, Hematoxylin and eosin. B, CD31 immunostain.
408
Immunohistology of Lung and Pleural Neoplasms
the BALT and remaining lung parenchyma, ready to react to exposure to antigens. Other cells are also involved in the immunologic response, such as dendritic macrophages (Langerhans cells). The respiratory epithelium overlying the BALT usually contains relatively few goblet and ciliated cells. The BALT-associated epithelial cells are surfaced by microvilli, and the epithelium is infiltrated by predominantly CD8-positive and occasionally CD4-positive lymphocytes. According to Swigris and colleagues,125 BALT does not express the secretory component of IgA and differs from other MALT such as that seen in the GI tract. Bienenstock and coworkers126,127 were the first to provide information on the morphologic and functional characteristics of BALT. The majority of nonneoplastic and neoplastic lymphoid infiltrates in the lung are related to BALT. In neoplasia, BALT proliferations can explain most neoplastic pulmonary lymphoid diseases. As stated previously, difficulty may be encountered in distinguishing between neoplastic and nonneoplastic lymphoid lesions. In many instances, molecular biology techniques such as flow cytometry are necessary to prove malignancy.128,129 A variety of recognizable benign conditions of the lung are associated with lymphoid infiltrates that seem haphazardly oriented. These conditions include usual interstitial pneumonia; desquamative interstitial pneumonia; cases of collagen vascular associated lung disease; nonspecific interstitial pneumonia, cellular phase; hypersensitivity pneumonitis; the early phase of sarcoidosis, Wegener granulomatosis; Churg-Strauss granulomatosis; microscopic polyarteritis nodosa; and infiltrates associated with certain drugs such as sulfasalazine. Detailed information concerning most lymphoproliferative diseases of the lung is provided in the references 130 through 135. Most pulmonary lymphoid lesions represent hyperplasias of BALT and neoplastic lymphoproliferative disorders that arise from BALT. Kradin and Mark136 were the first to discard the terms pseudolymphoma and lymphoid interstitial pneumonitis and replace them with “nodular and diffuse hyperplasias of BALT.” As discussed in detail by Koss,135 localized hyperplasias of BALT, also termed follicular bronchitis/bronchiolitis, is due to a proliferation of BALT in the region of small bronchi and bronchioles. Lymphoid hyperplasias are seen in a number of different clinical conditions, including chronic infection, congenital immune deficiency syndromes, obstructive pneumonias, and collagen vascular diseases. Pathologically, they are characterized by collections of lymphoid nodules, with or without germinal centers, in a peribronchial/peribronchiolar distribution. The term nodular lymphoid hyperplasia BALT has replaced pseudolymphoma and is characterized by reactive lymphoid proliferation that characteristically shows numerous lymphoid follicles, with large germinal centers; usually it occurs in middle-aged people, most of whom are asymptomatic. Approximately 10% to 15% of patients with nodular lymphoid hyperplasia have a collagen vascular disease, such as systemic lupus erythematosus, or an immune disease of uncertain etiology, and they frequently exhibit polyclonal gammopathy. Polytypic plasma cells are common. Marker studies
show a mixed population of CD4- and CD8-positive T cells. Most cases occur as solitary nodules, and they recur in as many as 15% of surgically excised cases. LYMPHOCYTIC INTERSTITIAL PNEUMONIA-PNEUMONITIS
Lymphocytic interstitial pneumonia-pneumonitis (LIP) is currently thought to be a diffuse hyperplasia of BALT,137 a response to a variety of stimuli that include a wide spectrum of autoimmune diseases; systemic immunodeficiency states; allogenic bone marrow transplantation; pulmonary alveolar microlithiasis; uncommon infections such as Legionella pneumonia, tuberculosis, Mycoplasma, and Chlamydia; dilantin use; and pulmonary alveolar proteinosis. Most patients who develop LIP are women between the ages of 40 and 70 years. They may come to medical attention with a variety of symptoms that may include fever, hemoptysis, arthralgias, weight loss, and pleuritic chest pain. Some have Sjögren syndrome or myasthenia gravis. The chest radiograph usually shows a diffuse reticular or reticulonodular infiltrate with occasionally nodular lesions. Some patients have hypergammaglobulinemia, and approximately 10% of adult patients will have hypogammaglobulinemia. If LIP patients have a monoclonal gammopathy, it is usually due to a coexistent lymphoma. A significant number of patients with LIP respond to steroids, although approximately 30% to 50% die within 5 years, frequently from infectious complications, some as a result of treatment. Some patients develop lymphoma and immunoblastic sarcomas. NODULAR LYMPHOID INFILTRATES OF UNCERTAIN NATURE
Some nodular lymphoid lesions of uncertain etiology that may be found in the lung include plasma cell granuloma, pulmonary hyalinizing granuloma, and benign lymphocytic angiitis and granulomatosis (BLAG). Plasma cell granuloma, also referred to as myofibroblastic tumors or inflammatory pseudotumors, occur predominantly in the lungs of younger individuals but have been observed in a wide variety of organs and tissues that include the trachea, thyroid, heart, stomach, liver, pancreas, spleen, lymph nodes, kidney, retroperitoneum, mesentery, bladder, pelvic soft tissue, breast, spinal cord meninges, and orbit.100 The term granuloma may be somewhat misleading, unless is it realized that this term has been used loosely to describe inflammatory tissue similar to that observed in Wegener and lymphomatoid granulomatosis (LYG). Histologically, these nodules are made up of an inflammatory infiltrate composed predominantly of plasma cells and a proliferation of myofibroblasts. Some studies suggest that plasma cell granulomas are neoplastic. Inflammatory pseudotomors are discussed in some detail below. Pulmonary hyalinizing granuloma occurs most frequently as single or, occasionally, multiple nodules. Some individuals are asymptomatic, whereas others have cough, shortness of breath, chest pain, or weight loss. Histologically, the dominant finding is one of dense lamellar collagen bundles oriented parallel to one
Primary Lung Neoplasms
another and, not infrequently, an associated lymphoid infiltrate, frequently in a bronchial distribution. BLAG was described in 1977 by Saldana and colleagues138 and by Israel and associates.139 This entity was reported as less common than Wegener granulomatosis and LYG. Most patients are middle-aged and in most instances have multiple nodular pulmonary lesions. Histologically, the nodules are composed of a dense lymphoid infiltrate with occasional giant cells, including multinucleated histiocytic giant cells, but without welldefined granuloma formation. Lymphoid infiltrates around arteries and veins are frequent, and lymphocytes invade vessel walls and occasionally produce occlusion of vascular lumina. Differential diagnoses include lymphocytic lymphoma and LYG when the lesions are multiple. LYMPHOMAS OF THE LUNG
Few neoplasms have received as much emphasis on classification and subtyping as lymphoma. Histology/ cytology, IHC, flow cytometry, and other molecular biology techniques have been used extensively for the classification of lymphoma. The guidelines for proper handling and recommendations for classification and reporting of lymphoid neoplasms were published in 2002,140 and IHC criteria for diagnosing such neoplasms was reported in detail in 2001.141 Current lymphoma classification and diagnostic issues can be found in Chapters 5 and 6. PULMONARY NON-HODGKIN LYMPHOMA
Most non-Hodgkin lymphoma that occurs in the lung is low-grade B-cell lymphoma thought to be derived from BALT. As previously stated, there can be considerable difficulty in differentiating a low-grade B-cell lymphoma from a nonneoplastic process. Routine IHC markers such as kappa and lambda light chains are of occasional value in separating neoplastic from nonneoplastic processes. Flow cytometric analysis and polymerase chain reaction (PCR) should be used to evaluate most cases. Clinically, low-grade B-cell lymphomas are associated with a good prognosis. T-cell lymphomas occur in the lung but are much less common than B-cell lymphomas.142 Occasional primary lymphomas in the lung show prominent plasma cell differentiation.143 The predominant criteria for diagnosing primary non-Hodgkin lymphomas of lung are 1) involvement of the lung or involvement of a lobar or mainstem bronchus, either unilaterally or bilaterally, with or without mediastinal lymph node involvement; and 2) no evidence of extrathoracic lymphoma at the time of diagnosis or for 3 months afterwards.144 Patients with primary non-Hodgkin lymphoma of the lung are usually older than 60 years, and 50% are asymptomatic. Most are found to have an abnormal chest radiograph. Symptomatic patients usually have fever, night sweats, and weight loss, and occasionally they have dyspnea on exertion, cough, and chest pain. A significant number of non-Hodgkin lymphoma lung patients have associated von Willebrand syndrome, erythema nodosum, or Sjögren syndrome.
409
Primary lymphomas of the lung comprise less than 1% of all primary pulmonary neoplasms, small cell lymphomas comprise approximately 80% to 90% of all primary pulmonary lymphomas, and marginal zone B-cell lymphoma represents more than 90% of the cases. MARGINAL ZONE B-CELL LYMPHOMA, MUCOSAL-ASSOCIATED LYMPHOID TISSUE TYPE
Marginal zone B-cell lymphoma of the MALT type is a low-grade extranodal lymphoma composed of small B-lymphoid cells with phenotypic features of marginal zone B lymphocytes. Most patients who develop marginal zone B-cell lymphoma are adults, although this lymphoma may occur in children, particularly in those infected with human immunodeficiency virus (HIV). Although most MALT lymphomas are suspected to arise as a result of chronic antigenic stimulation, such as that caused by Helicobacter pylori in cases of gastric MALT lymphoma, no specific etiologic agent has been found for marginal zone B-cell lymphomas in the lung. Most patients come to medical attention with nonspecific pulmonary complaints and have a mass less than 5 cm in diameter or multiple masses. Hilar lymphadenopathy is almost always absent. A subset of patients may have a synchronous lymphoma of the MALT type at another anatomic location in the body. The larger neoplasms are composed of small lymphocytes that show peripheral tracking along lymphatic pathways, and bronchial infiltration is often associated with the formation of lymphoepithelial lesions. Reactive germinal centers are frequently seen within the neoplasm, adjacent to the bronchovascular structures or at the periphery; the neoplastic population replaces the expanded marginal zones of the germinal centers. Cytologically, the predominant cells are small lymphocytes that are usually round, but they may be lymphoplasmacytic. Some cells may have clear cytoplasm and may be referred to as monocytoid cells. Scattered plasma cells may be present. The lymphocytes of marginal zone lymphoma characteristically express pan–B-cell markers such as CD20, Pax-5, and CD79a (Table 12-13). They are typically negative for CD5, CD10, CD23, Bcl-6, and Bcl-1/cyclin D1. CD43 is expressed in as many as 50% of lymphocytes, and there may be nuclear expression of Bcl-10. Light-chain restriction can be seen in plasma cells in approximately 25% of cases. MULTIPLE MYELOMA
Multiple myeloma, a terminal B-cell lymphoma, can occur in the lung as isolated pulmonary masses with sheets of immunoglobulin, typically light chains; immunoamyloid can be observed in some cases. Most patients have disseminated myeloma with part of a systemic process.145 Some cases of multiple myeloma are associated with asbestos exposure.146 CHRONIC LYMPHOCYTIC LEUKEMIA–SMALL CELL LYMPHOCYTIC LYMPHOMA
Lung involvement in chronic lymphocytic leukemia– small cell lymphocytic lymphoma (CLL-SLL) usually
−
+ S S − − − − + S − −
+
S
−
−
−
−
−
−
−
S
−
S
CD79a
CD43
CD5
CD10
CD23
CD15
CD30
Cyclin D1
Bcl-2
Bcl-10
EBV
Monoclonal light chain
S
−
S
−
−
−
−
S
S
+
+
S
−
−
S
−
−
−
−
S
S −
−
U
−
−
−
−
−
−
−
−
U
U
S
Lymphomatoid Granulomatosis
S
−
S
−
S
S
−
−
−
−
− −
S
S
S
Hodgkin Lymphoma
+
+
+
Follicular Lymphoma
Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative; U, uncertain. EBV, Epstein-Barr virus.
S
S
+
+
Pax-5
+
+
+
CD20
Mantle Cell Lymphoma
Marginal Zone B Cell
Antibody Directed Against
Chronic Lymphocytic Leukemia/ Small Cell Lymphocytic Lymphoma
TABLE 12-13 Immunohistochemical Features of Primary Lymphomas of Lung
U
U
U
S
−
−
−
−
U
S
R
+
+
+
Intravascular Lymphoma
−
S
U
U
U
S
−
−
−
−
S
−
−
−
Primary Effusion Lymphoma
−
S
U
U
U
−
−
−
−
−
S
+
+
+
PyothoraxAssociated Lymphoma
410 Immunohistology of Lung and Pleural Neoplasms
Primary Lung Neoplasms
occurs in patients with a long-standing history of the disease. In most cases, a biopsy is not necessary, because the diagnosis can often be made on clinical grounds. Patients often come to medical attention with progressive shortness of breath and cough and have interstitial infiltrates in chest radiographs, and some patients have endobronchial obstructions. Most patients are over 50, and the male/female ratio is usually 2 : 1. Histologically, the condition is characterized by a dense lymphoid infiltrate that follows bronchovascular bundles and/or is in a bronchiocentric distribution with a relative sparing of the remainder of the lung. The infiltrate is composed predominantly of a population of small, round lymphocytes with a mature chromatin pattern. Sometimes larger neoplastic cells are found, and if larger cells are present in sheets, this would suggest a composite diffuse large B-cell lymphoma. The cells of CLL-SLL are B lymphocytes that express B-cell markers CD20, Pax-5, and CD79a (see Table 12-13). They often show coexpression of CD5, CD43, CD23, and Bcl-2 and are negative for CD10, Bcl-6, Bcl-1, and cyclin D1. Ki-67 is usually low (~10% to 20%), and molecular studies may show clonality of the Ig heavy or light chains. The cells frequently have abnormal karyotypes. MANTLE CELL LYMPHOMA
The lung is an unusual site of involvement of mantle cell lymphoma, even in cases of advanced disease. This lymphoma is characterized by a monomorphic population of small to intermediate-sized lymphoid cells with mild to marked irregular nuclei and a relatively mature chromatin pattern without prominent nucleoli. Occasional epithelioid macrophages are associated with neoplastic lymphoid cells, but they do not aggregate into granulomas. A blastoid subtype has been described in which approximately 10% of cases have a fine chromatin pattern that resembles a lymphoblastic lymphoma. The neoplastic cells are similar to other small B-cell lymphomas in that they express pan–B-cell markers CD20, Pax-5, and CD79a (see Table 12-13). They also usually express CD5, CD43, Bcl-2, Bcl-1, and cyclin D1. They are negative for CD10, CD23, and Bcl-6, and Ki-67 studies usually show reactivity in approximately 20% to 50% of the cells. A high percentage of these cases show t(14;18) that involves the BCL2 gene. FOLLICULAR LYMPHOMA
Follicular lymphoma involvement of the lung is rare, and usually it is a widespread process often clinically diagnosed as infection. The follicular pattern observed in a lymph node involved by follicular lymphoma is the same as that seen in the lung. The neoplastic lymphoid cells express pan–B-cell markers CD20, Pax-5, CD79a, Bcl-2, Bcl-6, and CD10 but show no immunostaining for CD5, CD43, and CD23. Molecular studies show clonality of Ig heavy- and/or light-chain genes, and in approximately 90% of cases, t(14;18) involves the BCL2 gene.
411
PRIMARY PULMONARY HODGKIN LYMPHOMA
Primary pulmonary Hodgkin disease is uncommon and morphologically resembles Hodgkin disease in lymph nodes. Not infrequently, when Hodgkin disease primarily involves the lung, it is misdiagnosed as an inflammatory process or as an organizing pneumonia. In 1990, Radin147 reported on 61 cases of primary pulmonary Hodgkin disease. In the series were 36 females and 25 males, and the average age of the entire group was 42.5 years, with a range of 12 to 82 years. The most common histologic type of Hodgkin disease identified was nodular sclerosing, with the second most frequent being the mixed cellular type. The criteria used for diagnosing primary pulmonary Hodgkin disease included documentation of pulmonary parenchymal involvement that primarily affected the lung with minimal or no enlargement of hilar and mediastinal lymph nodes. The most frequent symptoms before diagnosis included cough, weight loss, chest pain, dyspnea, hemoptysis, fatigue, rash, night sweats, and wheezing. Physical examination was often normal. Bronchoscopic evaluation in 35 of 61 patients was normal in 18 and abnormal in 16. Radiologic abnormalities were dominated by nodular masses in 45 of 61 patients and pneumonic infiltrates in 13 of 61 patients. To diagnose primary pulmonary Hodgkin disease, the clinician must think of the diagnosis and identify the same histologic pattern seen in lymph nodes involved in Hodgkin disease. IHC can facilitate the diagnosis in that Reed-Sternberg cells are frequently CD15 and CD30 positive and may occasionally express CD20 (see Table 12-13). RARE PRIMARY LYMPHOMAS IN THE LUNG AND CHEST CAVITY
Several rare lymphomas exist in the lung, the most notable of which is LYG. Other rare lymphomas include intravascular lymphoma, primary effusion lymphoma, pyothorax-associated lymphoma, secondary lymphomas/leukemias, and Erdheim-Chester syndrome. Lymphomatoid Granulomatosis
Lymphomatoid granulomatosis (LYG) was first reported by Liebow and coworkers,148 whose publication contains the most precise pathologic description of the entity. Forty patients were included in the study, and more than 50% showed “B” symptoms frequently seen in patients with lymphoma. More than 50% of the patients died within a year of diagnosis. In 1979, Katzenstein and colleagues149 reviewed information concerning the clinicopathologic features of LYG. They noted that patients who had adverse outcomes included those younger than 25 years; patients with an increased white blood cell count, neurologic abnormalities, or hepatosplenomegaly; and those whose pulmonary infiltrates showed atypical lymphoid cells. Over the years, the diagnosis of LYG evolved, and the condition was thought to represent an angiocentric lymphoma, specifically, an angiocentric T-cell lymphoma based on IHC studies. Guinee and colleagues150 analyzed 10 cases of LYG by IHC and in situ hybridization (ISH) for CD20
412
Immunohistology of Lung and Pleural Neoplasms
Figure 12-18 Lymphomatoid granulomatosis is composed of a variegated lymphoid infiltrate (×100).
and Epstein-Barr virus (EBV) and by PCR for IgG heavy-chain gene rearrangement. The authors found that in all cases, the majority of small and medium-sized lymphocytes were CD45RO-positive T lymphocytes. A much smaller population of large atypical cells were CD20-positive B cells, and in each case, combined IHC and ISH confirmed EBV in the CD20-positive B cells. The authors concluded that the proliferating cell in LYG was a large B lymphocyte and was probably a manifestation of an EBV-associated disease. LYG continues to be somewhat of an enigma to clinicians and pathologists with respect to its exact nature and clinical course. Most patients who have the disease follow a rapidly downhill course and die, although some have responded rather dramatically with treatment by cyclophosphamide.151 Pathologically, LYG is composed of distinct nodules made up of a variegated lymphoid infiltrate with large, atypical lymphoid cells (Fig. 12-18) with a tendency to infiltrate pulmonary veins and to cause necrosis. The large atypical cells in LYG express B-cell antigen CD20 as shown in Figure 12-19. An update of the pathologic features of LYG was published by Colby in 2012.152 Key histologic features necessary for the diagnosis include a mixed
mononuclear cell infiltrate that shows vascular infiltration, appreciable numbers of T cells, and variable numbers of CD20-positive B cells that show positivity for EBV RNA in situ hybridization (EBER) by ISH. Dr. Colby stated that the current definition of LYG was more restrictive than that originally applied by Liebow and colleagues148 in the first large series and was also more restrictive than that used by Katzenstein and associates.149 In 2010, Katzenstein and others153 proposed that the criteria for LYG diagnosis, which are always present, include 1) a mixed mononuclear cell infiltrate that contains large and small lymphoid cells, often with plasma cells and histiocytes, that replaces the lung parenchyma and shows vascular infiltration; and 2) variable numbers of CD20-positive large B cells, often with atypia, present in a background of CD3-positive small lymphocytes. Supportive findings that are usually but not always present include 1) necrosis of the cellular infiltrate; 2) positive ISH for EBER; 3) multiple lung nodules evident radiologically, or 4) skin or nervous system involvement. Colby152 and Katzenstein and colleagues153 stated “probably the most controversial point is whether identification of EBER is necessary for the diagnosis of LYG. In [their] opinion, as sampling can be a problem in identifying EBER, negative ISH for EBER in a single biopsy specimen does not exclude LYG, provided that criteria 1 and 2 are present in the appropriate clinical setting (criterion 5).” Colby152 opined the criteria for the diagnosis of LYG will remain an area of contention between pulmonary pathology and hematopathology. Intravascular Lymphoma
Intravascular lymphomas are neoplasms of large B cells that occur in an extranodal distribution and are characterized by neoplastic cells in the lumens of small vessels, particularly capillaries. These have also been referred to as angioendotheliosis proliferans.154-160 The intravascular growth pattern is thought to be secondary to a defect in homing receptors on the neoplastic cells. These studies suggest that neoplastic B cells lack adhesion molecules. Intravascular lymphomas commonly involve CNS and skin but can occur as primary lymphomas in the lungs. They can be a challenging diagnosis unless considered as a possibility, and IHC studies are usually necessary to make the diagnosis. Primary Effusion Lymphoma
Figure 12-19 The large atypical cells in lymphomatoid granulomatosis express B-cell antigen CD20 (×400).
Primary effusion lymphoma (PEL) is a neoplasm of large B cells that presents as a serous effusion with no detectable tumor masses elsewhere in the body.161-164 PEL is known to be associated with human herpesvirus 8 (HHV-8, Kaposi sarcoma–associated herpesvirus [KSHV]), and it most frequently occurs in the setting of AIDS. Patients typically come to medical attention with pleural effusions in the absence of lymphadenopathy or organomegaly. Neoplastic cells are stated to mark for KSHV in all cases. The neoplastic cells are CD45 positive and negative for B-cell markers and surface immunoglobulin, and they often express CD30, CD38, or CD138.
Primary Lung Neoplasms
Pyothorax-Associated Lymphoma
Pyothorax-associated lymphoma (PAL) is a rare type of lymphoma that arises in patients with chronic pyothorax, often decades after the initial pleural injury.165-167 The disease was first described in Japan, and the largest series originated there. Clinical presentation includes effusion, chest pain, weight loss, and dyspnea. Men are more frequently affected than women, and patients typically do not have a history of HIV infection or immunosuppression. The cause of the pyothorax is usually tuberculosis, although the postulated pathogenesis includes chronic antigenic stimulation analogous to MALT lymphoma of the stomach. Gross findings include a mass, often 10 cm or greater, associated with pleural fibrosis with direct invasion of adjacent structures. The neoplastic cells are large B-lymphoblastic cells. Lymphoplasmacytic cells are found in a smaller number of cases. At autopsy, more than 50% of the patients had disease limited to the intrathoracic region, and the remaining patients had extrathoracic extension. The neoplastic cells characteristically are CD45, CD20, and CD79a positive. Some cells are CD138 positive, and most cells are CD3 negative. SECONDARY LYMPHOMAS THAT INVOLVE THE LUNG AND LEUKEMIC INFILTRATES
In all instances in which a lymphoma is identified in the lung, the patient should be evaluated to determine whether a known lymphoma lies outside of the lung. If so, that lymphoma should be reviewed and compared morphologically with the lymphoma found in the lung. Sometimes, a transformation of lymphoma occurs that can make it difficult to determine whether it is primary within or metastatic to the lung. Chronic lymphocytic leukemia (CLL) is the most common leukemia to infiltrate the lung, and it can be indistinguishable from primary or secondary small lymphocytic lymphomas. When Hodgkin disease secondarily involves the lung, it usually infiltrates along bronchovascular structures with neoplastic cells oriented around blood vessels. Although leukemic infiltrates in the lung are found histologically at autopsy in between 25% and 64% of cases, they are relatively infrequently found during the life of the patient. In patients with acute leukemia, nonlymphocytic-type leukemias more frequently infiltrate the lung than acute lymphocytic leukemias.
413
Erdheim-Chester disease involving the lung. Pulmonary involvement is fairly characteristic and is in a subpleural, interlobar, septal/bronchovascular distribution. Erdheim-Chester disease is a primary neoplastic histiocytic disorder, and the neoplastic cells show immunostaining for CD68 and factor XIIIa. S-100 protein has been identified in some cells, although studies to date have not identified Langerhans cell granules; these are generally treated as a histiocytic lymphoma. Pulmonary Langerhans Cell Histiocytosis (Pulmonary Histiocytosis X, Pulmonary Eosinophilic Granuloma)
A conceptual understanding of pulmonary Langerhans cell histiocytosis (PLCH), also referred to as histiocytosis X or eosinophilic granuloma, is based on an understanding of normal Langerhans cells,169 identified in 1869 by Paul Langerhans, that compose approximately 3% to 8% of cells in the epidermis. Normal Langerhans cells are large dendritic cells that process antigen and present it to T lymphocytes. These cells occur in a variety of organs, including epidermis, esophagus, anus/rectum, cervix, thymus, lymph nodes, and occasionally in normal lung. In the lung they are usually associated with BALT and are frequently located between epithelial cells. In 1961, Michael Birbeck identified unusual cytoplasmic inclusions in Langerhans cells known as Birbeck granules or Langerhans cell granules. PLCH occurs almost exclusively in cigarette smokers and occurs more frequently in younger patients. The individual histiocytic cells are much smaller than normal Langerhans cells and do not show extensive dendritic processes; they typically contain Langerhans cell granules and show immunostaining for S-100 protein (Fig. 12-20), CD1A, CD68, and CD31. The lesions first occur in a bronchial distribution and can progress from a cellular phase to a fibrotic phase. In some instances, the fibrotic phase is difficult to diagnose, because the number of histiocytic cells is few. Patients with PLCH can be asymptomatic, or they may have relatively severe symptoms with an elevated sedimentation rate, fever, and weight loss. On chest radiograph, the nodules can occasionally be large enough to suggest metastatic cancer. In rare cases, the disease presents as a single
Erdheim-Chester Disease
Identified by William Chester in 1930, Erdheim-Chester disease is a rare, nonfamilial, histiocytic disorder that primarily affects middle-aged and older adults and predominantly involves long bones of the extremities. The etiology of Erdheim-Chester disease is unknown. According to the report by Allen and associates,168 approximately 50% of patients have involvement of other tissues that may include skin, retroorbital and periorbital tissue, the pituitary-hypothalamic axis, heart, kidney, retroperitoneum, breast, skeletal muscle, and sinonasal mucosa. Approximately 20% have pulmonary involvement. This report lists 24 cases of
Figure 12-20 Langerhans cells show nuclear and cytoplasmic immunostaining for S-100 protein (×400).
414
Immunohistology of Lung and Pleural Neoplasms
Figure 12-21 Portion of a nodule of pulmonary Langerhans cell granulomatosis. Langerhans cells are smaller than mature alveolar macrophages and have highly convoluted nuclei and a small amount of pale cytoplasm. Note the associated inflammatory cells (×400).
Figure 12-23 This clear cell tumor of lung is composed of significantly more pleomorphic cells than the tumor seen in Figure 12-22 (×400).
nodule, although in most cases, multiple nodules are apparent with sparing of the lower lobes. Cystic change occurs frequently, and the diagnosis usually is suspected by high-resolution computed tomography (CT) scans. Transbronchial biopsy specimens may be sufficient to diagnose the disease, but in most instances, an open lung biopsy is necessary. The nodules of PLCH are composed of histiocytosis X cells admixed with varying numbers of lymphocytes, plasma cells, eosinophils, neutrophils, and cigarette smoker’s macrophages. Histiocytosis X cells are smaller than mature alveolar macrophages and mature Langerhans cells, and they contain extensively convoluted nuclei (Fig. 12-21).
Clear cell neoplasms of the lung occur as unencapsulated discrete nodules, often referred to as sugar tumors, and are a member of the PEComa family of tumors.170
PEComas include angiomyolipoma, lymphangioleiomyomatosis, clear cell tumor of the lung, clear cell myomelanocytic tumor of ligamentum teres/falciform ligament, and abdominopelvic sarcoma of perivascular epithelioid cells.171 These tumors coexpress human melanoma black 45 (HMB-45) and muscle markers. Histologically, PEComas are usually composed of cells that are relatively uniform in size, shape, and nuclear appearance (Fig. 12-22), although they can be more pleomorphic (Fig. 12-23). The neoplastic cells characteristically contain large amounts of glycogen in their cytoplasm that ultrastructurally is membrane bound. The neoplastic cells show immunostaining for vimentin and frequently express HMB-45 (Fig. 12-24) and S-100 protein. In some cases, the neoplastic cells express NSE, Leu-7, synaptophysin, and HMB-50, and they are keratin negative; they have also been reported to express MyoD1.172 As described by Gaffey and colleagues,173 some neoplastic cells contain melanosomes (Fig. 12-25), as demonstrated ultrastructurally.
Figure 12-22 This clear cell tumor of lung is composed of relatively uniform cells. The clear cytoplasm is the result of glycogen (×400).
Figure 12-24 The neoplastic cells of this clear cell tumor express human melanoma black 45 (HMB-45; ×400).
Clear Cell Neoplasm/Sugar Tumor and Perivascular Epithelioid Cell Tumors
Primary Lung Neoplasms
415
LAM than HMB-45. The authors further concluded that because podoplanin was involved in cell invasion, it may have functional implications in LAM disease progression. Even though this is an uncommon condition, the use of PLAP and D2-40 will be helpful in identifying LAM.
Sclerosing Hemangioma
Figure 12-25 The neoplastic cells of this clear lung neoplasm contain melanosomes (×16,000).
Lymphangioleiomyomatosis A rare, proliferative but nonneoplastic pulmonary condition, lymphangioleiomyomatosis (LAM), is briefly mentioned here, because the proliferative cells in this condition express HMB-45.174,175 This disease primarily affects women of reproductive age and is characterized by proliferation of atypical smooth cells that surround lymphatics and blood vessels with cystic space formation. These cells may express actin, vimentin, estrogen receptor (ER) protein, progesterone receptor (PR) protein, and HMB-45. Patients with LAM frequently have renal angiomyolipomas. Mandal and colleagues176 described LAM as a rare fatal lung disease of premenopausal women and stated that LAM lesions are characterized by infiltrating smooth muscle–like cells that cause cystic destruction of lungs that eventually requires lung transplant. They noticed vascular smooth muscle immunoreactivity for placental alkaline phosphatase (PLAP) and also noted that D2-40 reacted with vasculolymphatic endothelial channels, so they decided to evaluate LAM lesions for expression of PLAP and D2-40. The authors studied 21 patients with LAM with immunostains for SMA, HMB-45, PLAP, CD31, and D2-40. SMA was detected in most LAM cells, followed by D2-40, and PLAP and HMB-45 were detected in a lower number of cells. Predominantly D2-40–positive lesions also had abundant HMB-45–positive cells but only rare PLAP-positive cells. The authors concluded that PLAP was a novel marker for a subpopulation of LAM cells, immunoreacting significantly stronger than vascular or airway smooth muscle cells, and they stated that podoplanin (D2-40) was a novel marker for a large subpopulation of LAM cells, and that it may be a better diagnostic marker for
Sclerosing hemangioma is one of the most extensively studied rare pulmonary neoplasms, and it usually occurs as a round-oval solitary subpleural mass,177,178 the majority of which occur in relatively young women.179 Most sclerosing hemangiomas are histologically variegated and show cellular areas along with variably sized spaces that occasionally contain blood, sclerosis, and papillary structures. In the solid cellular regions, the neoplastic cells are round, oval, and slightly spindle shaped (Fig. 12-26). The spaces are usually lined by cuboidal or columnar cells that appear morphologically as epithelial cells and are often different than adjacent tumor cells. Most sclerosing hemangiomas contain varying types and numbers of inflammatory cells, especially mast cells. Dail100 reviewed several IHC studies of sclerosing hemangiomas and found that positive and negative IHC reactions of the neoplastic cells in the solid areas are contrasted to positive and negative IHC reactions reported for the lining cells in Table 12-14. In a recent case we evaluated, the lining cells and tumor cells in solid areas showed intense immunostaining for EMA (Fig. 12-27) and moderately intense cytoplasmic immunostaining for vimentin. Occasional lining cells showed low-intensity immunostaining for keratin. The lining cells and neoplastic cells in solid areas showed no immunostaining for actin, desmin, S-100 protein, HMB-45, CD31, and FVIII antigen. Ultrastructurally, the neoplastic cells in solid areas had epithelioid features with short microvillous processes and small intercellular junctions (Fig. 12-28). The neoplastic cells of sclerosing hemangiomas characteristically express TTF-1 (Fig. 12-29).180 An endobronchial variant has been described,181 as have cases associated with lymph node metastases.182
Figure 12-26 In the solid region of this sclerosing hemangioma, the majority of the neoplastic cells are round, oval, and occasionally slightly spindle shaped (×400).
416
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-14 Immunohistochemical Findings in Sclerosing Hemangiomas of Lung Cells in Solid Regions of Tumor
Cells Lining Spaces in Tumor
Positive Reactions
Negative Reactions
Positive Reactions
Negative Reactions
Vimentin
Keratin
Keratin
Vimentin
EMA
S-100 protein
Vimentin
S-100 protein
Keratin (rare)
EMA
Clara cell antigen
TTF-1
Surfactant apoprotein
CEA TTF-1
CEA, Carcinoembryonic antigen; EMA, epithelial membrane antigen; TTF-1, thyroid transcription factor 1.
Rhabdoid Tumor
Figure 12-28 Ultrastructurally, the central cells of sclerosing hemangioma exhibit epithelioid features with short microvillous processes and small intercellular junctions (×10,000).
Malignant rhabdoid tumors of the kidney were described in 1978 by Beckwith and Palmer183 as highly malignant tumors of infants and children initially thought to represent a variant of Wilms tumor. Similar neoplasms were described in extrarenal sites and in adults,184-197 and those that resemble rhabdoid tumors in the kidney but that occur in nonrenal sites are frequently designated as pseudorhabdoid tumors. They have a diverse IHC phenotype,184-197 although the majority of them express vimentin, and many coexpress vimentin and keratin. In 1996, Cavazza and coworkers198 described six lung tumors with rhabdoid morphology. These neoplasms were composed predominantly of large, round cells with ovoid nuclei, large nucleoli, and large eosinophilic globular inclusions that compress the nucleus toward one side of the cell (Fig. 12-30). These six primary rhabdoid tumors of lung were evaluated immunohistochemically with 17 antibodies in five cases and 18 antibodies in one case. The rhabdoid component of the tumor
immunostained for vimentin in all cases, with a high staining intensity in most cases. The cytoplasmic eosinophilic inclusions showed immunostaining for EMA and NSE in 5 of 6 cases, chromogranin and broad-spectrum keratin in 3 of 6 cases, CAM5.2 keratin in 2 of 6 cases, NFP in 2 of 6 cases, and Leu-7 and glial fibrillary acidic protein (GFAP) in 1 of 6 cases. Synaptophysin was focally positive in 3 of 6 cases, and CD34 was positive in 1 of 6 cases but did not stain the globular inclusions. Diffuse granular cytoplasmic immunostaining for myoglobin was observed in one case. The neoplastic cells showed no immunostaining for FVIII antigen, actin, desmin, S-100 protein, HMB-45, and light chain immunoglobulin (evaluated in one case). In my experience with six cases with a primary in the lung, five showed immunostaining for vimentin (Fig. 12-31), and one showed coexpression of vimentin and keratin. Miyagi and associates199 described three cases of primary lung rhabdoid tumor, and all were associated with an adenocarcinoma. The authors concluded that the
Figure 12-27 This sclerosing hemangioma is composed of cells that show relatively intense immunostaining for epithelial membrane antigen (×400).
Figure 12-29 The neoplastic cells of sclerosing hemangiomas characteristically express thyroid transcription factor 1.
Primary Lung Neoplasms
417
rhabdoid cells in these cases represented dedifferentiated components of an adenocarcinoma.
cells express keratin. These tumors can be invasive and resemble low-grade sarcomas.203,204 It has been reported that expression of tumor suppressor gene product p53 is helpful in differentiating sarcoma from inflammatory pseudotumor,205 although this marker has been controversial in differentiating other inflammatory conditions from neoplasms (e.g., fibrosing pleuritis vs. desmoplastic mesotheliomas). Cytogenetic clonal changes have been reported in inflammatory pseudotumor of lung.206 Yousem and colleagues207 described the chromosomal abnormalities of inflammatory pseudotumors of lung and reported that three of nine primary pulmonary inflammatory pseudotumors showed changes in the 2p23 and anaplastic lymphoma kinase (ALK) gene regions. The authors suggested that IHC detection of ALK might be helpful in predicting the future biologic behavior of inflammatory pseudotumors. Freeman and colleagues208 suggested ALK-1 expression could be useful in diagnosing inflammatory pseudotumors.
Inflammatory Pseudotumor
Desmoplastic Small Round Cell Tumor
Inflammatory pseudotumor of lung, also referred to as plasma cell granuloma of lung, represents less than 1% of all lung tumors.200 Most occur in patients younger than 40 years, and 15% arise in those aged 1 to 10 years.201,202 Inflammatory pseudotumors cause symptoms and signs of cough, chest pain, dyspnea, hemoptysis, clubbing, and fever. Radiographically, they are usually circumscribed but may be irregularly shaped. Macroscopically, they are yellowish white and well circumscribed, and they can infiltrate normal lung tissue and cause its destruction. Histologically, inflammatory pseudotumors are composed of mature plasma cells; macrophages, including multinucleated histiocytic great cells; lymphocytes; mast cells; neutrophils; and spindle cells. The differential pathologic diagnoses usually include sclerosing hemangioma, malignant fibrous histiocytoma, malignant plasmacytoma, and reactive lymphoid proliferation. Immunohistochemically, the plasma cells show polyclonal expression of light-chain immunoglobulin. The spindle cells usually stain as myofibroblasts and express vimentin and actin; in rare cases, the spindle
Desmoplastic small round cell tumor (DSRCT) occurs primarily in the abdominal cavity.209 The neoplasms characteristically show immunostaining for desmin (dotlike pattern), Wilms tumor 1 (WT1), keratin, NSE, CD99, and actin. They also show the EWSR1-WT1 gene fusion transcript. DSRCT was reported primary in the lung by Syed and coworkers.210 Ultrastructurally, this tumor showed intracytoplasmic whorls of intermediate filaments, presumably desmin. DSRCT was also reported as a primary neoplasm in the pleura.211
Figure 12-30 This primary rhabdoid tumor of lung is composed of large cells with large, globular eosinophilic inclusions (×400).
Epithelial-Myoepithelial Neoplasm Epithelial-myoepithelial neoplasms have been reported primary in the lung.212-215 These neoplasms are composed of an inner epithelial cell layer that immunostains for keratin, CEA, and EMA and an outer cell layer of myoepithelial cells that immunostains for S-100 protein and actin.
Granular Cell Tumor Granular cell neoplasms are thought to be derived from Schwann cells and may occur as solitary pulmonary neoplasms.216 The neoplastic cells typically express S-100 protein, NSE, vimentin, and actin.
Salivary Gland Neoplasm Salivary gland–like neoplasms rarely occur in the lung. They show the same IHC profile as those that arise in salivary glands217 and are thought to arise from minor salivary glands in the bronchial mucosa.
Primary Intrapulmonary Thymoma Figure 12-31 The globular inclusions in this primary rhabdoid tumor of lung show intense immunostaining for vimentin (×200).
Moran and associates218 reported on eight cases of primary intrapulmonary thymoma in which no evidence of mediastinal masses was found radiographically
418
Immunohistology of Lung and Pleural Neoplasms
or at surgery. The masses varied from 0.5 to 10 cm in diameter. Five were located close to the hilum, and three were in a subpleural location. The masses were composed of mixtures of lymphocytes and epithelial cells separated by fibrous bands, and the epithelial cells immunostained for keratins and EMA.
Pulmonary Meningothelial Nodules Minute pulmonary meningothelial-like nodules (MPMNs), previously referred to as minute chemodectoma-like bodies, are most frequently found incidentally in the lung. They are composed of spindle-shaped cells and form nodules centered around small veins. Ionescu and colleagues219 reported on 16 cases that yielded 33 separate MPMNs and 10 cases of meningioma. The cells that form MPMNs showed immunostaining for vimentin in 96.6% of cases, EMA in 33.3% of cases, and S-100 protein in 3% of cases. All cases were negative for cytokeratin and synaptophysin. They found that the MPMNs lacked mutational damage, consistent with a reactive origin. In contrast, the four cases with multiple MPMNs (MPM-omatosis) showed genotypic findings suggestive of transition between reactive and neoplastic transformation. The 10 meningiomas evaluated showed the highest frequency of loss of heterozygosity. Mukhopadhyay and colleagues220 evaluated 400 consecutive surgical lung biopsies of various types to further evaluate meningothelial-like nodules in surgical lung biopsies, lobectomies, and pediatric autopsies to clarify their incidence, distribution, and relation to age and underlying disease and to shed potential light on their origin. Tissue sections were immunostained for PR protein, CD56, EMA, TTF-1, CD99, CD34, CD31, and Ki-67. Meningothelial-like nodules were roundly distributed in alveolar septa and were rarely present in scars. No relation to venules was apparent, although common in the nodules were small vessels that appeared to be entrapped. Immunostains were stated to have been positive for PR in 14 of 14 cases, CD56 in 14 of 14 cases, and EMA in 10 of 10 cases; and they were reported negative for TTF-1 in 0 of 4 cases, CD99 in 0 of 11 cases, CD34 in 0 of 6 cases, and CD31 in 0 of 6 cases. Ki-67 was focally positive in 2 of 12 cases. The authors concluded that the incidence of meningothelial-like nodules in their study was higher than previously appreciated. The presence in nearly half of the extensively sampled lobectomy specimens suggested they may be present in all lungs if sufficiently sampled. The absence of meningothelial-like nodules in patients younger than 20 years suggests they are not congenital rests. Staining for CD56 was novel but had been reported in meningotheliomas, thus supporting the concept that meningothelial-like nodules were of meningothelial origin. They suggested that a more appropriate term for these would be meningothelial nodules. The lesions were stated to have no clinical significance.
Placental Transmogrification of Lung Brief mention is made of placental transmogrification of the lung. It occurs in association with lipomatosis221 and
bullous emphysema222 and is composed of placental villuslike structures in the lung parenchyma. The epithelium surfacing the placentoid structures shows immunostaining for TTF-1 in most cases, the stromal cells express vimentin and are nonreactive for TTF-1, and mast cells are common in the stromal tissue.
Differential Diagnosis and Pitfalls of Lung Neoplasms The lung is the site of numerous metastatic neoplasms, and pathologists must be acutely aware of this.223,224 Metastatic neoplasms to lung are more common than primary tumors. The problem with differentiating primary from metastatic lung neoplasms is compounded by similar histologic appearances of primary and metastatic lung neoplasms. Adenocarcinoma as a group is the most difficult neoplasm from which to differentiate primary tumors from metastatic disease. Metastatic tumors to the lung always have to be considered when making a diagnosis of primary lung cancer, even when considering solitary pulmonary nodules. As discussed by Wang and colleagues in 1995,225 antibodies against CK7 and CK20 are helpful in distinguishing primary from metastatic carcinoma, although they are not specific. Many GI tract cancers, a few renal cell carcinomas (RCCs), gynecologic neoplasms, and bladder neoplasms express CK7. TTF-1 is the most specific antibody in identifying pulmonary adenocarcinoma, but it is negative in as many as 25% to 40% of cases of primary pulmonary adenocarcinoma. Variability has been observed, especially with mucin-producing pulmonary adenocarcinomas, mucinous bronchioloalveolar carcinomas, and the goblet-cell variant of primary mucinous carcinomas of the lung. Some primary pulmonary adenocarcinomas, especially those that are mucin producing, may express CK20 and CDX-2. The immunophenotype of large cell undifferentiated neoplasms of lung is unpredictable, but most are carcinomas that coexpress keratin and vimentin, although some express only vimentin. Some large cell neoplasms look like carcinomas but, in fact, are not. Another area of potential confusion is related to observations that non-NE non–small cell carcinomas, as determined by histologic appearances, express NE markers by IHC. Pelosi and colleagues226 studied the prevalence of transactivating (TA) p63 and non-TA p63 (p40) isoforms in 20 pulmonary adenocarcinomas using p63 in paraffin sections with 1A4 clone, recognizing all p63 isoforms, and p40 polyclonal antibody, recognizing all non-TA p63 isoforms. Independent of growth factors, p63 immunoreactivity with clone 1A4 was found in 15% of pulmonary adenocarcinomas (range 10% to 70% of tumor cells), whereas none exhibited p40 immunostaining, revealing a strong prevalence of TA isoforms. Pulmonary squamous carcinomas were always diffusely positive for both 1A4 and p40 antibodies. The authors concluded that the absence of p40 immunoreactivity in pulmonary adenocarcinomas, paralleling a strong prevalence of TA isoforms, might be a useful diagnostic tool
Differential Diagnosis and Pitfalls of Lung Neoplasms
when the distinction from pulmonary SCCs was crucial for neoadjuvant therapy, such as in the setting of poorly differentiated tumors and/or small biopsies. The bottom line from this report226 is that p63, but not p40, can be found in lung adenocarcinomas.
TABLE 12-16 Immunohistochemical Reactivities of SCC, Adenocarcinoma, and SCLC for CK5/6, TTF-1, and p63 Markers
SCC (n = 39) Positive (n)
AC (n = 10) Positive (n)
SCLC (n = 28) Positive (n)
Squamous Cell Carcinomas of Lung
CK5/6
31
2
2
SCCs of lung show a variety of histologic forms and can be poorly differentiated. Small cell squamous carcinomas of lung can be confused with small cell NECs, and these two neoplasms are contrasted in Table 12-15. The main difference is that small cell squamous carcinomas do not express NE markers, usually do express HMW keratin, and show no immunostaining for TTF-1, whereas small cell NECs typically express NE markers, do not show immunostaining for HMW keratin but rather show punctate immunostaining for LMW keratin, and express TTF-1 in a high percentage of cases. Small cell squamous carcinomas are usually positive for p63, whereas in our experience, SCLC is usually negative for p63 (although it has been reported in NE lung neoplasms). Squamous carcinomas frequently show spindle cell features. The neoplastic spindle squamous cells often coexpress keratin and vimentin. Some spindle cell squamous carcinomas express predominantly vimentin and relatively small amounts of keratin. Kargi and associates227 reported on the diagnostic value of TTF-1, CK5/6, and p63 in the classification of lung carcinomas. The authors evaluated bronchoscopic
TTF-1
0
4
28
32
0
0
TABLE 12-15 Immunohistochemical Features of Small Cell Squamous Carcinoma of Lung and Small Cell Neuroendocrine Carcinoma of Lung Small Cell Squamous Cancer
Small Cell Neuroendocrine Cancer
Low-molecular-weight keratin
S
+*
High-molecular-weight keratin
S
−
CK5/6
S
−
CK7
−
R
CK20
R
R
Synaptophysin
−
+
Chromogranin A
−
S†
Thyroid transcription factor 1
N
+
+/−
−/+
Antibody Directed Against
p63
*Pattern of staining is usually punctate using antibody 35βH11. † Pattern of staining is usually punctate. Reactivity: +, Almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative. CK, Cytokeratin.
419
p63
AC, Adenocarcinoma; CK, cytokeratin; SCC, squamous cell carcinoma; SCLC, small cell lung carcinoma; TTF-1, thyroid transcription factor 1.
biopsies of 77 lung cancers in which the morphology of the tumor was stated to have been easily studied. All cases were immunostained for p63, CK5/6, and TTF-1; the results of their study are shown in Table 12-16. Of the 39 SCCs, 32 were positive for p63, 31 were positive for CK5/6, 27 were positive for p63 and CK5/6, and 36 were positive for p63 or CK5/6. With respect to SCLCs, 2 of 28 were positive for CK5/6, and 2 of 28 showed p63 or CK5/6 positivity. Of the 10 adenocarcinomas, 2 were CK5/6 positive, and 2 of 20 expressed p63 or CK5/6 positivity. The authors concluded that to achieve the most accurate histologic typing of lung cancer possible, TTF-1 in combination with p63 and CK5/6 might be useful components of analyzing poorly differentiated lung carcinomas in biopsy tissues. Nonaka228 evaluated 150 primary lung adenocarcinomas to investigate the utility of ΔNp63 (p40) to distinguish between pulmonary adenocarcinomas and SCC and stated that it was important to make this distinction because different treatment regimens were available. Nonaka used the following panel of immunostains in his study: p63 and CK5/6 for squamous markers and TTF-1 and Nap-A for adenocarcinoma markers. The typical immunoprofile for adenocarcinoma was TTF-1 positive or negative and p63 negative. The typical immunoprofile for SCC was TTF-1 negative and p63 positive. The study included 3 BAC/in situ and 35 acinar, 2 papillary, 40 solid, 58 mixed, and 12 mucinous primary lung adenocarcinomas and 35 SCCs. Twentysix adenocarcinomas (17%) contained p63-positive neoplastic cells to a variable extent with a diffuse (3+/4+) reaction seen in 12 tumors (8%). In fact, p63 expression was also seen in all subtypes of adenocarcinoma except for the mucinous type, and p40 was negative in all adenocarcinomas. All SCCs were positive for p63 and p40, predominantly in a diffuse fashion, and 116 adenocarcinomas (77%) were positive for TTF-1 to a variable extent, whereas all SCCs were negative for TTF-1. In addition, p63 expression was seen in 23 TTF-1–positive adenocarcinomas (20%), and p40 was negative in all TTF-1–positive tumors. Nonaka concluded that p63 was not uncommonly seen in adenocarcinomas, whereas p40 was specific for SCC and is as sensitive as p63. Nonaka stated that the presence of p63-positive cells in poorly differentiated lung adenocarcinoma may be erroneously interpreted as evidence of squamous cell
420
Immunohistology of Lung and Pleural Neoplasms
differentiation, therefore p40 can potentially be a more reliable marker for SCC. Brown and colleagues229 pointed out that the distinction of lung adenocarcinoma from other types of primary lung cancers is important because of new treatment modalities for adenocarcinoma. In their study, the authors utilized a cocktail of two nuclear antibodies, TTF-1 and p40, to evaluate 183 lung cancers morphologically (93 adenocarcinomas and 90 SCCs). Of the 90 SCCs, 83 stained positive with p40 (sensitivity 92%, specificity 92%), whereas 72 of 93 adenocarcinomas stained for TTF-1 (sensitivity 77%, specificity 100%). One case stained for both and was reclassified as an adenosquamous carcinoma. The authors concluded that the TTF-1/p40 cocktail was very effective in distinguishing lung adenocarcinoma from SCC on small specimens on a single slide. Whithaus and colleagues48 evaluated 197 adenocarcinomas and 66 SCCs for Nap-A, CK5/6, p63, and TTF-1 to find the most cost-effective panel with which to distinguish lung adenocarcinoma from SCC of the lung. For adenocarcinomas, the authors found that Nap-A had an 83% sensitivity and 98% specificity, whereas TTF-1 had a 60% sensitivity and 98% specificity for adenocarcinoma. For SCCs, CK5/6 had a 53% sensitivity and 96% specificity, whereas p63 had a 95% sensitivity and 86% specificity for SCC. The authors concluded the best panel for distinguishing adenocarcinoma from SCC was Nap-A and p63, which showed a specificity of 94% and a sensitivity of 96%.
Primary Versus Metastatic Squamous Cell Carcinoma Distinguishing primary carcinoma from metastatic SCC of the lung can be difficult. Amador-Ortiz and associates230 studied 27 patients to see if they could determine whether an SCC of the lung was a primary or metastatic neoplasm in patients with a concurrent or prior extrapulmonary SCC. In most cases, the prior SCC was found to originate from the head and neck or lower gynecologic tract. Also, because a significant proportion of these tumors were HPV related, the authors used a panel of immunostains that included p16 and p63, as well as ISH for high-risk HPV, to determine the relationship between the pulmonary and extrapulmonary tumors. Of the 27 cases studied, 14 were males (mean age 58 years, range 42 to 77 years) with SCC of the lung and an extrapulmonary SCC; of these, 23 cases were from the head and neck, and 4 were gynecologic tract carcinomas (3 cervix, 1 endometrium). In 9 cases (33%; 3 cervical, 6 head and neck), the pulmonary and extrapulmonary tumors had positive HPV status, and the lung SCCs were considered metastases; whereas 6 of the SCCs (22%, all head and neck) had different HPV status, and the lung SCCs were determined to be of primary origin. In three cases (2 head and neck, 1 endometrium), both extrapulmonary and pulmonary tumors were negative for HPV with divergent p53, suggesting primary pulmonary origin. In nine cases (all head and neck), both sites were HPV negative and showed similar p53 staining. A definitive classification
of primary versus metastatic neoplasms could not be determined by IHC alone. However, the authors were able to determine metastasis in 67% of cases (100% gynecologic and 61% head and neck). The authors concluded that a panel of immunostains with p16, p53, and HPV ISH is a useful tool to distinguish between primary and metastatic pulmonary SCC. A problem with this study is that there have been reports of a significant number of primary lung cancers that express HPV.
Basaloid Carcinoma Basaloid carcinoma is a relatively rare lung neoplasm231 that may be confused with NEC. Basaloid carcinomas are composed predominantly of nests of relatively small, undifferentiated cells with extensive necrosis and palisading of the peripheral cell layer (Fig. 12-32). They may show squamous and glandular differentiation, although the degree of this differentiation is usually poorly developed. The glandular component is often composed of small cells. Basaloid carcinoma can be confused with small cell carcinoma, atypical carcinoid, and large cell NEC. Basaloid carcinomas usually express LMW and HMW keratins and do not express NE markers. Expression of TTF-1 and 34βE12 (cytokeratins 1, 5, 10, and 14) was evaluated by Sturm and associates72 in basaloid and large cell NECs. TTF-1 expression was not observed in basaloid carcinomas, and expression of HMW keratin (34βE12) was observed in only one large cell NEC. Basaloid carcinoma is contrasted with small cell carcinoma, atypical carcinoid, and large cell NEC in Table 12-17. Nonaka and Chiriboga232 studied basal cell differentiation in 108 cases of stage 1 primary lung adenocarcinoma by IHC with tissue microarrays with immunostains for p63, TTF-1, CK7, CK20, 34βE12 (HMW keratin), CK14, CK17, surfactant apoprotein A (SP-A), surfactant apoprotein C (SP-C), Clara cell protein 16 (CC16), and CD208 (dendritic cell lysosomal-associated membrane protein [DC-LAMP]). The results are given in their report. They concluded that p63-positive cells
Figure 12-32 This basaloid carcinoma of lung is composed of undifferentiated, relatively small cells and often resembles primary pulmonary neuroendocrine carcinomas (×200).
Differential Diagnosis and Pitfalls of Lung Neoplasms
421
TABLE 12-17 Immunohistochemical Features of Basaloid Carcinoma of Lung Compared with Small Cell Carcinoma, Atypical Carcinoid, and Large Cell Neuroendocrine Carcinoma of Lung Antibody Directed Against
Basaloid Carcinoma
Small Cell Carcinoma
Atypical Carcinoid
Large Cell Neuroendocrine Carcinoma
LMW keratin (35ßH11)
S
+
S
S
HMW keratin (34βE12)
S
−
S
R
CK5/6
S
−
−
R
CK7
S
R
R
R
CK20
R
−
−
−
Synaptophysin
−
+
S
S
Chromogranin A
−
S
+
+
CEA
S
S
S
S
TTF-1
R
+
S
S
Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative. CEA, Carcinoembryonic antigen; CK, cytokeratin; HMW, high molecular weight; LMW, low molecular weight; TTF-1, thyroid transcription factor 1.
were not uncommon components in lung adenocarcinomas, and most of them showed a CK14-negative, CK17-positive immunophenotype, which corresponded to a basal cell (reserve cell) of the distal airway (type B basal cell). In fact, p63- and/or 34βE12-positive cells in lung carcinoma did not always indicate squamous cell differentiation, and the possibility of a tumor with a basal cell phenotype should be considered. Crapanzano and colleagues233 studied the cytologic, histologic, and IHC findings of pulmonary carcinomas with basaloid features and found that basaloid carcinoma and basaloid SCC showed overlapping features with SCLC and large cell NEC in cytologic and histologic specimens. Unlike small cell carcinomas, basaloid carcinomas and basaloid squamous carcinomas lacked prominent nuclear molding and showed greater numbers of tightly cohesive clusters, and they demonstrated palisading of cells along the periphery of the nests. Positive p63, positive HMW keratin, and negative TTF-1 were helpful in distinguishing basaloid carcinomas and basaloid squamous carcinomas from SCLCs and large cell NECs.
Mucinous Forms of Primary Pulmonary Adenocarcinoma Pulmonary adenocarcinomas are currently the most common primary lung cancer234,235 and show a wide range of differentiation. In most cases, pulmonary adenocarcinomas show more than one histologic pattern. Several mucinous forms of primary pulmonary adenocarcinoma exist, including a cystic mucinous form and signet-ring adenocarcinoma. Histologically, it is often difficult to differentiate a primary mucinous pulmonary adenocarcinoma from a metastatic adenocarcinoma from the GI tract, such as colon. Ultrastructurally, it is usually impossible to differentiate these neoplasms with respect to their site of origin. With the passage of time, some IHC reactions become less specific. Lau and coworkers39 evaluated TTF-1,
CK7, and CK20 expression in 48 nonmucinous, 12 mucinous, and 7 mixed histology bronchioloalveolar carcinomas. The 12 mucinous bronchioloalveolar carcinomas were TTF-1-negative, and a trend was noted toward absence of TTF-1 expression in the mucinous component of bronchioloalveolar carcinomas of mixed histology. In addition, 63 of 67 bronchioloalveolar cell carcinomas (94%) were CK7 positive, with no difference in expression among the different subtypes, and the three bronchioloalveolar carcinomas that were CK20 positive exhibited a mucinous morphology. These results indicated that mucinous bronchioloalveolar carcinomas were frequently TTF-1 negative and could express CK20. Simsir and colleagues40 evaluated bronchioloalveolar cell carcinomas: six mucinous, four nonmucinous, and six with focal mucinous differentiation. Of the mucinous bronchioloalveolar carcinomas, four of six (67%) were CK7 and CK20 positive and TTF-1 negative. All four nonmucinous bronchioloalveolar carcinomas were CK7 positive and CK20 negative, and half were TTF-1 positive. The six mixed bronchioloalveolar carcinomas were diffusely positive for CK7 and focally positive for CK20; five (83%) were TTF-1 positive. The authors concluded that mucinous and mixed bronchioloalveolar carcinomas have an immunophenotype different from that of conventional pulmonary adenocarcinoma. A variety of adenocarcinomas show mucus production. With respect to the lung, certain adenocarcinomas produce mucin, including bronchioloalveolar cell carcinomas; thus it can be challenging to differentiate a primary mucus-producing pulmonary adenocarcinoma from an adenocarcinoma that also produces mucus elsewhere in the body. Likewise, CK7 and CK20 can be seen in some primary pulmonary adenocarcinomas and other primary adenocarcinomas such as pancreatic adenocarcinoma. Amaro and colleagues236 tried to determine whether Nap-A and TTF-1 were of any value in differentiating a mucus-producing primary pulmonary adenocarcinoma from an adenocarcinoma elsewhere in
422
Immunohistology of Lung and Pleural Neoplasms
the body. Nap-A is an aspartic proteinase that appears to be involved in the maturation of surfactant protein B (SP-B), and it has been shown to be superior in sensitivity and comparable in specificity to TTF-1 for primary lung adenocarcinomas. However, the question remained whether Nap-A retained its usefulness for pulmonary adenocarcinomas with mucinous differentiation, the variants of which are notorious for being morphologically and histochemically difficult to distinguish from metastatic adenocarcinomas (usually colorectal adenocarcinomas). The authors studied a total of 40 cases of mucinous adenocarcinoma (21 lung primary, 12 colorectal/appendiceal, and 7 ovarian) and found that all seven cases of mucinous lung adenocarcinomas that were TTF-1 positive were also positive for Nap-A; however, an additional four cases were positive for Nap-A but not for TTF-1. The authors concluded that Nap-A demonstrated greater sensitivity (0.52 vs. 0.33) and was nearly equivalent in specificity (0.94 vs. 1.0) compared with TTF-1 when applied to primary pulmonary adenocarcinomas with mucinous differentiation in comparison to mucinous tumors of the appendix/colon and ovary. Another concern in all mucinous adenocarcinomas is that CDX-2, which is often thought to be very specific for GI tract carcinomas, can be seen in primary pulmonary adenocarcinomas, specifically those that are mucin producing.
Pulmonary Adenocarcinoma Versus Metastatic Colon Cancer The IHC profile of pulmonary adenocarcinoma is contrasted with metastatic colonic adenocarcinoma in Table 12-18. In general, primary pulmonary adenocarcinomas typically express CK7 and TTF-1 and do not express CK20. Metastatic colonic adenocarcinomas to lung typically express CK20 and CDX-2 and show no expression of CK7 or TTF-1. As stated previously,
TABLE 12-18 Immunohistochemical Features of Primary Mucinous Pulmonary Adenocarcinomas vs. Metastatic Colonic Adenocarcinomas Primary Pulmonary Mucinous Tumors
Metastatic Colonic Mucin-Producing Adenocarcinoma
CK5/6
R
−
CK7
S
S
CK20
S
S
CEA
+
+
SP-A
S
−
Antibody Directed Against
TTF-1
S
−
CDX-2
R
S
Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative. CEA, Carcinoembryonic antigen; CK, cytokeratin; S-PA, surfactant protein A; TTF-1, thyroid transcription factor 1.
TTF-1 is the most specific marker in differentiating primary pulmonary adenocarcinomas from adenocarcinomas of other sites. Approximately 60% to 75% of primary pulmonary adenocarcinomas express TTF-1. Caution is encouraged, however, because mucin-producing pulmonary adenocarcinomas can express CDX-2.
Pulmonary Adenocarcinoma Versus Breast Carcinoma Some peripheral pulmonary adenocarcinomas with acinar and papillary patterns with abundant extracellular mucin may express gross cystic disease fluid protein 15 (GCDFP-15) in addition to TTF-1. Striebel and colleagues237 reviewed 211 adenocarcinomas and found that 5.2% expressed GCDFP-15, a specific marker used in breast pathology; in addition, 81% percent of these cases also expressed TTF-1, but none expressed hormone receptors. Caution must be exercised in the hunt for breast metastasis in the lung, because GCDFP-15 has a very high specificity for breast carcinoma. Differentiating a primary breast cancer from a lung adenocarcinoma often presents a diagnostic challenge because of overlapping morphologic features and IHC profiles. Lin and associates238 stated that TTF-1, Nap-A, and estrogen receptors (ERs) were the recommended panel of markers for differentiating breast carcinoma from lung adenocarcinoma, although the authors stated these three markers were not entirely sensitive and specific for differentiating a lung primary from a breast primary. The purpose of their study was to reevaluate the expression of an extensive panel of biomarkers, including GATA3 and Trefoil factors 1 and 3 (TFF1 and TFF3), by using a single immunostaining system. The authors evaluated the expression of epithelial markers, mucin gene products, tumor suppressor genes, transcription factors, and tumor-associated proteins in 146 cases of breast carcinoma (98 ductal carcinomas and 48 lobular carcinomas) and 111 cases of lung adenocarcinoma. Of the breast cancer cases, 95% immunostained for GATA3, 88% for TFF3, 86% for ER, and 77% for TFF1. The breast cancer cases showed no immunostaining for TTF-1 or Nap-A. Of the lung adenocarcinoma cases, 78% stained for TTF-1, 77% for Nap-A, 22% for TFF3, and 5% for TFF1. The lung cancer cases showed no immunostaining for GATA3 and ER. The authors concluded that TTF-1, Nap-A, GATA3, ER, TFF1, and TFF3 were the most effective diagnostic panel for distinguishing lung adenocarcinoma from breast carcinoma. In our opinion, the findings suggest that a combination of TTF-1 and Nap-A (positive in lung cancer, negative in breast cancer) and GATA3 and ER (positive in breast cancer, negative in lung cancer) would be the most effective panel.
Primary Pulmonary Adenocarcinoma Versus Metastatic Renal Cell Carcinoma Distinguishing primary pulmonary adenocarcinoma from metastatic renal cell carcinoma (RCC) can be difficult. Renal tumors frequently metastasize to the pleura
Differential Diagnosis and Pitfalls of Lung Neoplasms
and produce a pseudomesotheliomatous cancer. One of the authors (S.P.H.) observed a case of a patient who had a significant history of asbestos exposure. The patient also had hyaline pleural plaque characteristic of plaque caused by asbestos, and a significant number of asbestos bodies were identified in lung tissue. The initial diagnosis by the treating hospital was mesothelioma, and the macroscopic features of the neoplasm were characteristic of a typical mesothelioma. Subsequently, however, it was brought to the attention of the treating hospital pathologist that the patient had an RCC resected from his right kidney. The pathologists were subsequently concerned the tumor had the features of a RCC. The tumor did, in fact, represent a pseudomesotheliomatous lung cancer, specifically, a metastatic kidney cancer. Primary pulmonary adenocarcinoma can have a clear cell pattern and other patterns similar to RCC. Pereira and colleagues239 studied 88 NSCLCs (34 squamous, 42 adenocarcinomas, 8 large cell carcinomas, and 4 large cell NECs) with 52 renal cell carcinomas (25 clear cell, 10 chromophobe, and 17 papillary). The authors interpreted the stain as positive if 5% or more of the malignant cells stained, regardless of the intensity: TTF-1 and Pax-2 nuclear staining, RCC and CK7 cytoplasmic staining, and Nap-A granular cytoplasmic staining were evaluated. They found that TTF-1 was very specific for lung, whereas RCC and Pax-2 were very specific for kidney. In this study, CK7 and Nap-A were not helpful in the differentiation of lung versus kidney cancer; with respect to CK7, 40% of the squamous cell lung carcinomas stained positive. SCC of the lung is thought to arise from the basal hyperplasia that then undergoes squamous metaplasia, and then squamous cell dysplasia, and becomes SCC in situ. However, Trump and associates240 suggest that it arises from metaplasia of mucous columnar epithelial cells. Arcila and colleagues241 studied Nap-A in 77 lung carcinomas and 509 nonpulmonary malignancies; specifically, 50 lung adenocarcinomas, 23 lung SCCs, 4 lung NE neoplasms, 160 colonic adenocarcinomas, 81 gastric adenocarcinomas, 29 cervical adenocarcinomas, 52 endometrial adenocarcinomas, 57 breast adenocarcinomas, 50 clear cell renal carcinomas, 46 papillary RCCs, and 34 marginal zone lymphomas. Nap-A was positive in 37 of 50 lung adenocarcinomas (74%) and 13 of 46 papillary RCCs (28%). All pulmonary SCCs, NE neoplasms, and nonpulmonary malignancies were negative for Nap-A. Strong reactivity was also found in type II pneumocytes and renal tubular cells. This study confirmed previous reports that Nap-A showed robust expression in the majority of lung adenocarcinomas and in a subset of papillary RCCs but in no other extrapulmonary malignancies.
Metastatic Carcinoma Versus Primary Pulmonary Adenocarcinoma Ye and colleagues242 studied 29 resected metastatic carcinomas of the lung that included 7 colonic carcinomas, 10 RCCs, 3 papillary or Hürthle cell carcinomas of the thyroid, 1 endocervical adenocarcinoma, 1 ovarian endometrioid carcinoma, 1 prostatic adenocarcinoma, 2
423
hepatocellular carcinomas, 2 adrenocortical carcinomas, and 2 breast carcinomas. In this study, 3 of 7 metastatic colonic adenocarcinomas showed weak to moderate, patchy nuclear staining for TTF-1 in 5% to 20% of tumor cells. In addition, 1 of 10 clear RCCs, 1 ovarian carcinoma, and 1 prostatic adenocarcinoma also exhibited 5% to 30% of tumor cells to be weakly to moderately positive for TTF-1. All cases were negative for Nap-A. Of the lung adenocarcinomas, 104 of 121 (86%) showed staining for Nap-A and 98 of 121 (81%) showed staining for TTF-1 with no statistically different sensitivity between the two. Papillary RCCs were positive for Nap-A, and more than 80% of the tumor cells showed moderate to strong immunostaining in 12 of 15 cases (80%), whereas all other renal epithelial neoplasms were negative for Nap-A. This was the first time TTF-1 was detected in both clear cell RCCs and prostatic adenocarcinomas metastatic to the lung. The authors concluded that combined TTF-1 and Nap-A immunostains were more powerful in separating primary lung adenocarcinomas from metastases.
Neuroendocrine Neoplasms Pathologists and clinicians continue to extensively evaluate NE neoplasms. Cooper and colleagues243 evaluated 77 patients retrospectively who underwent surgical resection. Of the 77 neoplasms, 50 were typical carcinoids, 5 were atypical carcinoids, 9 were large cell NECs, 4 were classified as mixed large cell–small cell NECs, and 9 were small cell NECs. Follow-up was obtained for 62 of 77 patients for an average of 38.1 months (range 2 to 132 months), and 8 of 13 deaths were disease related: 4 in patients with large cell NEC; 2 in small cell carcinoma patients; 1 in an atypical carcinoid patient; and 1 death in a patient with mixed small cell–large cell carcinoma. The mean disease-free intervals for patients with NE neoplasms were typical carcinoid, 41.3 months; atypical carcinoid, 20 months; large cell NEC, 25 months; and small cell NEC, 48 months. The authors acknowledged the limitations of the study and the controversial role of surgery in high-grade NECs. Lyda and Weiss244 immunostained 142 primary lung carcinomas for B72.3, 34βE12 (CKs 1, 5, 10, and 14), CK7, CK17, synaptophysin, and chromogranin to determine the utility of NE markers and epithelial markers in diagnosing primary lung cancers. They found that 84% (37/44) of large cell and small cell NECs were chromogranin positive; 58% (21/36) of small cell carcinomas and 100% (6/6) of large cell NECs were synaptophysin positive; 5% (2/43) were keratin 34βE12 positive; 9% (4/44) were CK7 positive; and 5% (2/37) of small cell carcinomas and 50% (3/6) of large cell NECs were B72.3 positive. Among 98 non-NECs, 5% (5/98) were chromogranin positive; 3% (3/98) were synaptophysin positive; 97% (95/98) were positive for 34βE12 or CK7; and 99% (97/98) were positive for either 34βE12, CK7, or B72.3. An antibody panel consisting of CK7, 34βE12, chromogranin, and synaptophysin separated 132 of 141 (94%) tumors into distinct groups. Sturm and colleagues245 evaluated 227 NE proliferations and tumors for TTF-1. Immunostaining was
424
Immunohistology of Lung and Pleural Neoplasms
detected in 85.5% (47/55) of SCLCs; in 49% (31/64) of large cell carcinomas; and in 0 of 15 NE hyperplasias, 23 tumorlets, 27 typical carcinoid, and 23 atypical carcinoids. In 95% (19/20) of combined SCLCs and large cell NECs, TTF-1 was expressed in the NE and non-NE components of the tumor. The authors concluded their findings challenging the concept of a spectrum of NE neoplasms and suggested the findings lent credence to the alternative hypothesis of a common derivation for SCLCs and NSCLCs. Sturm and colleagues’245 conclusion is difficult to accept, because there is abundant biochemical, histochemical, and ultrastructural evidence of the association among various NE lung neoplasms. In addition, as discussed previously, others have found TTF-1 expression in typical carcinoids and atypical carcinoids. Barbareschi and colleagues246 evaluated CDX-2 expression in routine samples of 20 normal endocrine/ NE tissues and 299 samples of well-differentiated NE tumors and high-grade NECs from different sites. CDX-2 was expressed at high levels in 81% of intestinal NECs. Somewhat unexpectedly, CDX-2 was seen in 39% of NECs of other sites. Reactivity was found to have frequently overlapped TTF-1 expression, suggesting deregulated expression of homeobox genes in NECs. The authors concluded that these findings show a limited diagnostic role for CDX-2 in NECs because of its frequent expression in non-GI tumors. Lin and colleagues247 studied the value of CDX-2 and TTF-1 expression in separating metastatic NE neoplasms of unknown origin in a study of 155 primary NE tumors, including 60 pulmonary, 60 GI, 30 pancreatic, and 5 NE tumors from other sites. In addition, they evaluated 13 metastatic NE tumors, including 11 GI and 2 pulmonary tumors. CDX-2 was expressed in 47% (28/60) of gastrointestinal NE tumors. Of those that expressed CDX-2, 11 of 11 were appendiceal NE tumors, 12 of 14 (86%) were small intestinal, 3 of 4 (75%) were colonic, 2 of 11 (18%) were rectal, and none of the 20 were gastric. TTF-1 was observed in 43% (13/30) of pulmonary carcinoid tumors and in 90% (27/30) of pulmonary SCLCs. In contrast to the study by Barbareschi and coworkers,246 Lin and colleagues247 concluded CDX-2 expression to be highly specific in identifying NE tumors of intestinal origin, and TTF-1 expression is helpful in identifying NE tumors of pulmonary origin. Proliferation marker Ki-67 (MIB-1 clone) has been used to determine low-grade versus high-grade NECs as reported by Lin and others.248 The authors found that when MIB-1 immunoreactivity was considered, all lowgrade NE neoplasms showed immunoreactivity of less than 25% of the neoplastic cells, in contrast to highgrade NE neoplasms, which showed MIB-1 immunoreactivity in more than 50% of the neoplastic cells. This observation was further expanded upon by Pelosi and colleagues,249 who observed seven patients with typical or atypical carcinoid tumors that were overdiagnosed as SCLCs in bronchial biopsy specimens. The authors studied bronchial biopsies from nine consecutive SCLC patients histologically and immunohistochemically (for cytokeratins, chromogranin A, synaptophysin, Ki-67/MIB-1, and TTF-1). The typical
carcinoid tumors were centrally or peripherally located and were composed of tumor cells with a granular or coarse nuclear chromatin pattern, intense chromogranin A and synaptophysin reactivity, and a low (<20%) Ki-67/MIB-1 labeling index. The carcinoid tumors’ stroma contained thin-walled blood vessels. The SCLCs were in a central location, had finely dispersed nuclear chromatin, and showed less intense immunostaining for chromogranin A and synaptophysin and a high (>50%) Ki-67/MIB-1 labeling index. The authors concluded that overdiagnosis of carcinoid tumors as SCLC in small, crushed bronchial biopsies remained a significant problem. Careful evaluation of H&E-stained sections was the most important tool in arriving at the correct diagnosis, and evaluation of the tumor cell proliferation index by Ki-67/MIB-1 was the most useful ancillary technique for detection. Non–small cell primary lung neoplasms may show NE differentiation that can cause confusion, if the neoplasm being evaluated is considered histologically to have features suggestive of a non-NE tumor. Visscher and colleagues250 evaluated 56 poorly differentiated non–small cell primary lung neoplasms with monoclonal antibodies directed against chromogranin A, synaptophysin, S-100 protein, keratin, vimentin, and neurofilament antigen. These neoplasms had no histologic features of NE differentiation. Using frozen, unfixed tissue sections, 29% (5/17) of large cell undifferentiated carcinomas and 21% (4/19) of adenocarcinomas showed immunostaining for chromogranin A or synaptophysin. Diffuse intense immunostaining for synaptophysin was observed in two large cell undifferentiated carcinomas and one poorly differentiated adenocarcinoma. Of the poorly differentiated squamous carcinomas, 1 of 20 (5%) expressed synaptophysin. Of interest, 58.8% (10/17) of large cell undifferentiated carcinomas and 52.6% (10/19) of poorly differentiated adenocarcinomas expressed vimentin or neurofilament antigen. The authors concluded that immunohistologic evidence of NE differentiation was observed in a significant number of large cell undifferentiated carcinomas and poorly differentiated adenocarcinomas and was accompanied by heterogeneous intermediate filament expression. Linnoila and associates251 evaluated 113 surgically resected primary lung neoplasms with antibodies against chromogranin A, Leu-7, NSE, serotonin, bombesin, calcitonin, adrenocorticotropic hormone (ACTH), vasopressin, neurotensin, CEA, keratin, vimentin, and neurofilament by using formalin-fixed, paraffin-embedded (FFPE) sections. They observed that the majority of typical carcinoids and small cell carcinomas expressed multiple NE markers in a high percentage of tumor cells and that approximately 50% of NSCLCs contain subpopulations of tumor cells that express NE markers. Occasional NSCLCs showed immunostaining patterns indistinguishable from small cell carcinoma. NE markers were found more frequently in large cell undifferentiated carcinomas and adenocarcinomas than in squamous carcinomas. Mooi and colleagues252 evaluated 11 resected primary lung neoplasms classified as large cell carcinoma or SCC
Differential Diagnosis and Pitfalls of Lung Neoplasms
but showing some microscopic resemblance to bronchial carcinoid and small cell carcinoma. All cases were NSE and PGP9.5 positive, indicative of NE differentiation. Bombesin and chromogranin were positive in two cases each, and C-terminal peptide was expressed in five cases. In six of seven cases evaluated by electron microscopy, dense core NE granules were observed. Based on the published photographs of the neoplasms, it could be argued that the neoplasms reported represent mixed NE and non-NE tumors. Wick and colleagues253 compared 12 large cell carcinomas of lung showing NE differentiation with 15 large cell pulmonary neoplasms showing no NE differentiation. The large cell neoplasms showing NE differentiation would have been classified by Travis and colleagues’69 criteria as large cell NECs and not as large cell undifferentiated carcinomas showing focal NE differentiation. Of interest and potential importance, the large cell neoplasms with NE differentiation had a significantly worse prognosis than those that did not show NE differentiation. The authors suggested that IHC and electron microscopy are necessary to diagnose such neoplasms, and they concluded that these were probably underdiagnosed. Some medical oncologists suggest that all large cell carcinomas of lung be evaluated for NE differentiation because of potential differences in chemotherapeutic treatment. Loy and colleagues254 evaluated 66 neoplasms that had been examined ultrastructurally and with a battery of NE markers that included NSE, chromogranin A, Leu-7, and synaptophysin and with non-NE marker B72.3. They studied 11 small cell carcinomas, 4 lowgrade NECs, 2 large cell carcinomas with NE differentiation, 26 adenocarcinomas, 10 SCCs, and 11 large cell undifferentiated carcinomas. Of these, 4 of 10 squamous carcinomas, 3 of 26 adenocarcinomas, and 1 of 11 large cell undifferentiated carcinomas showed immunostaining for chromogranin A. None of the 10 squamous carcinomas, 4 of 26 adenocarcinomas, and none of the 11 large cell undifferentiated carcinomas showed immunostaining for Leu-7. In addition, 6 of 10 squamous carcinomas, 15 of 26 adenocarcinomas, and 7 of 11 large cell undifferentiated carcinomas showed immunostaining for NSE; and 6 of 10 squamous carcinomas, 16 of 26 adenocarcinomas, and 7 of 11 large cell undifferentiated carcinomas showed immunostaining for synaptophysin. Overall, 79% (34/47) of carcinomas without NE features expressed at least one NE marker, and all NECs expressed at least one NE marker. Xu and colleagues255 used IHC analysis to study novel protein K homology domain–containing protein overexpressed in cancer (KOC) in NE lung cancers. KOC, a member of the insulin-like growth factor (IGF) mRNA-binding protein family, was expressed during embryogenesis and in certain malignancies. The authors studied NE lung neoplasms, and 10 SCLCs exhibited strong cytoplasmic staining, 9 with diffuse positivity and 1 with focal positivity; 14 large cell NECs expressed KOC, 9 of which exhibited strong and diffuse cytoplasmic staining, and 5 cases showed focal immunoreactivity. In contrast, no KOC was detected in 21 typical and atypical carcinoids, except for a single atypical carcinoid
425
with oncocytic features. Although SCLCs exhibited a strong and diffuse staining pattern more frequently than large cell NECs, the difference did not reach statistical significance. The authors concluded that their findings of equivalent IGF-2 expression in KOC-positive SCLC and large cell NEC, and KOC-negative carcinoid tumors suggested different regulatory mechanisms in the control of IGF-2 expression in these tumors. This perhaps could be another way for pathologists to avoid the trap of misdiagnosing typical carcinoids as SCLC or atypical carcinoids. Schleusener and colleagues256 used IHC to evaluate 107 patients with stage IIIa, IIIb, and IV NSCLCs—62 adenocarcinomas, 22 SCCs, 18 large cell carcinomas, and 5 adenosquamous carcinomas—with antibodies against keratin, synaptophysin, Leu-7, and chromogranin A. Keratin was used as a control and was positive in 99.1% of cases, and 35% of adenocarcinomas, 41% of squamous carcinomas, and 33% of large cell carcinomas expressed at least one NE marker. Somewhat surprising was the finding of increased survival in patients whose tumors expressed one or more NE markers. However, no correlation was found between NE markers and response to chemotherapy. The bottom line for pathologists is that lung neoplasms not classified by histologic criteria as NE neoplasms may express NE markers by IHC. A summary of these studies that shows the frequency of expression of chromogranin A, synaptophysin, NSE, and Leu-7 is shown in Figure 12-33.
Small Cell Lung Cancer Versus Merkel Cell Carcinoma Ralston and colleagues257 evaluated mammalian achaetescute complex homolog 1 (MASH-1) as a marker in differentiating SCLC from Merkel cell carcinoma. The authors pointed out that achaete-scute complex–like 1 (ASCL1), also known as MASH-1 or human achaetescute complex homolog 1 (HASH-1), was a basic helixloop-helix transcription factor crucial for NE cell differentiation. MASH-1 was reported to be expressed in some NE neoplasms, including SCLC. The authors studied 30 cases of Merkel cell carcinoma and 59 cases of SCLC that were immunostained for MASH-1 and TTF-1 antibodies. Of the 59 SCLCs, 49 (83%) expressed MASH-1 in a nuclear staining pattern, whereas out of 59 SCLCs, 43 (73%) expressed TTF-1 in a nuclear staining pattern. MASH-1 was stated to be completely negative in all 30 Merkel cell carcinomas, whereas TTF-1 expression was seen in 3% (1/30) of Merkel cell carcinomas. The authors concluded that MASH-1 was a useful adjunct for differentiating small cell carcinoma of the lung from Merkel cell carcinoma. Rajagopalan and colleagues258 studied the utility of CD99 (MIC-2) in the diagnosis of Merkel cell carcinoma, previous studies of which reported rates of expression that ranged from 13% to 55%. When specified, a membranous or cytoplasmic staining pattern was considered significant, and the authors found paranuclear dotlike staining in several cases of Merkel cell carcinoma, including cases that lacked CK20
Immunohistology of Lung and Pleural Neoplasms
Percent positive
426
80 70 60 50 40 30 20 10 0 Adenocarcinoma
Figure 12-33 Summary of expression of neuroendocrine (NE) markers in histologically diagnosed non-NE neoplasms. NE expression in non-NE lung neoplasms is usually focal and of low intensity.
expression. All 14 cases of Merkel cell carcinoma showed at least focal CD99 staining, with both membranous and paranuclear dotlike staining patterns, and 12 of 14 cases stained for CK20 with the characteristic dotlike pattern. In addition, 4 of 7 cases of pulmonary small cell carcinoma showed CD99 staining, and 2 cases showed a finely granular dotlike staining pattern. The authors concluded that the unusual pattern of paranuclear dotlike expression of CD99 seen in 14 cases of Merkel cell carcinoma, two of which did not express CK20, may be useful in differentiating Merkel cell carcinoma from other cutaneous malignancies, especially when CK20 expression is limited or absent. Bobo and associates259 assessed the usefulness of several IHC stains in distinguishing between Merkel cell carcinoma and small cell carcinoma of the lung using antibodies for CK7, CK20, NSE, chromogranin, synaptophysin, neurofilaments, TTF-1, CD56, S-100 protein, vimentin, CD117, and ErbB-2 oncoprotein. All 13 cases of Merkel cell carcinoma were positive for CK20 and negative for TTF-1, whereas 11 of 13 SCLCs were positive for TTF-1, and all but one SCLC was negative for CK20. Of 13 Merkel cell carcinomas, 12 were positive for neurofilaments, whereas all SCLCs were negative. With respect to the remaining antigens tested, no significant differences were noted between the two neoplasms. The findings suggested that a set of three IHC stains—CK20, neurofilament protein (NFP), and TTF-1—be used in distinguishing between Merkel cell carcinoma and SCLC. Lau and coworkers260 reviewed the usefulness of TTF-1 IHC staining in the diagnosis of neoplastic conditions, and based on published studies to date, TTF-1 was found to be a very useful reagent in distinguishing small cell carcinoma of the lung from Merkel cell carcinoma and other neoplasms. Leech and others261 came to a similar conclusion when investigating whether IHC staining for CK20 and TTF-1 was useful in distinguishing Merkel cell carcinoma from metastatic SCLC. In this study, 90% (10/11) of Merkel cell carcinomas stained positively for CK20 and showed no immunostaining for TTF-1, whereas 10 SCLCs showed no immunostaining for CK20, and 10 of 10 showed strong positive staining for TTF-1. Leech and colleagues261 concluded that the use of both CK20 and TTF-1 can
Squamous cell carcinoma
Neuron-specific enolase Synaptophysin Chromogranin A
Large cell undifferentiated carcinoma Leu7 Combined
reliably distinguish between Merkel cell carcinoma and metastatic small cell carcinoma of the lung. KEY DIAGNOSTIC POINTS Immunohistologic Pitfalls of Primary vs. Metastatic Carcinoma in Lung • CDX-2 expression may be seen in primary mucinous “colloid” pulmonary carcinomas, which may also express CK20 but lack CK7 and TTF-1. Clinical correlation and imaging may be critically necessary in these situations. • GCDFP-15 may be expressed in up to 5% of primary lung adenocarcinomas. These tumors often have abundant extracellular mucin, and they typically express TTF-1 and synaptophysin. Caution is in order in the hunt for metastatic breast carcinoma in this situation. • Alpha inhibin may be seen in nearly 25% of primary lung carcinomas. Additional panel markers, especially keratins, will obviate this pitfall. • Some clones of estrogen receptor may demonstrate positive immunostaining in primary lung adenocarcinoma. Estrogen receptor should not be used as a diagnostic application in this setting.
Lymphomas, Sarcomas, and Melanomas Lymphoepithelioma-like carcinoma, a subtype of primary large cell undifferentiated carcinoma, may occur as a primary lung cancer composed of large anaplastic cells and may be confused with lymphomas and other neoplasms.262 Most express LMW and HMW keratin and show ultrastructural features of epithelial differentiation that include desmosomes and intracellular tonofilaments. Some express EBV and Bcl-2.263 Giant cell carcinoma of lung is a subtype of large cell undifferentiated carcinoma composed of at least 40% of cells greater than 40 µm in diameter.264 They usually coexpress keratin and vimentin and must be differentiated from metastatic sarcomas and melanomas. Some show cytoplasmic immunostaining for CEA. Some large cell anaplastic lymphomas have an epithelioid appearance and show immunostaining for keratin.265 Most Ki-1 (CD30)-positive anaplastic lymphomas express EMA.266 Some large cell anaplastic
Differential Diagnosis and Pitfalls of Lung Neoplasms
427
TABLE 12-19 Immunohistochemical Features of Various Types of Large Cell Undifferentiated Neoplasms Involving Lung Antibody Directed Against
Large Cell Undifferentiated Carcinoma of Lung
Giant Cell Carcinoma of Lung
Lymphoepithelioma like Carcinoma of Lung
Large Cell Neuroendocrine Carcinoma
Malignant Melanoma
Anaplastic Lymphoma
AE1/AE3
+
+
S
S
R
R
LMW keratin
+
+
S
S
R
R
HMW keratin
S
S
S
S
R
R
CK7
S
S
S
S
R
−
CK20
−
−
−
−
−
−
Vimentin
S
S
S
S
+
S
EMA
S
S
S
S
R
S
CEA
S
S
S
S
R
R
S-100 Protein
R
R
R
R
S
R
HMB-45
−
−
−
−
S
−
CD30
−
−
−
−
R
S
CD20
−
−
−
−
−
S
NSE
S
S
R
+
S
R
SYN
R
R
R
+
R
−
CGA
R
R
R
S
R
−
CEA, Carcinoembryonic antigen; CGA, chromogranin A; CK, cytokeratin; EMA, epithelial membrane antigen; HMW, high molecular weight; HMB-45, human melanoma black 45; LMW, low molecular weight; NSE, neuron-specific enolase; SYN, synaptophysin. Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative.
lymphomas have ultrastructural features suggestive of epithelial differentiation.267 A list of large cell undifferentiated neoplasms and their immunophenotypes is shown in Table 12-19. Chu and associates268 recently reported finding significant numbers of nonhematopoietic epithelioid neoplasms that express T/natural killer (NK) cell antigens, although few were of diagnostic significance (Table 12-20). As known by most surgical pathologists, malignant melanomas may show significant variability in differentiation. Nearly all show immunostaining for S-100 protein and vimentin. Approximately 50% immunostain for human melanoma black 45 (HMB-45) antibody, and most stain for Pan Melanoma antibody. Rare melanomas immunostain for keratin,269 which can cause diagnostic confusion.
CDX-2 Immunoreactivity CDX-2 transcription factor is characteristically expressed in GI adenocarcinomas.270 Rossi and colleagues271 evaluated 13 primary mucinous (colloid) carcinomas of the lung. All 11 goblet-cell–type mucinous carcinomas strongly immunostained for CDX-2 and MUC2. Eight reacted with TTF-1, six with CK20, nine with CK7, and two with MUC5AC. The two signet-ring mucinous carcinomas immunostained for TTF-1, CK7, and MUC5AC but did not immunostain for CDX-2 and CK20. The authors concluded that because goblet-cell–type
mucinous carcinomas strongly immunostained for CDX-2, MUC2, and CK20, the differential diagnosis with metastatic colorectal carcinoma was challenging and required appropriate clinical correlation. Mazziotta and colleagues272 evaluated a number of different types of adenocarcinomas for CDX-2 expression, including 84 lung adenocarcinomas. Of these 84 lung cancers, 10 showed areas of fairly strong immunoreactivity for CDX-2; 7 of the lung cancers were adenocarcinomas, 3 were large cell undifferentiated carcinomas, and 3 of 7 adenocarcinomas and 1 large cell carcinoma were positive for TTF-1 and CK7 and were negative for CK20. In 7 of 8 cases evaluated, gene expression of CDX-2 was identified. The authors concluded that CDX-2 was a relatively specific marker for tumors with intestinal differentiation with the caveat that CDX-2 expression could be seen in some primary adenocarcinomas, large cell carcinomas of the lung, and mucinous carcinomas of the ovary.
Thyroid Transcription Factor 1 Immunoreactivity The pathologist must be aware of cytoplasmic TTF-1 immunoreactivity in other neoplasms. Bejarano and Mousavi273 evaluated 361 neoplasms from 29 organ sites that included primary and metastatic neoplasms. Twenty-three tumors (6.4%) showed cytoplasmic staining for TTF-1. In 13 of these cases, the primary site of origin was established with certainty: 7 were lung
428
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-20 Frequencies of CD2, 3, 4, 5, 7, 8, 56, and 138 Expression in 447 Nonhematopoietic Neoplasms with Epithelioid Features No. of Positive Cases Tumor Type
Total
CD2
CD3
CD4
CD5
CD7
CD8
CD56
CD138
Lung Lung adenocarcinoma
21
0
0
0
2
8
0
1
11
Small cell carcinoma
6
0
0
0
0
0
0
6
0
Colon
10
0
0
0
5
6
0
0
9
Pancreatic carcinoma
13
0
0
0
6
9
0
0
4
Gastric adenocarcinoma
15
0
0
0
0
3
0
0
2
Hepatocellular carcinoma
25
0
0
0
0
0
0
0
15
Cholangiocarcinoma
14
0
0
0
12
13
0
3
13
Prostate carcinoma
18
0
0
0
4
0
0
1
6
Renal cell carcinoma
19
0
0
0
1
0
0
2
12
Transitional cell carcinoma
24
0
0
0
5
9
0
0
22
Breast (ductal and lobular)
26
0
0
0
7
3
0
1
18
Ovarian carcinoma
24
0
0
0
3
8
0
2
10
Endometrial adenocarcinoma
10
0
0
0
0
3
0
1
9
Squamous cell carcinoma
26
0
0
0
0
2
0
0
25
Basal cell carcinoma
20
0
0
0
0
2
0
0
14
Follicular adenoma
24
0
0
0
0
0
0
17
10
Papillary carcinoma
9
0
0
0
0
0
0
9
2
17
0
0
0
0
0
0
15
2
Adrenal cortical neoplasm
20*
0
0
0
2
1
0
18
7
Neuroendocrine carcinoma
9
0
0
0
0
0
0
9
3
10
0
0
0
0
0
0
9
2
9
0
0
0
0
0
0
9
2
8
0
0
0
0
0
0
0
0
Germ cell tumor
14
0
0
0
0
0
0
0
0
Malignant melanoma
20
0
0
0
2
8
0
1
1
Salivary gland tumor
11
0
0
0
1
1
0
0
3
Malignant mesothelioma
16†
0
0
0
2
4
0
0
0
Epithelioid sarcoma
10
0
0
0
3
7
0
2
0
0 (0.0)
0 (0.0)
0 (0.0)
Gastrointestinal Tract
Genitourinary Tract
Female Reproductive System
Skin
Thyroid Tumors
Medullary carcinoma Neuroendocrine Tumors
Carcinoid tumor Merkel cell carcinoma Miscellaneous Thymoma
Total
448
55 (12.3%)
87 (19.5%)
0 (0.0)
106 (23.7%)
202 (45.2%)
From Chu PG, Arber DA, Weiss LM: Expression of T/NK-cell and plasma cell antigens in nonhematopoietic epithelioid neoplasms: an immunohistochemical study of 447 cases. Am J Clin Pathol 2003120:64-70. *An additional 10 cases of adrenocortical carcinoma were studied for CD56 expression. Of 30 cases, 25 were CD56 positive. † An additional 5 cases of malignant mesothelioma were studied for CD138 expression. Of 21 cases, 1 was CD138 positive.
Differential Diagnosis and Pitfalls of Lung Neoplasms
429
carcinomas (3 primary lung adenocarcinomas, 1 primary large cell carcinoma, 1 metastatic small cell carcinoma to the liver, 1 metastatic adenocarcinoma to a neck lymph node, and 1 metastatic adenocarcinoma to thigh soft tissue); 3 were colonic adenocarcinomas (2 metastatic to vertebrae and 1 to lung); 1 was a metastatic breast ductal adenocarcinoma to the femur; 1 was metastatic laryngeal SCC to liver; and 1 meningioma involved the orbit bone. Occasional cytoplasmic immunostaining for TTF-1 was observed in several different types of neoplasms but was a nonspecific finding and should be disregarded for diagnostic purposes.
S-100 Protein Immunoreactivity A few primary pulmonary adenocarcinomas may show low-intensity immunostaining for S-100 protein.274 However, it is more common in primary pulmonary adenocarcinomas, especially nonmucinous bronchioloalveolar cell carcinomas, to show S-100 protein– positive dendritic cells admixed with the neoplastic cells (Fig. 12-34).275 These S-100 protein–positive cells represent Langerhans cells (Fig. 12-35), and adenocarcinomas may secrete a factor that is chemotactic for the Langerhans cells.276 Langerhans cells, however, can be seen in a wide variety of neoplasms and nonneoplastic pulmonary conditions.277 Dorion and colleagues278 studied the utility of S-100 protein in differentiating lung adenocarcinoma from papillary and follicular carcinoma of the thyroid. They found that pulmonary adenocarcinoma showed nuclear and cytoplasmic staining for S-100 protein in 31 of 39 cases, whereas all cases of papillary and follicular carcinomas of the thyroid were negative for S-100 protein. The authors, however, did not mention that Langerhans cells are S-100 protein positive, can frequently be seen in pulmonary adenocarcinomas, and can easily be misinterpreted as cancer cells.
Figure 12-35 Ultrastructurally, the S-100 protein–positive cells represent Langerhans cells, which are antigen-presenting processing macrophages that contain peculiar cytoplasmic organelles referred to as Langerhans cells granules or Birbeck granules (×20,000).
Not infrequently, nonmucinous bronchioloalveolar cell carcinomas show intranuclear inclusions that are period
acid–Schiff (PAS)-diastase positive (Fig. 12-36).279-282 The intranuclear PAS-positive inclusions immunostain for the apoprotein portion of the surfactant (Fig. 12-37). When examined ultrastructurally, these intranuclear inclusions consist of 45-nm-diameter tubules that attach to the inner nuclear membrane (Fig. 12-38). Antibodies against surfactants are commercially available and have been evaluated in diagnosing pulmonary adenocarcinomas. Surfactant antibodies are not 100% specific for surfactant-producing pulmonary adenocarcinomas. Bejarano and colleagues23 used IHC markers to distinguish between primary NSCLC and metastatic breast carcinoma. They studied 57 primary NSCLCs, including 46 adenocarcinomas and 51 adenocarcinomas of the breast. They found surfactant
Figure 12-34 This nonmucinous bronchioloalveolar cell carcinoma shows numerous S-100 protein–positive dendritic cells admixed with the tumor cells (×400).
Figure 12-36 This nonmucinous bronchioloalveolar cell carcinoma shows intranuclear periodic acid–Schiff (PAS)-positive inclusions (×400).
Surfactant Antibodies
430
Immunohistology of Lung and Pleural Neoplasms
Inhibin-Alpha Immunoreactivity
Figure 12-37 The intranuclear inclusions in nonmucinous bronchioloalveolar cell carcinomas immunostain for the apoprotein portion of surfactant A (×400).
proteins A and B and TTF-1 in 49%, 53%, and 63% of non–small cell pulmonary carcinomas, respectively, and in 54%, 63%, and 76% of primary pulmonary adenocarcinomas, respectively. Squamous cell lung carcinomas rarely stained with these antibodies. Fifty-one breast carcinomas showed no immunostaining for TTF-1 and surfactant B, although four breast cancers immunostained for surfactant A. As shown in Table 12-9, most recent studies of primary pulmonary adenocarcinomas have shown that those that express TTF-1 usually express surfactant protein A (SP-A). This finding is not unexpected, because TTF-1 is a transcription factor for surfactant in primary pulmonary adenocarcinomas.
Figure 12-38 Ultrastructurally, the intranuclear inclusions are composed of 45-nm-diameter tubules that connect to the inner nuclear membrane (×20,000).
Tigrani and Weydert283 studied expression of alphainhibin (INHA) in 48 primary pulmonary non–small cell carcinomas by IHC. Inhibin is a glycoprotein hormone known to be expressed in carcinomas of adrenocortical and germ cell origins. The authors found INHA expression in 9 of 48 cases. The extent of expression was limited to less than 25% of the tumors. The authors concluded that primary lung carcinoma was not excluded by a positive staining for INHA. The extent of positive staining in lung carcinoma, however, appeared to be limited to the minority of cells in the given tumor. Zhang and coworkers284 studied expression of INHA in non–small cell carcinoma of the lung with a focus on adenocarcinoma. Their results were similar to those from the study by Tigrani and Weydert.283
Hormone Receptors Carcinomas of breast frequently metastasize to pleura and lung, often many years after the initial diagnosis.224 Ollayos and others285 evaluated the sensitivity of estrogen receptor (ER) protein by IHC in known cases of adenocarcinoma of colon, pancreas, and lung. Fortythree colon adenocarcinomas and 18 pancreatic adenocarcinomas showed no nuclear immunostaining for ER protein, whereas 3 of 42 primary pulmonary adenocarcinomas immunostained for it. Canver and colleagues286 studied sex hormone receptor expression by IHC in 64 non–small cell lung cancers. Specimens were stated to have been acetone fixed. They found no immunostaining for sex hormone receptors in normal lung; however, 62 of 64 NSCLCs showed nuclear immunostaining for ER protein. Immunostaining for progesterone receptor (PR) protein was not found in 50 cases (78%) and was weakly positive in 14 cases (22%). Bacchi and coworkers287 reported expression of nuclear ER and PR proteins in a few pulmonary carcinoids and small cell carcinomas. DiNunno and associates288 evaluated 248 consecutive cases of stage I and II NSCLCs for estrogen and progesterone receptors using FFPE tissue, but no nuclear or cytoplasmic expression of these receptors was found. The authors concluded that finding estrogen and progesterone receptors in a lung carcinoma was supportive of a nonpulmonary metastatic carcinoma to the lung. In contrast, Dabbs and associates289 evaluated 45 resected primary pulmonary adenocarcinomas, of which 25 were nonmucinous bronchioloalveolar carcinomas and 20 were moderately differentiated adenocarcinomas not otherwise specified, for estrogen and progesterone receptors using monoclonal antibodies to two different clones (6F11 and 1D5) on FFPE tissue. Nuclear ER expression was seen in 56% of the nonmucinous bronchioloalveolar carcinomas and in 80% of the primary pulmonary adenocarcinomas not otherwise specified with antibody clone 6F11. No ER immunostaining was observed with antibody clone 1D5, and no progesterone expression was detectable in any neoplasm. The authors concluded that further study was warranted to discern the nature of the 6F11 clone
Theranostic Applications in Lung Neoplasms
antiestrogen antibody, and the clinical significance and ramifications of ER in pulmonary adenocarcinomas remains unknown. Selvaggi and colleagues290 evaluated 130 primary lung carcinomas (60 squamous, 48 adenocarcinoma, and 22 large cell undifferentiated) for ERBB2 expression. Six of 60 squamous carcinomas, 6 of 48 adenocarcinomas, and 3 of 22 large cell undifferentiated carcinomas expressed ERBB2. On multivariate analysis, ERBB2 expression and extent of tumor were an independent factor for disease-related survival. The median survival time (85 weeks versus 179 weeks) and overall survival rate were significantly lower in patients with more than 5% ERBB2-positive tumor cells.
Germ Cell Markers The mediastinum contains a variety of neoplasms, including those of germ cell origin. Difficulty can be encountered when determining whether a neoplasm is primary in the lung, invading the mediastinum, or primary in the mediastinum and invading lung. In addition, thymic carcinomas show significant variability in differentiation, including cases that show germ cell differentiation. This problem can be compounded, because primary lung cancers have been reported to express germ cell markers. Yoshimoto and colleagues291 reported a case of a poorly differentiated mucin-producing primary pulmonary neoplasm with tumor cells that showed immunostaining for CEA, α-fetoprotein (AFP), and human chorionic gonadotropin (hCG). Autopsy tissue showed these substances in different tumor cells, which the authors interpreted to suggest the lung cancer consisted of at least three clones of cancer cells with different phenotypes. Kuida and colleagues292 found hCG expression in 4 of 11 primary lung cancers. The application of TTF-1 would potentially help clarify some cases with respect to a primary mediastinal or pulmonary origin of the neoplasm. Trophoblastic expression was evaluated in 40 NE lung neoplasms, 29 primary pulmonary adenocarcinomas, 20 SCCs, and 1 adenosquamous carcinoma for hCG and its derivatives, luteinizing hormone (LH, LHβ), follicle stimulating hormone (FSH, FSHβ), placental lactogen (PL), and growth hormone (GH-227).293 Trophoblastic hormone immunoreactivity was found in 31% (28/90) of all lung carcinomas but primarily in typical carcinoids. Rare primary lung neoplasms that resemble hepatocellular carcinoma, referred to as hepatoid carcinomas, may express high concentrations of AFP and abnormal prothrombin.294
Thymic Carcinomas Thymic carcinomas can have a morphology essentially identical to that of lung cancers, therefore it can be difficult to distinguish thymic carcinomas from poorly differentiated lung carcinomas. Asirvatham and colleagues295 evaluated archived cases of proven thymic carcinoma (n = 13) and poorly differentiated lung carcinoma (n = 15) for intensity and proportion of
431
expression of Pax-8 (nuclear), CD117 (membranous), and CD5 (membranous) with the interpreters kept blind to the diagnoses. Staining in less than 10% of cells was interpreted as negative. Pax-8 was positive in 69.2% (9/13) of thymic carcinomas and 6.7% (1/15) of lung carcinomas. The intensity varied from weak to strong granular nuclear staining in 30% to 100% of cells. A single lung carcinoma positive for Pax-8 showed focal squamous differentiation. Although CD117 was positive in 84.6% (11/13) of thymic carcinomas, a significant proportion of lung carcinomas (26.6%) was also positive; in addition, 53.8% of thymic carcinomas and no lung carcinomas were positive for CD5, and 46.1%, 53.8%, and 69.2% of thymic carcinomas were dual positive for combinations of CD5/Pax-8, CD117/CD5, and CD117/Pax-8, respectively. None of the lung carcinomas showed dual positivity for any of these combinations. The authors concluded that Pax-8 was a sensitive marker for distinguishing thymic carcinoma and was an important addition to the diagnostic panel for differentiating thymic carcinoma from poorly differentiated lung carcinoma. Adding Pax-8 to CD117 and CD5 increased the diagnostic yield for thymic carcinoma, especially when at least two of the three markers were positive. However, triple negativity did not exclude the diagnosis of thymic carcinoma. Lee and colleagues296 studied CD24 expression in 267 consecutive cases of NSCLC by IHC using a tissue microarray technique correlated with clinicopathologic parameters, including the seventh tumor-nodemetastasis (TNM) classification. CD24 expression was demonstrated in 33% (87/267) of cases and was associated with advanced new pathologic stage (35/81 [43%] vs. 52/186 [28%]; P = .016), and it was higher histologically in adenocarcinoma than in SCC (64/165 [39%] vs. 20/88 [23%]; P = .023). CD24 expression in NSCLC was associated with advanced, newly proposed TNM stage and adenocarcinoma histology, as well as disease progression and cancer-related death, indicative of aggressive tumor behavior.
Theranostic Applications in Lung Neoplasms More than 50 protein markers have been assessed by IHC for their prognostic value in NSCLC patients.297 To date, none is sufficiently robust for adoption in patient care. More recent studies conducted on tumor samples of patients involved in large phase III placebocontrolled randomized adjuvant chemotherapy trials have revealed optimism about some IHC markers. The International Adjuvant Lung Trial (IALT) demonstrated that IHC assessment of excision repair crosscomplementation group 1 (ERCC1) expression in NSCLC before chemotherapy is an independent predictor of the effect of adjuvant chemotherapy.298 ERCC1 is 1 of 16 genes that encode the proteins of the nucleotide excision repair complex. Patients with completely resected NSCLC and ERCC1-negative tumors showed a substantial benefit from adjuvant cisplatin-based
432
Immunohistology of Lung and Pleural Neoplasms
chemotherapy, compared with patients with ERCC1positive tumors.298,299 The significance of epidermal growth factor receptor (EGFR) protein expression in NSCLC in predicting response to tyrosine kinase inhibitors (TKIs) remains contradictory, and a standard method has not yet been adopted.300-302 Recently reported results of comparative analysis of EGFR protein expression in NSCLC by IHC using the Dako EGFR pharmDx kit (scoring percent of positive tumor cells) and Zymed monoclonal antibody clone 31G7 (combined scoring of percent of positive cells and intensity of staining) suggested that the Dako PharmDx kit and percentage of positive staining may provide more accurate prediction of gefitinib treatment on survival.304 Recent studies suggest that EGFR immunopositivity may play a role as a selection criteria for cetuximab-based therapy, but this has to be further validated. Several markers—ERBB2, Ki-67, p53, and Bcl-2— were suggested to be important as prognostic markers by meta-analysis.297 Cyclin E, VEGFA, Cdkn2a, Cdkn1b, and β-catenin are promising candidates but need further study in large, randomized clinical trial samples by using standardized assays and scoring systems.297
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications A high likelihood of response to EGFR TKIs in lung adenocarcinoma patients correlates with somatic mutations in exons 18 through 21 of the TK domain of the EGFR gene.304-306 The most common are in-frame deletions in exon 19 (45%), followed by a point mutation (CTG to CGG) in exon 21 at nucleotide 2573, which results in substitution of leucine by arginine at codon 858 (L858R; 41%). Other less common mutations associated with sensitivity to EGFR TKIs include G719 mutations in exon 18 and L861 mutations in exon 21. Exon 19 deletions are also associated with better survival independent of EGFR TKI treatment.307 These data suggest that EGFR mutations in addition to predictive value have prognostic significance. Direct DNA sequencing is the most common method of mutational analysis, but other, more sensitive methods summarized in Table 12-19 are also used in a clinical practice.308 These assays can be performed on fresh, frozen, and archival FFPE tissue, including surgical resection specimens or FNA biopsies.309,310 Resistance mutation D790M in exon 20 has been identified in as many as 50% of patients who were initially sensitive to TKIs.311 Resistance is also associated with MET oncogene amplification, which could be detected by fluorescence in situ hybridization (FISH) in only 3% of primary naïve lung adenocarcinomas and in approximately 21% of EGFR TKI–treated adenocarcinomas that developed resistance.312,313 KRAS mutations of codon 12 are a negative predictor of lung adenocarcinoma response to EGFR TKI therapy and are an adverse prognostic factor.314,315 HER2 (3%) and BRAF (1%) mutations are rarely identified in lung adenocarcinomas but are mutually exclusive with EGFR mutations.316
Development of new targeted therapies has resulted in clinical testing in lung adenocarcinomas beyond EGFR mutational status, and some clinical laboratories are putting into practice comprehensive mutational profiles for lung adenocarcinoma (Fig. 12-39). EGFR mutations are frequently associated with increased EGFR gene copy numbers, and discussion is still ongoing about the most appropriate clinical testing for establishing EGFR status in lung adenocarcinoma, particularly gene copy-number analysis (FISH or chromogenic in situ hybridization [CISH]).317-321 The main issue with EGFR FISH is lack of standardized interpretation criteria. Conflicting results regarding prediction of tumor response to EGFR TKIs were demonstrated in multiple studies by using the University of Colorado scoring system for EGFR FISH (Table 12-21).317,319,320 Some studies also used qPCR. Increased EGFR copy number detected by FISH may be a predictor of patient response to cetuximab.322 Recent literature also explored CISH as a method of choice for detection of EGFR copy number in FFPE tissue.323 The results showed good correlation with FISH analysis, however, chromosome 7 polysomy cannot be readily distinguished from EGFR amplification. The importance of distinguishing polysomy from gene amplification is still uncertain. Several recent abstracts/articles have discussed theranostic applications in primary lung cancer. Barletta and colleagues324 studied 125 consecutive patients with lung adenocarcinomas treated at the Brigham and Women’s Hospital between 1997 and 1999. The authors found that TTF-1 protein expression identified by IHC was highly correlated with TTF1 gene amplification and was associated with a better overall survival in patients with high levels of TTF-1 expression. TTF1 gene amplification was a predictor of poor outcome. Their results indicated that TTF-1 expression by IHC and TTF1 gene amplification by FISH could be significant prognostic factors for patients with lung adenocarcinomas. Beasley and colleagues325 studied hypoxia-induced factor 1-alpha (HIF-1a) in 187 adenocarcinomas, 90 SCCs, 70 NSCLCs not otherwise specified, 40 typical carcinoids, 5 atypical carcinoids, 11 large cell NECs, and 39 small cell carcinomas. The authors found that HIF-1 was expressed in the majority of pulmonary NECs regardless of grade and was seen more frequently in NECs than in NSCLCs. The finding of HIF-1 in the majority of typical carcinoids suggested that HIF-1 was not necessarily an independent marker for aggressive behavior in NECs. In contrast, low-level expression of HIF-1 in adenocarcinomas/BAC carcinomas compared with NSCLCs was reflective of the less aggressive behavior of adenocarcinoma/BAC adenocarcinoma. However, a high level of expression of HIF-1 in NECs could indicate a role for targeted therapy. Beheshti and colleagues326 studied TTF-1 positivity as a predictor of EGFR mutation and treatment response in pulmonary adenocarcinomas. The authors found that strong, extensive TTF-1 staining was a statistically significant and sensitive predictor of EGFR mutation and response to TKI treatment. Weak or absent TTF-1 staining had a very high negative predictive value for
Theranostic Applications in Lung Neoplasms
433
Pulmonary adenocarcinoma
KRAS mutation
EFGR
HER2 mutation
BRAF mutation
PIC3CA mutation FGFR4 mutation HER4 mutation ?
MEK inhibitor
?
Mutation + amplification HER2 inhibitor
?
Gefitinib/erlotinib Acquired resistance to gefitinib/erlotinib T790M
MET amplification
Irreversible EGFR-TKI
MET inhibitor + EGFR-TKI
?
D761Y?
?
Figure 12-39 Comprehensive mutational profiles for lung adenocarcinoma. EGFR-TKI, Epidermal growth factor receptor-tyrosine factor kinase.
treatment response. The authors concluded that in centers where EGFR analysis was not available, a pathologist’s interpretation of weak/absent TTF-1 (score <2+) might help exclude patients from EGFR testing and/or TKI therapy. Kundu and colleagues327 evaluated alpha-methylacylCoA racemase (AMACR) in tissue microarrays made from 240 NSCLCs. AMACR expression in poorly differentiated large cell carcinomas of the lung was statistically associated with a poor 5-year survival in early stage patients with prior, presumably curative resection. The authors suggested AMACR expression may help stratify these patients into a category of increased risk of death from their cancer despite resection. Increased AMACR expression in lung cancers might serve as a target for molecular radiology and therapy. Perner and colleagues328 evaluated TTF1 amplification with respect to TTF-1 overexpression in 198 lung adenocarcinomas using FISH. Significantly higher TTF-1 expression was found in adenocarcinomas with TTF1 amplification compared with adenocarcinomas without amplification, with a trend toward patients with TTF1 amplification having a worse outcome than patients without it.
Vohra and colleagues329 studied IMP3 expression in 247 primary NSCLCs (187 adenocarcinomas, 90 SCCs) by IHC. IMP3 is an oncofetal RNA-binding protein and member of the insulin growth factor family, and it has been proven useful as a biomarker of disease progression in RCC. IMP3 immunostaining was found in 98 adenocarcinomas (52.4%) and 69 SCCs (76.6%). IMP3 expression in greater than 30% of tumor cells showed a strong positive trend in predicting a better 5-year survival in early stage SCCs of the lung. The authors stated this marker could be useful both in stratifying SCCs in studies of new therapies and as a potential target for molecular therapies. Cutz and colleagues330 used IHC for evaluation of epidermal growth factor and downstream effectors such as extracellular signal-regulated kinase (ERK). The authors studied 44 patients who received curative chemoradiation therapy and found the radiographic response was 50.15 ± 15% at 6 weeks post therapy with an overall survival of 14.13 ± 9.6 months. The authors observed a statistically significant negative correlation between membrane and cytoplasmic p-EGFR and overall survival. Cytoplasmic p-Erk levels correlated negatively with radiographic response. The study
434
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-21 Methods for Detecting EGFR Mutations in Lung Cancer Specimens Technique
Sensitivity (% Mutant DNA)
Mutations Identified
Comprehensive Detection of Deletions and Insertions?
Direct sequencing
25
Known and new
Yes
PCR-SSCP
10
Known and new
Yes
TaqMan PCR
10
Known only
No
Loop-hybrid mobility shift assay
7.5
Known only
Yes
Cycleave PCR
5
Known only
Yes
PCR-RFLP and length analysis
5
Known only
Yes
MALDI-TOF MS-based genotyping
5
Known only
No
PNA-LNA PCR clamp
1
Known only
No
Scorpions ARMS
1
Known only
No
dHPLC
1
Known and new
Yes
Single-molecule sequencing
0.2
Known and new
Yes
Mutant-enriched PCR
0.2
Known only
No
SMAP
0.1
Known only
No
From Pao W, Ladanyi M: Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method. Clin Cancer Res 2007;13:4954-4955. ARMS, Amplified refractory mutation system; dHPLC, denaturing high-performance liquid chromatography; MALDI-TOF MS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry; PCR, polymerase chain reaction; PNA-LNA, peptide nucleic acid–locked nucleic acid; RFLP, restriction fragment length polymorphism; SMAP, smart amplification process; SSCP, single-strand conformation polymorphism.
suggested that p-EGFR levels, and not total EGFR levels as measured by IHC, served as an independent poor prognostic factor in a subset of patients. IHC scores of cytoplasmic p-Erk appeared to predict poor radiographic response to curative chemoradiation therapy in locally advanced NSCLCs. Li and colleagues331 studied overexpression of NRF-2 and p53 in pulmonary papillary adenocarcinomas by IHC on tissue microarrays. The authors found that NRF-2 and p53 were strongly expressed in papillary adenocarcinoma cancer cells and that overexpression of NRF-2 correlated with p53 expression and was independent of tumor size and stage. The authors concluded that overexpression of NRF-2 by virtually all cancer cells in papillary adenocarcinoma differed from results in other NSCLCs and could be linked to poor prognosis and poor chemotherapeutic response. Luu and associates332 studied the prognostic value of aspartyl (asparaginyl) hydroxylase (AAH) in 375 lung carcinomas using tissue microarrays studied by IHC. Expression of AAH was associated with a poor prognosis for bronchioloalveolar cell carcinoma and SCC, and a strong trend was observed for poor prognosis for large cell undifferentiated carcinoma. Shahjahan and associates333 studied ProEx C, a biomarker reagent that contains antibodies to minichromosome maintenance protein 2 (MCM2) and topoisomerase II A (TOP2A), used to detect aberrant S-phase induction in cells. The authors studied 289 NSCLCs by IHC and found that ProEx C was expressed in more than two thirds of NSCLCs, and strong expression was associated with a longer 5-year survival in
certain cellular subtypes. The findings suggested a role in tumor progression of these cancer cells and might be a potential basis for targeted therapy. Sienko and colleagues334 evaluated expression of galectin-4 in 264 stage I and II NSCLCs by IHC. Galectin-4 was stated to be a member of the galectin family of binding proteins (β-galactosides) that play a major role in regulation of cell differentiation, proliferation, and apoptosis. The study showed that overexpression of galectin-4 was associated with a trend toward worse prognosis in early stage NSCLCs. They stated galectin-4 could prove to be a useful prognostic marker in NSCLCs and for therapeutic targets. Song and colleagues335 studied c-Met expression in pulmonary NE tumors; c-Met was stated to be a tyrosine kinase receptor that played an important role in tumor growth, invasion, metastasis, and drug resistance. The authors studied 44 carcinoids, 35 SCLCs, and 9 large cell NECs by IHC; c-Met expression was frequently observed in all three categories of NE lung tumors, supporting the potential use of c-Met as a therapeutic target in tumors, including SCLC and large cell NEC. Haque and colleagues336 studied tissue microarrays constructed from 43 SCLCs immunostained with E-cadherin and pan-cadherin. E-cadherin and pancadherin expression were seen in 40% of tumors, and a significant inverse correlation was found between E-cadherin and pan-cadherin in the expression and presence of metastasis at diagnosis. Reduced expression or lack of expression of E-cadherin and pan-cadherin in SCLCs was associated with metastasis and,
Theranostic Applications in Lung Neoplasms
consequently, a higher stage and poorer prognosis. Cadherin-mediated adhesion might be a potential therapeutic target for control of SCLC metastasis. Homan and colleagues337 studied downregulation of GADD45A protein expression in NSCLCs. Decreased expression of GADD45A was observed in 60% (69/115) of NSCLCs, including 66% of SCCs, 51% of adenocarcinomas, and 64% of BACs. Downregulation was noted in 100% of cases in which death occurred within 1 year of diagnosis and in 78% within 5 years versus 40% of cases of death beyond 5 years. The authors concluded that expression of GADD45A was significantly decreased in NSCLCs compared with adjacent nonneoplastic bronchial epithelium. SCCs of higher stage and those with decreased survival showed a downregulation of GADD45A protein expression. The results showed evidence for an association between altered GADD45A expression and tumorigenesis. Malik and coworkers338 studied MST1R, a member of the MET protooncogene family of tyrosine kinase receptors, in 175 cases of primary and metastatic lung cancer by IHC. MST1R was widely expressed and constitutively phosphorylated in all histotypes of lung cancer with a potential role as a novel therapeutic target. A positive correlation between MST1R/pMST1R expression levels, higher tumor stage, and more regional spread of tumors suggests MST1R was likely involved with a more malignant state through its proliferative, motile, and morphologic effects. MST1R provided prognostic information and helped to identify patients who required adjuvant therapy for SCLC and more extensive resection for NSCLC patients. CD117 (c-Kit) is a transmembrane tyrosine kinase receptor that has been immunolocalized in various neoplasms, the most notable of which is gastrointestinal stromal tumors (GISTs), in which c-Kit is felt to be a relatively specific and sensitive IHC marker. If a GIST expresses CD117, it is usually treated with imatinib. CD117 has been evaluated in lung and pleural neoplasms.339-342 Lonardo and associates339 evaluated c-Kit expression in primary lung cancer and mesothelioma using two antibodies with and without heat-induced epitope retrieval (HIER). Positive reactivity was observed predominantly in SCLCs and infrequently in other cancers. With the Dako antibody, 7 of 33 mesotheliomas showed immunostaining. Expression of c-Kit in SCLC suggested it plays an important role in the biology of this malignancy and could be targeted in subsets of patients for therapy by using c-Kit inhibitors. In the report by Pelosi and colleagues,340 membranous CD117 immunostaining was observed in 19 of 88 adenocarcinomas (22%) and 15 of 113 SCCs (13%). Cytoplasmic labeling was seen in 28 adenocarcinomas and 8 SCCs. In the tumors that showed membranous immunostaining for CD117, immunoreactivity was associated with a higher proliferative fraction and with features of more aggressive tumor behavior, including higher stage, size, and grade in addition to occurrence of clinical symptoms and other changes. Immunoreactive tumors exhibited increased levels of Bcl-2, cyclin E, ERBB2, p27, and fascin. CD117 immunoreactivity
435
identified a peculiar subset of stage I adenocarcinomas and SCCs of lung that could have prognostic relevance in patients whose tumors express CD117. The authors also suggested that targeting the CD117 pathway could be a novel therapeutic strategy in treating a subset of primary lung cancers. A study by Casali and colleagues341 investigated c-Kit protein overexpression in large cell NECs, because it had been observed in SCLCs and was associated with a poor prognosis. They used a polyclonal c-Kit antibody and evaluated 33 patients who had undergone radical resection. Overall, 1-, 3- and 5-year survival rates were 79%, 58%, and 51% in tumors that expressed c-Kit. Survival analysis was stated to show no difference for any clinicopathologic features except for CD117 immunostaining. The 1- and 3-year survival rates were 91% and 82%, respectively, for CD117-negative large cell NECs, and 72% and 44%, respectively, for CD117positive neoplasms. CD117 expression was associated with an elevated recurrence rate (60% vs. 23% for CD117-positive and CD117-negative large cell NECs, respectively). These authors concluded that c-Kit protein was frequently expressed in large cell NEC and represented a negative prognostic factor. Butnor and coworkers342 evaluated 61 lung/pleural cancers that included 11 small cell carcinomas, 4 large cell NECs, 22 SCCs, 23 adenocarcinomas, 11 pulmonary typical carcinoid tumors, 19 pleural malignant mesotheliomas, and 6 localized pleural fibrous tumors using a polyclonal c-Kit antibody. SCLCs demonstrated c-Kit staining in 82% of cases, nearly all of which demonstrated moderate to intense immunoreactivity. Immunostaining was observed in 25% of large cell NECs, and focal staining was observed in 9% of SCCs and in 17% of adenocarcinomas. Typical pulmonary carcinoid tumors showed no reactivity. Moderately intense immunostaining was noted in 50% of localized fibrous tumors of the lung, and malignant mesotheliomas were nonreactive for c-Kit in 95% of cases. The authors concluded that the high frequency of c-Kit immunostaining in SCLC could have important potential therapeutic implications. Maddau and associates343 evaluated the expression of p53 and Ki-67 in NSCLC using IHC detection. Overexpression of p53 was associated with a significantly worse patient outcome in stage I disease, whereas no excess risk was evident in stage II and III disease. The same pattern was observed with Ki-67 expression. Excess risk in stage I cases with p53 and Ki-67 overexpression was observed only in adenocarcinoma. Kobayashi and colleagues344 evaluated endogenous secretory receptor for advanced glycation endproducts in 182 NSCLC surgical specimens. Endogenous secretory receptor for advanced glycation endproduct expression in cytoplasm was reduced or absent in 75% (137/182) of carcinomas in contrast to normal lung tissue, and mRNA expression was also suppressed in the cancer cells. Among patients with low expression of the cytoplasmic secretory receptor, the overall survival rate was significantly lower than that of patients with normal expression. Cytoplasmic end ogenous secretory receptor for advanced glycation end product expression has
436
Immunohistology of Lung and Pleural Neoplasms
the potential to be a prognostic factor for predicting outcome of curative surgery in patients with NSCLC. Allen and coworkers345 studied the expression of glutathione S-transferase (GST)-π and glutathione synthase in 201 NSCLCs by IHC with antibodies against GST-π and GSH2 using standard immunostaining techniques. Nuclear staining with GST-π in greater than 10% of cells was closely associated with decreased survival in stage I and II SCCs (n = 40). Cytoplasmic staining showed a similar trend that did not reach statistical significance. No significant correlation between GST-π staining and survival was determined for other histologic types of NSCLC. Cytoplasmic GSH2 staining in greater than 80% of tumor cells was associated with a trend toward improved survival for stage I adenocarcinoma (p = .08) but did not show a relationship to survival for other histologic types. GST-π expression predicted prognosis in stage I and II squamous cell lung cancer, and GSH2 expression may indicate better survival in early stage adenocarcinoma of the lung. Manipulation of GST-π and GSH2 had the potential basis for treatment of some NSCLCs. Lee and colleagues346 studied the significance of extranodal expression of regional lymph nodes in surgically resected NSCLC. The authors studied 199 NSCLC patients who were found to have regional lymph node involvement after resection. Histologic examination included tumor cell type, grade of differentiation, vascular invasion, regional lymph node metastasis emphasizing the number and station of lymph node involvement, the presence or absence of extranodal extension, and the IHC of p53 expression. Extranodal extension was significantly higher in women and in adenocarcinoma, advanced stages, tumors with vascular invasion, or p53 overexpression. Multivariate analysis of survival, presence and total number of lymph nodes with extranodal extension, tumor stage, and p53 expression were significant prognostic factors. Fukuoka and associates31 studied desmoglein 3, a desmosomal protein of the cadherin family, in primary NSCLCs and NE tumors by IHC analysis. Negative IHC staining with desmoglein 3 was associated with a shorter survival for all lung cancer patients, regardless of histologic type (5-year survival of 20.9% vs. 49.5%, P < .001). In patients with atypical carcinoid tumors that lacked desmoglein 3 expression, the 5-year survival rate was 0% compared with 36.8% for cases that showed expression of desmoglein 3. Desmoglein 3 status indicated a poor prognosis in lung cancers and portended a more aggressive behavior for atypical carcinoid tumors. Aviel-Ronen and colleagues347 studied glypican-3 expression in lung cancer at the protein and mRNA levels and correlated these findings with clinical, histologic, and genomic characteristics such as RAS mutation status. They performed these studies using IHC on tissue microarrays in 97 patients. Glypican-3 immunostaining was stated to have been negative in all normal lung tissues and was positive in 23% of lung carcinoma samples. High protein and mRNA expression was associated with squamous histology. Among smokers, glypican-3 mRNA expression was reduced in adenocarcinoma patients and elevated in those with SCC. Patients
with tumors that stained positively for glypican-3 smoked significantly more than patients with tumors that stained negatively. No association was found between glypican-3 expression and patient outcome. The authors concluded that glypican-3 was overexpressed in cancerous versus normal lung tissue and that adenocarcinoma and SCC had differential expression of glypican-3, with a predilection for SCC patients who smoked. Glypican-3 expression in SCC as an oncofetal protein was stated to render it a potential candidate marker for early detection of squamous cell lung cancer. LaPoint and associates348 evaluated coexpression of c-Kit and Bcl-2 in small cell carcinoma and large cell NEC of the lung. Using a polyclonal antibody against c-Kit and a monoclonal antibody against Bcl-2, the authors found that 100% (7 of 7) SCLCs were positive for c-Kit and Bcl-2. Of 14 large cell NECs, 7 (50%) immunostained for c-Kit and 9 of 14 (64%) expressed Bcl-2. All cases of high-grade NECs (SCLCs and large cell NECs) that expressed c-Kit coexpressed Bcl-2. In contrast, all typical and atypical carcinoids were negative for c-Kit, and only 6.3% (1/16) of typical carcinoids and 16.7% (1/6) of atypical carcinoids stained positive for Bcl-2. Progressive increase of c-Kit and Bcl-2 expression and coexpression from carcinoid tumors and atypical carcinoids to large cell NECs and SCLCs was noted. High-grade NECs were more likely coexpress c-Kit and Bcl-2 when compared with carcinoid tumors. The high expression and coexpression of these two molecules in high-grade NECs suggested they may be involved in the carcinogenic pathway and that therapeutic targeting for c-Kit and Bcl-2 molecules might be beneficial in the management of patients with highgrade NECs. However, c-Kit mutations were not part of this study. Meyronet and associates349 pointed out that highgrade NECs show unfavorable outcome and should receive multimodal therapy. The authors evaluated 123 NE lung tumors and 41 randomly selected non-NE tumors. CRMP5 expression in tumors, metastases, and healthy lung tissue was assessed using immunostaining methods. Strong and extensive CRMP5 expression was seen in 98.6% of high-grade NE lung tumors, including SCLC and large cell NE cancer, but not in pulmonary SCC or pulmonary adenocarcinomas. In contrast, the majority of low-grade NE lung tumors were negative for CRMP5 staining, although weak CRMP5 expression was seen in some, with two different staining patterns. Their findings suggest CRMP5 is a novel marker for routine pathologic evaluation of lung tumor surgical samples in distinguishing between highly aggressive NEC and other lung cancers. Yousem and colleagues350 evaluated 220 adenocarcinomas of lung lacking KRAS and EGFR mutations and identified 10 adenocarcinomas with BRAF V600E mutations. All BRAF V600E mutations were heterozygous. A slight female predilection (6 : 4) was reported in these elderly patients, whose age averaged 67 years, and they were found to have a greater than expected incidence of intralobar satellite nodules and N2 nodal involvement. The adenocarcinomas were described as being largely of mixed type with a high incidence of
Pleural Neoplasms
437
papillary (80%) and lepidic growth (50%). Adenocarcinomas with this clinicopathologic phenotype might be worthwhile investigating for BRAF V600E mutations as more genetically oriented drug therapies emerge.
Pleural Neoplasms Neoplasms of the pleura are relatively common, with more metastatic than primary tumors involving the pleura.224,351 The differentiation of primary from metastatic pleural neoplasms has received extensive attention in the discipline of IHC. In part, this is due to an increased understanding of the biology of the serosal membranes and the pathology/pathobiology of mesotheliomas. The celomic cavity develops relatively early in embryogenesis and gives rise to the pleural, peritoneal, and pericardial cavities by partitioning membranes that divide it.352 Mesotheliomas arise from the serosal tissue of the body cavities. The biology and morphology of the normal pleura has been extensively studied. In the resting state, the pleura is composed of a layer of relatively flattened mesothelial cells separated from the underlying connective tissue components by a basement membrane (Fig. 12-40). The sub–basement membrane cells are spindle shaped and are associated with elastic tissue and collagen that is best seen in elastic tissue–stained sections. Epithelial mesothelial cells and pleural spindle cells show a marked reaction to injury
and an increase in number and size (Fig. 12-41). The pleural spindle cells are interesting in that they express keratin, vimentin, actin, and calretinin by IHC (Fig. 12-42), and ultrastructurally they have the appearance of myofibroblasts (Fig. 12-43). In 1986 we353 extensively studied the pleura and its reaction to injury and related the histologic, IHC, and ultrastructural features of mesotheliomas to the reactive pleural cells. Although doubted by some, there is evidence that epithelial mesothelial cells are derived from a proliferation of
Figure 12-40 In the resting state, the normal pleura is composed of a slightly flattened layer of mesothelial cells with underlying spindle cells, collagen, and elastic tissue (×200).
Figure 12-42 The proliferating myofibroblasts of the pleura immunostain keratin, vimentin, actin, and calretinin. This photograph shows immunostaining for calretinin (×400).
Figure 12-41 When injured, the pleura shows hypertrophy and hyperplasia of lining mesothelial cells and a proliferation of underlying spindle cells that have features of myofibroblasts (×400).
438
Immunohistology of Lung and Pleural Neoplasms
Figure 12-43 These proliferating spindle cells have ultrastructural features of myofibroblasts (×40,000).
sub–basement membrane spindle cells that differentiate into epithelial-mesothelial cells. We named these cells multipotential-subserosal cells. Except for a cohort of insulators studied by Selikoff and Seidman,354 pleural mesotheliomas account for approximately 90% of all mesotheliomas and show a wide range of histologic differentiation, although they can be grouped into four major subtypes: 1) epithelial, 2) sarcomatoid (fibrous, sarcomatous), 3) biphasic, and 4) desmoplastic, a variant of a sarcomatoid mesothelioma. IHC is the predominant technique used for accurately diagnosing mesotheliomas and differentiating them from primary lung cancers that invade the pleura and from primary lung cancers and neoplasms outside of the chest cavity that metastasize to pleura. The four major histologic types of pleural mesothelioma represent a marked oversimplification of what exists. For example, approximately 20 different epithelial subtypes have been defined (Box 12-2; Fig. 12-44, A-W). A well-differentiated papillary form of mesothelioma exists in the peritoneal cavity and pleural cavity that is important to recognize, because unlike other mesotheliomas, it is usually clinically benign or has a low malignant potential.355-357 In addition, serosal membranes are extremely reactive tissues and show a variety of changes when injured and can be misinterpreted as malignant.358 IHC may not be helpful diagnostically in differentiating atypical reactive pleural processes from malignant ones, but it is often helpful in identifying cells in these processes as either epithelial-mesothelial cells or as pleural spindle cells.359 As discussed later in this chapter, IHC studies have been used to separate benign from malignant processes, such as atypical mesothelial hyperplasia from
epithelioid mesothelioma, and whether mesothelial cells are reactive or neoplastic.360,361 There are also a significant number of reports in the literature that have tried to determine whether a serosal membrane cell is mesothelial or nonmesothelial, the most recent of which was by Anttila, published in 2012.362 A variety of antibodies have been used to understand and help diagnose mesotheliomas. They can be divided into three main categories: 1) antibodies that are relatively specific for mesothelial cells and mesotheliomas, which when positive serve as a positive marker for mesotheliomas; 2) antibodies that show no reaction for mesothelial cells or mesotheliomas and when negative serve as a negative marker for mesotheliomas; and 3) other antibodies that may react with mesothelial cells and mesotheliomas but are relatively nonspecific. The selection of a panel of antibodies to evaluate a suspected mesothelioma varies depending on the type of differentiation the suspected mesothelioma shows. Antibodies frequently used in diagnosing suspected mesotheliomas are listed and characterized in Table 12-22. At the eleventh International Conference of the International Mesothelioma Interest Group held in Boston, Massachusetts, in September 2012, Le Stang and colleagues363 presented a meta-analysis based on routinely used IHC markers for the diagnosis of pleural epithelioid malignant mesothelioma. They incorporated the results of 99 papers published in PubMed and medicine electronic literature databases between 1979 and 2010. All in all, 3037 malignant mesotheliomas and 3165 lung adenocarcinomas were studied. Markers for EMA, human milk fat globule protein 2 (HMFG-2), Text continued on p. 447
Box 12-2 EPITHELIAL MESOTHELIOMA SUBTYPES Adenoid cystic Adenomatoid Bakery roll Clear cell Deciduoid Gaucher-like Glandular/acinar Glomeruloid Histiocytoid/epithelioid In association with excess amounts of hyaluronic acid or proteoglycan In situ Macrocystic Microcystic Mucin positive Placentoid Pleomorphic Poorly differentiated Rhabdoid Signet ring Single file Small cell Tubulopapillary Well-differentiated papillary
Pleural Neoplasms
A
B
C1
C2
D1
D2
E1
E2 Figure 12-44 Histologic variants of epithelial mesothelioma are shown in parts A through W.
439
440
Immunohistology of Lung and Pleural Neoplasms
F1
F2
F3
G
H1
H2
I
J1 Figure 12-44, cont’d
Pleural Neoplasms
J2
K1
K2
L
M1
M2
N1
N2 Figure 12-44, cont’d
441
442
Immunohistology of Lung and Pleural Neoplasms
N3
O1
O2
P
Q
R1
R2
S Figure 12-44, cont’d
Pleural Neoplasms
T
U1
U2
V
W1
W2 Figure 12-44, cont’d
443
444
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-22 Antibodies Used to Confirm, Eliminate, or Classify Suspected Mesotheliomas Antibody Directed Against
Clone
Keratin
AE1/AE3
Keratin
Characteristics of Antigens Recognized
Immunogen
Manufacturer
Dilution
Keratins: Moll numbers 1-5, 6, 8, 9, 10, 14-16, 18
Human epidermal keratin
Dako
1 : 200
MAK-6
Keratins: Moll numbers 8, 14-16, 18, and 19
Extracellular antigen from MCF-tissue culture and from human sole epidermis
Zymed
1 : 100
Keratin
5D3
Keratins: Moll numbers 8 & 18
Colorectal carcinoma cell line
BioGenex
1 : 100
Keratin
35βH11
Keratins: Moll number 8
Hep3B hepatocellular carcinoma cell line
Dako
1 : 50
Keratin
34ßE12
Keratins: Moll numbers 1, 5, 10, and 14
Human stratum corneum keratin
Dako
1 : 100
CK5/CK6
D5/16B4
Keratins: Moll numbers 5, 6, and, to a slight degree, 4
Purified cytokeratin 5
Biocare Medical
1 : 100
CK7
OV-TL 12/30
Keratins: Moll number 7
OTN 11 ovarian carcinoma cell line
Cell Marque
NA
CK20
Ks20.8
Keratins: Moll number 20
Villi of human duodenal mucosa
Cell Marque
NA
Vimentin
Vim3B4
Intermediate filament 57 kD
Vimentin from bovine eye lens
Dako
1 : 100
α-Actin
1A4
α-Smooth muscle isoform of actin
N-terminal decapeptide of human α-smooth muscle actin
Cell Marque
NA
MSA
HHF-35
42-kD protein in preparations of purified skeletal muscle actin and extracts of aorta, uterus, diaphragm, and heart
SDS extracted protein fraction of human myocardium
Cell Marque
NA
Desmin
NCLDE-R-11
53-kD intermediate filament in muscle cells, recognizing 18-kD rod piece of molecule
Desmin purified from porcine stomach
Ventana
NA
Calretinin
—
29-kD calcium-binding protein
Human recombinant calretinin
Cell Marque
NA
Mesothelioma antigen
HBME-1
Antigen present in membrane of mesothelial cells
Suspension of human mesothelial cells from malignant epithelial mesothelioma
Dako
1 : 400
Thrombomodulin
1009
Transmembrane glycoprotein of 75 kD molecular weight containing 6 repeated domains homologous with epidermal growth factor
Recombinant thrombomodulin
Dako
1 : 50
EMA
E29
250-400 kD glycoprotein of milk fat globule protein family
Delapidated extract of human milk fat
Ventana
NA
HMFG-2
115D8
MAM-6 mucus glycoprotein of >400 kD in glycocalyx of epithelial cells
Purified HMFG protein
BioGenex
1 : 25
N-cadherin
389
Transmembrane glycoprotein involved in calciumdependent cell adhesion
Intracellular domain of chicken N-cadherin
Zymed
1 : 100
pCEA
—
CEA and CEA-like proteins including nonspecific cross-reacting substance and biliary glycoprotein
Human CEA isolated from metastatic colonic adenocarcinoma
Ventana
NA
Pleural Neoplasms
445
TABLE 12-22 Antibodies Used to Confirm, Eliminate, or Classify Suspected Mesotheliomas—cont’d Antibody Directed Against
Clone
Characteristics of Antigens Recognized
CD15 (LeuM1)
C3D-1
TAG
Immunogen
Manufacturer
Dilution
3-Fucosyl-N-acetyllactosamine
Purified neutrophils from normal human peripheral blood
Ventana
NA
B72.3
Tumor-associated glycoprotein of a wide variety of human adenocarcinomas
Membrane-enriched fraction of metastatic breast carcinoma
Cell Marque
NA
Human epithelial antigen
VU-1D9
34- and 49-kD glycoproteins on the surface and in cytoplasm of most epithelial cells, except squamous epithelium, hepatocytes, and parietal cells
MCF-7 cell line
Ventana
NA
Thyroglobulin
2H11+6E1
Thyroglobulin
Thyroglobulin from human thyroid glands
Cell Marque
NA
TTF-1
8G7G3/1
40-kD member of NKx2 family of homeodomain transcription factors
Mouse ascites
Cell Marque
NA
PSA
ER-PR8
33-kD prostate-specific antigen
Purified human PSA
Ventana
NA
PAP
PASE/4LJ
52-kD human prostatic acid phosphatase
Purified PAP from human seminal plasma
Ventana
NA
Human epithelialrelated antigen
MOC-31
40-kD transmembrane glycoprotein present on most normal and malignant epithelial cells
Neuraminidase-treated cells from small cell carcinoma cell line
Dako
1 : 50
Lewis Y antigen
BG8-F3
Difucosylated tetrasaccharide found on type 2 blood group oligosaccharide
SK-LU-3 lung cancer cell line
Signet
1 : 40
E-cadherin
4A2C7
Transmembrane glycoprotein in calcium-dependent cell adhesion
Recombinant protein of human E-cadherin
Ventana
NA
GCDFP-15 (BRST-2)
D6
Pathologic secretion of breast composed of several glycoproteins including 15-kD monomer protein
GCDFP-15
Signet
1 : 50
Estrogen receptor protein
1D5
66-kD protein member of nuclear hormone receptor family that acts as ligandactivated transcription factor
Human recombinant estrogen receptor protein
Ventana
NA
Erbb2 oncoprotein
—
190-kD protein product of Erbb2 protooncogene
Synthetic human Erbb2 oncoprotein peptide
Dako
1 : 500
HLA, CD45
DAKOLCA
Five or more high-molecularweight glycoproteins on the surface of the majority of human leukocytes
Human peripheral blood lymphocytes maintained in T-cell growth factor
Ventana
NA
CD20 Human B-lymphocyte antigen
L26
33-kD nonglycosylated membrane-spanning protein
Human tonsil B lymphocyte
Ventana
NA
CD3 Human T-lymphocyte antigen
—
Intracytoplasmic portion of CD3 antigen
Synthetic human CD3 peptide
Ventana
NA
Continued
446
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-22 Antibodies Used to Confirm, Eliminate, or Classify Suspected Mesotheliomas—cont’d Antibody Directed Against
Clone
Characteristics of Antigens Recognized
Immunogen
Manufacturer
Dilution
CD30 Ki-1 antigen
Ber-H2
120-kD transmembrane glycoprotein
Co–cell line cells
Ventana
NA
Bcl-2 oncoprotein
124
25-kD integral protein localized in mitochondria that inhibits apoptosis
Synthetic peptide sequence amino acids 41-54 of Bcl-2 protein
Ventana
NA
NSE
—
Gamma subunit of enolase
NSE isolated from human brain
Dako
1 : 400
Chromogranin A
LK2H10
Member of secretogranin/ chromogranin class of proteins in secretory granules of endocrine and neuron cells
C-terminal 20-kD fragment of chromogranin A
Ventana
NA
Synaptophysin
—
38-kD membrane component of neuron synaptic vesicles
Synthetic human synaptophysin peptide coupled to ovalbumin
Cell Marque
NA
S-100 protein
—
S-100 proteins A and B
S-100 protein isolated from cow brain
Ventana
NA
Melanoma antigen
HMB-45
Neuraminidase-sensitive oligosaccharide side chain of glycoconjugate in immature melanosomes
Extract of pigmented melanoma metastases from lymph nodes
Dako
1 : 200
CD34
My10
105- to 120-kD single-chain transmembrane glycoprotein associated with human hematopoietic progenitor cells
CD34 antigen
Ventana
NA
CD31
JC/70A
100-kD glycoprotein in endothelial cells and 130-kD glycoprotein in platelets
Membrane preparation of spleen from patient with hairy cell leukemia
Cell Marque
NA
Factor VIII antigen
—
Human von Willebrand factor
von Willebrand factor isolated from human plasma
Cell Marque
NA
Mesothelin
5B2
Amino acids present in the mesothelin molecule
Mouse myeloma
Novo Castra
NA
WT1
6F-H2
Suppressor gene on chromosome 11p13
Tissue culture supernatant of mouse antibodies
Cell Marque
NA
D2-40
D2-40
Oncofetal antigen M2A
Mouse ascites
Signet
NA
SP-A
PE-10
Surfactant A
Surfactant apoproteins isolated from lung lavages of patients with alveolar proteinosis
Dako
1 : 100
CDX-2
CDX2-88
Homeobox family of intestinespecific transcription factor, regulates proliferation and differentiation of intestinal epithelial cells
Full-length CDX-2
BioGenex
NA
p63
4A4
Human p63 protein, a member of the p53 family
Mouse monoclonal antibody
Cell Marque
1 : 100
Antigens were obtained by heat-induced epitope retrieval (HEIR). EMA, Epithelial membrane antigen; GCDFP-15, gross cystic disease fluid protein 15; HLA, human leukocyte antigen; HMFG-2, Human milk fat globule protein 2; MSA, muscle-specific actin; NA, not applicable; NSE, neuron-specific enolase; PAP, prostatic acid phosphatase; pCEA, polyclonal carcinoembryonic antigen; PSA, prostate-specific antigen; SP-A, surfactant apoprotein A; TAG, tumor-associated glycoprotein; TTF-1, thyroid transcription factor 1.
Pleural Neoplasms
vimentin, AE1/AE3 keratin, thrombomodulin, mesothelin, calretinin, HBME-1, WT1, CK5/6, E-cadherin, N-cadherin, D2-40, CEA, BerEP4, CD15, MOC-31, TTF-1, and Bg8 (blood group 8) were evaluated on lung adenocarcinomas, epithelioid mesotheliomas, and the epithelioid component of biphasic mesotheliomas for the systematic analysis. Markers performed on cytology specimens were not analyzed. Publications that analyzed fewer than 10 tumors, abstracts, or case reports were excluded. Because of high variation among publications as to the percentage of staining considered positive, the sensitivity and specificity were calculated at five cutoffs: 1%, 10%, 25%, 50%, and 75%. Clopper and Pearson’s method was used to calculate 95% confidence intervals,363 and the results of their study are shown in Table 12-23. These results suggest the most sensitive panel to be used in the differential diagnosis of epithelioid mesothelioma versus metastatic adenocarcinoma includes CK5/6, monoclonal CEA (mCEA), MOC-31, WT1, and Bg8 evaluated at 1% staining cells cutoff; thrombomodulin, D2-40, and B72.3 at 10% of tumor cells cutoff; and calretinin and BerEP4 at 25% cutoff. In 2012, Betta and others360 reviewed the IHC features of 286 consecutive malignant mesotheliomas (211 epithelioid, 47 mixed with a predominant epithelioid component, and 28 sarcomatoid) from 459 cases of pleural pathology (67 pleural metastatic carcinomas and 106 benign serosal lesions) diagnosed between 2003 and 2009. The summary of their results is shown in Table 12-24. The authors also evaluated the sensitivity and specificity for mesothelioma and carcinoma markers, comparing the data between their series360 and that reported in the latest literature (King et al. 2006,364 Yaziji et al. 2006,365 Kushitani et al.,366 and Klebe et al. 2009367; Table 12-25).
Biology of Antigens and Antibodies We use a battery of antibodies for evaluating mesothelial proliferative lesions, including reactive and neoplastic processes. Keratin antibodies, with the exception of CK5/6, are generally not used to differentiate an epithelial mesothelioma from another neoplasm or from a reactive process, but they are used to identify the extent of a neoplastic or reactive mesothelial cell process. The antibodies we use to differentiate a well-differentiated or moderately well-differentiated epithelial mesothelioma from a pulmonary adenocarcinoma or nonpulmonary adenocarcinoma include AE1/AE3 cytokeratin, CK5/6, CK7, CK20, vimentin, EMA, HBME-1, calretinin, mesothelin, WT1, D2-40, caldesmon, CEA, Leu-M1, B72.3, BerEP4, and TTF-1. Yaziji and colleagues365 evaluated 133 neoplasms by IHC, including 65 epithelial mesotheliomas, 22 lung adenocarcinomas, 27 ovarian serous carcinomas, 24 breast carcinomas, and 5 gastric carcinomas. Calretinin was found to have the best sensitivity for mesothelioma (95%) followed by HBME-1 (84%), WT1 (78%), CK5/6 (76%), mesothelin (75%), vimentin (69%), and thrombomodulin (68%). Thrombomodulin had the best specificity for mesothelioma (92%), followed by CK5/6 (89%), calretinin (87%), vimentin (84%), and HBME-1
447
(48%). When ovarian carcinomas were excluded from the analysis, the specificity of mesothelin and WT1 for the diagnosis of mesothelioma increased to 90% and 81%, respectively. The authors found the sensitivity of the nonmesothelial antigens for adenocarcinoma was organ dependent, with Bg8 performing best in the breast cancer group (96%), and BerEP4, Bg8, and MOC-31 performing best in the lung cancer group (100%). The specificity of the nonmesothelial antigens for adenocarcinoma was 98% for Bg8 and CEA, 97% for CD15, 95% for BerEP4, and 87% for MOC-31. A statistical analysis technique that used logic regression analysis identified a three-antibody IHC panel that included calretinin, Bg8, and MOC-31, which provided more than 96% sensitivity and specificity for distinguishing epithelioid mesothelioma from adenocarcinoma. Marchevsky368 also reviewed the application of IHC to the diagnosis of malignant mesothelioma by including two or more epithelial markers used to exclude the diagnosis of carcinoma—such as monoclonal and polyclonal CEA, BerEP4, B72.3, CD15, MOC-31, TTF-1, and Bg8—and two or more mesothelial markers used to confirm the diagnosis, such as CK5/6, calretinin, HBME-1, WT1, mesothelin, thrombomodulin, and podoplanin (D2-40). These data are summarized in Table 12-26. With Bayesian statistics King and colleagues364 showed the use of panels composed of only two antibodies, one mesothelial and one epithelial, such as WT1 and TTF-1, or two epithelial epitopes, such as MOC-31 and TTF-1, provided the best odds ratio for the differential diagnosis between epithelial mesothelioma and pulmonary adenocarcinoma. The immunohistogram of a well-differentiated to moderately well-differentiated epithelial mesothelioma is shown in Figure 12-45. A comparison of the IHC profile of well-differentiated to moderately welldifferentiated epithelial mesothelioma and pulmonary adenocarcinoma is shown in Table 12-27. POSITIVE MARKERS
Keratins are found in nearly 100% of mesotheliomas. Keratin antibodies are used primarily to 1) identify neoplastic mesothelial cells; 2) determine invasion in suspected mesotheliomas; 3) diagnose sarcomatoid mesotheliomas; and 4) differentiate sarcomatoid mesothelioma from sarcoma, localized fibrous tumors of the pleura, and other neoplasms that are usually keratinnegative. LMW and HMW keratins are detectable in most mesotheliomas, especially LMW cytokeratins (CAM5.2, 35βH11). We use AE1/AE3 keratin as a broad-spectrum keratin screening antibody. Cytokeratin expression in mesotheliomas has been reviewed by Henderson and colleagues369 and by Ordonez.370 In 1985, Blobel and associates371 reported that normal and neoplastic mesothelial cells express CK7, CK8, CK18, and CK19—cytokeratins typically seen in simple epithelial cells and in adenocarcinomas. They found that some epithelial mesotheliomas contained CK4, CK14, CK16, and CK17. In a previous publication,372 it was reported that adenocarcinomas contained CK7, CK8,
448
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-23 Immunohistochemical Markers for the Diagnosis of Pleural Epithelioid Malignant Mesothelioma 1% Cells Cutoff Sensitivity (%)
95% CI
Specificity (%)
10% Cells Cutoff 95% CI
Sensitivity (%)
95% CI
Specificity (%)
95% CI
Mesothelial Markers AE1/AE3 keratin
100
97-100
1
0-5
—
—
—
CK5/6
86
83-90
87
83-91
81
74-87
93
84-98
HBME-1
86
82-89
35
28-42
85
80-90
79
70-86
Calretinin
94
92-96
82
79-85
87
82-90
93
91-95
Vimentin
62
57-66
78
72-82
64
57-71
75
70-79
EMA
85
81-88
1
0-4
84
91-93
0
0-7
WT1
83
78-87
93
90-96
67
56-77
95
83-100
D2-40
95
90-97
63
61-73
80
73-87
93
88-96
Mesothelin
91
87-95
49
43-54
77
67-86
59
53-66
HMFG-2
48
37-58
14
9-20
—
Thrombomodulin
76
72-79
80
77-83
86
N-cadherin
71
63-79
72
63-79
—
CEA
89
86-92
95
94-97
82
pCEA
89
85-92
93
91-95
mCEA
89
86-92
97
CD15 (LeuM1)
76
72-79
BerEP4
98
B72.3
75
MOC-31
—
—
—
—
90
86-93
—
—
77-85
99
97-100
74
67-80
99
96-100
95-99
87
82-91
99
96-100
92
90-94
72
66-77
94
90-97
96-100
89
86-91
96
78-100
93
87-97
71-79
91
88-93
80
72-87
100
98-100
95
90-98
95
91-98
94
88-97
88
80-94
TTF-1
79
74-83
100
99-100
75
70-79
100
98-100
Bg8
95
89-99
92
87-96
82
75-88
98
92-100
E-cadherin
88
83-92
68
61-74
—
—
—
80-90 —
Glandular Markers
—
/A
CK
AE 1
5/ 6 CK 7 CK 20 TT F HB -1 M Ca E-1 lre ti M es nin ot he lin W T1 D2 -4 0 EM HM A FG -2 CD p15 CE A (L eu M 1) B7 2. Be 3 rE P4
100 90 80 70 60 50 40 30 20 10 0 E3
Percent positive
CEA, Carcinoembryonic antigen; CI, confidence interval; EMA, epithelial membrane antigen; HMFG-2, human milk fat globule (protein) 2; mCEA, monoclonal CEA; pCEA, polyclonal CEA; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor 1.
Figure 12-45 Immunohistogram of a well to moderately differentiated epithelial mesothelioma. CK, Cytokeratin; TTF-1, thyroid transcription factor 1; HMFG, human milk fat globule 1; EMA, epithelial membrane antigen; CEA, carcinoembryonic antigen.
Pleural Neoplasms
25% Cells Cutoff Sensitivity (%)
95% CI
Specificity (%)
50% Cells Cutoff 95% CI
Sensitivity (%)
95% CI
Specificity (%)
08
92
85-96
1
449
75% Cells Cutoff 95% CI
Sensitivity (%)
95% CI
Specificity (%)
0-8
93
80-99
0
95% CI
96
92-99
1
76
70-81
98
95-100
63
58-69
100
98-100
54
46-62
100
97-100
91
85-95
40
28-52
80
70-88
56
44-68
68
58-78
70
58-80
93
89-95
94
91-97
83
79-86
95
93-97
69
63-75
100
98-100
41
34-47
85
78-90
16
11-22
98
94-100
0
0-4
100
97-100
79
74-84
2
0-5
63
57-69
11
7-15
38
31-45
30
22-37
74
67-80
100
97-100
51
44-58
100
97-100
32
24-41
100
96-100
78
66-88
100
89-100
52
44-60
100
97-100
34
25-44
100
94-100
80
74-86
77
70-84
65
58-72
87
80-93
48
38-58
98
91-100
26
13-43
27
14-42
12
5-23
52
39-65
3
0-14
84
70-94
51
45-57
95
90-98
28
23-33
99
96-100
10
6-16
100
97-100
58
49-57
79
70-86
37
29-47
85
77-91
16
9-24
98
93-100
81
76-85
98
97-100
69
64-73
100
99-100
52
45-59
100
99-100
76
70-82
98
96-99
68
62-75
100
98-100
37
25-50
100
98-100
89
83-94
86
80-91
69
62-75
100
98-100
58
50-66
100
97-100
68
61-74
99
97-100
50
43-57
100
99-100
32
25-39
100
99-100
99
95-100
98
95-99
97
94-99
100
98-100
58
50-65
100
98-100
64
57-51
98
96-99
35
29-42
100
99-100
15
11-21
100
99-100
94
87-99
96
92-99
81
71-89
100
97-100
40
29-51
100
97-100
69
62-76
100
98-100
56
50-62
100
98-100
28
21-36
100
98-100
85
70-95
100
98-100
81
68-91
100
97-100
45
29-62
100
96-100
71
64-78
86
81-91
51
43-58
93
89-96
24
18-32
96
91-98
CK18, and CK19, and squamous carcinomas showed a more complex cytokeratin pattern, containing both simple epithelial cytokeratins (CK7, CK8, CK18, and CK19) and stratified epithelium-type keratins, specifically CK5/6. In 1989, Moll and coworkers373 used antibody AE14 to demonstrate CK5 reactivity in 12 of 13 epithelial and biphasic mesotheliomas, but such reactivity was found in 0 of 21 pulmonary adenocarcinomas. They concluded that CK5 was a helpful marker in distinguishing between pulmonary adenocarcinomas and epithelial-biphasic mesotheliomas. The AE14 antibody, unfortunately, did not work on paraffin-embedded tissue, and it was not until 1997 that Clover and associates374—using commercial monoclonal antibody D5/16B4, which reacted with CK5 and CK6 in FFPE
0-15
tissue—obtained reactivity in 23 of 23 epithelial mesotheliomas. Reactivity was observed in 18.5% (5/27) of pulmonary adenocarcinomas. In 80% (4/5) of pulmonary adenocarcinomas, the reactivity was described as weak or equivocal; in one, it was described as patchy. Using the same antibody as Clover and colleagues,374 Ordonez370 found positive staining in 40 of 40 epithelial mesotheliomas, 15 of 15 squamous carcinomas of lung, and 0 of 30 pulmonary adenocarcinomas. Focal or weak reactivity was noted in 15.1% (14/93) of nonpulmonary adenocarcinomas; specifically 33.3% (10/30) of ovarian adenocarcinomas, 20% (2/10) of endometrial adenocarcinomas, 5.6% (1/18) of breast carcinomas, 14.3% (1/7) of thyroid carcinomas, 0 of 10 renal carcinomas, 0 of 10 colonic adenocarcinomas, and 0 of 8 prostatic
450
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-24 Immunohistochemical Markers for Malignant Mesothelioma Versus Pleural Metastatic Adenocarcinoma Malignant Mesothelioma (Positive/Total No. of Cases)
Pleural Metastatic Adenocarcinoma (Positive/ Total No. of Cases)
Sensitivity (%)
Specificity (%)
Positive Predictive Value (%)
Mesothelial Markers Keratin 5
138/139
7/36
99
81
95
Calretinin
248/252
4/36
98
89
98
WT1
43/44
5/18
98
72
90
Podoplanin (D2-40)
27/30
0/18
90
100
100
Carcinoma Markers pCEA
0/245
31/34
100
95
100
CD15 (LeuM1)
4/53
20/27
92
74
83
13/219
29/31
94
94
69
MOC-31
0/39
16/21
100
76
100
TTF-1
0/44
20/31
100
65
100
BerEP4
pCEA, Polyclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor protein 1.
adenocarcinomas. Cytokeratin profiles of epithelial mesothelioma, pulmonary adenocarcinoma, and SCC of lung are contrasted in Table 12-28. Kahn and associates375 reported a difference in the pattern of keratin distribution in benign and malignant mesothelial cells compared with adenocarcinomas. In mesothelial cells, they observed keratin filaments in a perinuclear or peripheral distribution, whereas in adenocarcinomas, they showed an arborizing pattern. Their report was followed by a more extensive study,376 in which 10 adenocarcinomas, 10 typical carcinoids, and 4 mesotheliomas were evaluated for keratin intermediate filament distribution by using three monoclonal and three polyclonal antibodies against keratin. When they allowed the diaminobenzidine color reaction to proceed for less than 2 minutes, they observed a weblike pattern of reactivity in adenocarcinomas, a punctate crescent pattern in carcinoids, and perinuclear staining pattern in mesotheliomas. Although the perinuclear distribution of keratin intermediate filaments is common in epithelial mesotheliomas, it is not seen in all cases, and some pulmonary adenocarcinomas show a perinuclear distribution of keratin. We do not use the “distribution pattern” of keratin diagnostically. Although 100% of epithelial mesotheliomas express keratin, the percentage of sarcomatoid mesotheliomas reported in the literature to express keratin is variable. Montag and colleagues377 detected keratins in 16 of 16 sarcomatoid mesotheliomas, findings identical to Battifora,378 who observed keratins in 100% of over 20 sarcomatoid mesotheliomas examined. In contrast, some investigations have failed to detect keratin in more than 40% of sarcomatoid mesotheliomas,371,379-385 and the US-Canadian Mesothelioma Panel recognizes cases of keratin-negative sarcomatoid mesothelioma. In our experience with large samples of well-fixed autopsy tissue, keratin expression in sarcomatoid mesotheliomas
can be highly variable. Some regions of a sarcomatoid mesothelioma show intense immunostaining for keratin, and other regions of the same tumor show no keratin staining. This result has been observed with broadspectrum keratin antibodies (AE1/AE3) and with antibodies against LMW keratins (35βH11, CAM5.2). Lucas and colleagues386 immunostained 20 mesotheliomas with sarcomatoid components (10 biphasic and 10 sarcomatoid) for pancytokeratin, CK5/6, calretinin, WT1, thrombomodulin, and SMA. They compared the immunophenotypic profile of these tumors with 24 high-grade sarcomas, 10 pulmonary sarcomatoid carcinomas, and 16 epithelioid mesotheliomas. The 10 pulmonary sarcomatoid carcinomas were also immunostained for TTF-1. Their results are shown in Table 12-29. They concluded 1) there was a decrease in epithelial and mesothelial epitopes in sarcomatoid mesothelioma; 2) a wide immunophenotypic overlap existed among sarcomatoid mesothelioma, sarcoma, and sarcomatoid carcinoma; 3) cytokeratin and calretinin had the most value in differentiating sarcomatoid mesothelioma from sarcoma; 4) with the exception of SMA, all other markers studied showed a similar distribution in sarcomatoid mesothelioma and sarcomatoid carcinoma, including frequent calretinin and thrombomodulin expression in both tumors; 5) because cytokeratin expression can be absent in sarcomatoid mesothelioma, the distinction between it and sarcoma is arbitrary; and 6) IHC plays a more limited role in the differential diagnosis of sarcomatoid tumors versus epithelioid neoplasms, and the macroscopic distribution of the neoplasm must be correlated with the microscopic and IHC findings. Attanoos and colleagues387 evaluated 31 sarcomatoid mesotheliomas and a spectrum of other spindle cell neoplasms with antibodies directed against cytokeratin, thrombomodulin, calretinin and CK5/6. Cytokeratin
—
Podoplanin (D2-40)
72
80
93
72
CD15 (LeuM1)
BerEP4
MOC-31
TTF-1
—
100
93
90
93
95
96
85
85
Specificity (%)
—
92
74
51
63
—
78
95
76
Sensitivity (%)
—
87
95
97
98
—
62
87
89
Specificity (%)
Yaziji et al365
—
—
—
—
93.2
—
98.8
95.5
70
Sensitivity (%)
—
—
—
—
98
—
84.3
66.7
58.8
Specificity (%)
Kushitani et al366
pCEA, Polyclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor protein 1.
83
pCEA
Carcinoma Markers
82
77
Calretinin
WT1
83
Keratin 5
Mesothelioma Markers
Sensitivity (%)
King et al364
92.9
—
82.4
68.2
100
—
77.8
98.2
96.6
Sensitivity (%)
52.9
—
83.3
73.1
63
—
—
81.5
57.9
Specificity (%)
Klebe et al367
TABLE 12-25 Sensitivity and Specificity for Mesothelioma and Carcinoma Markers: Comparison Among Most Recent Data
100
100
94
92
100
90
98
98
99
Sensitivity (%)
36
38
13
36
17
100
72
89
81
Specificity (%)
Betta et al360
Pleural Neoplasms
451
452
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-26 Immunostains in Malignant Mesothelioma and Other Malignant Neoplasms Antibody
Epithelial Mesothelioma (%)
Sarcomatoid Mesothelioma (%)
Adenocarcinoma (%)
Squamous Cell Carcinoma (%)
Renal Cell Carcinoma (%)
Epithelial Markers pCEA
5
0
mCEA
3
—
81
BerEP4
10
0
80
40
B72.3
7
0
80
87
—
CD15 (LeuM1)
7
0
72
30
25-100
MOC-31
7
0
93
97
0-75
0
Lung: 72 Other: Negative
7
0
93
CK5/6
83
13
14.9
Calretinin
82
88
15
HBME-1
85
—
57
TTF-1 Lewisy-Bg8
Negative
83
7 —
Negative 80
0 — 0-58
— 0-33
Mesothelial Markers
Thrombomodulin
61
13
20
WT1
77
13
4
100
0
—
0
36 (weak)
0%
—
Mesothelin D2-40 Podoplanin
86-100 86%-93%
100
5
40
10
—
—
—
0-32
Negative
0-4
27 — 15%
— — —
CK, Cytokeratin; mCEA, monoclonal carcinoembryonic antigen; pCEA, polyclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor 1.
was expressed in 77% (24/31) of sarcomatoid mesotheliomas, 29% (9/31) expressed thrombomodulin, 39% (12/31) expressed calretinin, and 29% (9/31) expressed CK5/6. Broad-spectrum cytokeratin and thrombomodulin were expressed in 22% (2/9) of sarcomas not otherwise specified, and 11% (1/9) expressed CK5/6. As might be expected, 100% of synovial sarcomas expressed broad-spectrum keratin but showed no immunostaining for thrombomodulin, calretinin, and CK5/6; in addition, 67% (2/3) of angiosarcomas expressed thrombomodulin, which is not surprising, because thrombomodulin stains normal endothelial cells. The authors concluded that combination of a broad-spectrum cytokeratin with calretinin resulted in a high sensitivity (77% for AE1/ AE3 keratin) and high specificity (100% for calretinin) for sarcomatoid mesothelioma. The mesothelial markers thrombomodulin and CK5/6 were not useful alone in diagnosing sarcomatoid mesothelioma. This study also found that 3% of sarcomatoid mesotheliomas did not express broad-spectrum keratin, which would again support the notion that cytokeratin-negative sarcomatoid mesotheliomas exist. We have not found the expression of broad-spectrum keratin (AE1/AE3) noted by Lucas and associates386 in sarcomas, nor have we observed the same degree of calretinin expression in sarcomatoid mesotheliomas, sarcomas, or sarcomatoid carcinomas. In our experience,
broad-spectrum keratin is only rarely found in sarcomas (<1% of cases). Calretinin expression is found in a maximum of 20% of sarcomatoid mesotheliomas and is not seen in sarcomas or in sarcomatoid carcinomas. Similar immunoreactions have been observed by others as well. Vimentin is observed in normal epithelial cells and in a variety of carcinomas.18,388-390 In our experience, vimentin is expressed in all sarcomatoid mesotheliomas, usually intensely, and in most poorly differentiated and transitional mesotheliomas. Churg391 reported vimentin expression in two alcohol-fixed epithelial mesotheliomas, one of which was a tubulopapillary variant. Jasani and colleagues392 observed vimentin expression in 75% of 44 malignant mesotheliomas of all histologic types. However, 46% of 24 pulmonary adenocarcinomas also demonstrated vimentin by IHC analysis. Mullink and colleagues393 found it more common for epithelial mesotheliomas than for pulmonary adenocarcinomas to coexpress keratin and vimentin. We have observed that 30% to 45% of epithelial mesotheliomas coexpress keratin and vimentin. Calretinin is a reproducible positive marker of epithelial mesothelioma. It is a calcium-binding protein similar to S-100 protein394,395 that has a molecular weight of 29 kD and is found in the central and peripheral nervous system and in a wide spectrum of
Pleural Neoplasms
TABLE 12-27 Comparison of the Immunohistochemical Profile of Well- to Moderately Differentiated Epithelial Mesotheliomas and Well- to Moderately Differentiated Pulmonary Adenocarcinomas
Antibody Directed Against
Well- to Moderately Differentiated Epithelial Mesothelioma
Well- to Moderately Differentiated Pulmonary Adenocarcinoma
AE1/AE3
+
+
LMW keratin (35βH11)
+
+
HMW keratin (34βE12)
+
S
CK5/6
S
R
CK7
+
+
CK20
S
S
Vimentin
S
S
HBME-1
S*
Calretinin
+/−
R
Caldesmon
+
N
EMA
S*
S†
CEA
R
+
CD15 (LeuM1)
R
S
BerEP4
S
S
B72.3
R
S
TTF-1
−
S
WT1
+
−
Mesothelin
+
−
D2-40
+
−
HMFG-2
S
S
R ‡
453
sarcomatoid mesotheliomas and in the sarcomatoid component of 5 biphasic mesotheliomas. Studying cells in serous fluids, Barberis and coworkers398 found immunostaining for calretinin in 8 of 8 epithelial mesotheliomas and noted low-intensity immunostaining in 23.1% (3/13) of adenocarcinomas. They did not state whether the staining was cytoplasmic and nuclear. Leers and colleagues399 observed calretinin immunostaining in 20 of 20 epithelial mesotheliomas and noted weak immunostaining in 4.8% (1/21) of adenocarcinomas, however, the location of the staining was not specified. Ordonez400 compared two commercially available calretinin antibodies and found calretinin immunostaining in 21.1% (8/38) of epithelial mesotheliomas and focal, generally weak immunostaining in 9% (14/155) of adenocarcinomas of various types using a calretinin antibody obtained from Zymed Laboratories. In contrast, 73.7% (28/38) of epithelial mesotheliomas and 3.9% (6/155) of adenocarcinomas showed immunostaining for calretinin using a calretinin antibody from Chemicon International. In our experience using a calretinin antibody from Biocare, 94.3% (198/210) of epithelial mesotheliomas showed immunostaining for calretinin. As with S-100 protein, the immunostaining pattern for calretinin was cytoplasmic and nuclear (Fig. 12-46). Reactive multipotential-subserosal spindle cells typically express calretinin in a cytoplasmic and nuclear distribution. Additional studies401-404 have shown a high degree of specificity and sensitivity for calretinin in differentiating epithelial mesotheliomas from pulmonary adenocarcinomas and other epithelioid neoplasms. The use of the ME1 antibody was initially reported by O’Hara and colleagues405 in 1990. ME1 is a monoclonal antibody generated from the mesothelial cell line SPC111, and it reacts with normal mesothelial cells and malignant epithelial mesotheliomas. Their antibody was only useful on frozen-section tissue and showed immunostaining of 40 of 40 epithelial mesotheliomas. Nineteen well-differentiated and moderately differentiated primary pulmonary adenocarcinomas failed to stain with the ME1 antibody, but one poorly differentiated
*Cell membrane distribution. † Cytoplasmic distribution ‡ Nuclear and cytoplasmic immunostaining
nonneural cells, including steroid-producing cells of ovaries and testes, fat cells, renal tubular epithelial cells, eccrine glands, thymic epithelial cells, and mesothelial cells. Gotzos396 found calretinin immunostaining in 7 of 7 epithelial mesotheliomas and in the epithelial component of 15 of 15 biphasic mesotheliomas. They found no immunostaining in sarcomatoid components of biphasic mesothelioma and also found no immunostaining in a single case of sarcomatoid mesothelioma. The four lung adenocarcinomas evaluated showed no immunostaining for calretinin. Doglioni and associates397 found calretinin immunostaining in 44 of 44 epithelial mesotheliomas and observed focal staining in 9.5% (28/294) of adenocarcinoma of various origins. Doglioni and colleagues397 found positive staining in 3 of 3
Figure 12-46 Most well and moderately differentiated epithelial mesotheliomas and a few sarcomatoid mesotheliomas show cytoplasmic and nuclear immunostaining for calretinin (×400).
454
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-28 Cytokeratin Profiles of Epithelial Mesothelioma, Pulmonary Adenocarcinoma, and Squamous Cell Carcinoma of Lung Cytokeratin Moll Number
Molecular Weight
Isoelectric pH
Primary Pulmonary Adenocarcinoma
Epithelial Mesothelioma
Primary Pulmonary Squamous Cell Carcinoma
1
68 kD
7.8
—
—
—
2
65.5 kD
7.8
—
—
—
3
63 kD
7.5
—
—
—
4
59 kD
7.3
—
—
S
5
58 kD
7.4
—
S
S
6
56 kD
7.8
—
S
S
7
54 kD
6.0
S
S
R
8
52.5 kD
6.1
S
S
S
9
64 kD
5.4
—
—
—
10
56.5 kD
5.3
—
—
—
11
56 kD
5.3
—
—
—
12
55 kD
4.9
—
—
—
13
54 kD
5.1
—
—
—
14
50 kD
5.3
—
S
S
15
50 kD
4.9
—
—
S
16
48 kD
5.1
—
—
S
17
46 kD
5.1
N
+/−
+/−
18
45 kD
5.7
+/−
+/−
−/+
19
40 kD
5.2
+/−
+/−
+/−
20
46 kD
5.2
R
R
R
Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative.
pulmonary adenocarcinoma showed intense immunostaining. As reviewed by Sheibani and coworkers,406 Battifora produced an ME1 monoclonal antibody designated HBME-1, which worked on FFPE tissue. As reported by these authors, this antibody showed immunostaining of a relatively high percentage of epithelial mesotheliomas in a cell membrane distribution (Fig. 12-47), although the intensity of this reaction varied from one case to
another. The Dako Product Specification Sheet states that 89.5% (17/19) of epithelial mesotheliomas showed immunostaining for HBME-1. The antibody also reacted with 38% (19/50) of adenocarcinomas. The specification sheet suggests using the antibody at a dilution of 1 : 100. In our experience, the antibody should be used at a much greater dilution (1 : 7500 in SPH’s laboratory). As reported by Henderson and associates,369 they
TABLE 12-29 Immunohistochemically Positive Mesotheliomas and Sarcomas Based on Histologic Type Pan-CK (%)
CK5/6 (%)
Calretinin (%)
WT1 (%)
TM (%)
SMA (%)
Epithelioid mesothelioma
100
100
100
69
81
50
Biphasic mesothelioma, epithelioid component
100
40
90
60
90
20
Biphasic mesothelioma, sarcomatoid component
90
10
60
20
50
60
Sarcomatoid mesothelioma
70
0
70
10
70
60
Sarcoma
17
4
17
4
38
58
Sarcomatoid carcinoma
90
0
60
0
40
10
From Lucas DR, Pass HI, Madan NV, et al: Sarcomatoid mesothelioma and its histological mimics: a comparative immunohistochemical study. Histopathology 2003;42:270-279. CK, Cytokeratin; Pan-CK, pancytokeratin; SMA, smooth muscle actin; TM, thrombomodulin; WT1, Wilms tumor 1.
Pleural Neoplasms
Figure 12-47 Most well to moderately differentiated epithelial mesotheliomas show thick cell membrane immunostaining for HBME-1 (×400).
use HBME-1 antibody in a dilution between 1 : 5000 and 1 : 15,000. They found that when lower dilutions (higher concentrations) were used, the cytoplasm of many mesotheliomas stained, and a significant number of adenocarcinomas showed cytoplasmic immunostaining. These findings suggest that HBME-1 should be used at a high dilution to be effective for differentiating epithelial mesotheliomas from other neoplasms. Of interest, HBME-1 reacts with respiratory epithelium and occasionally shows cell membrane staining of primary pulmonary SCCs. EMA and human milk fat globule protein 2 (HMFG2) are similar glycoproteins of high molecular weight (250 to 400 kD) and are known as human milk fat globule proteins. These glycoproteins are found in milk fat and in a variety of normal and neoplastic epithelial cells. Antibodies against EMA and HMFG-2 are of use in diagnosing epithelial mesotheliomas in that the majority of epithelial mesotheliomas show immunostaining in a cell membrane distribution (only anti-EMA is currently available). This is different than most adenocarcinomas and other carcinomas that usually show cytoplasmic immunostaining. We407 detected EMA in 78% (50/64) of epithelial mesotheliomas, in 62% (37/60) of adenocarcinomas, and in 42% (8/19) of SCCs. Walz and Koch408 demonstrated EMA expression in 75% (33/44) of epithelial mesotheliomas, and Wick and associates409 found immunostaining in 84% (43/51). As stated, the antigen is concentrated in the cell membrane, and in most well-differentiated to moderately well-differentiated epithelial mesotheliomas, it produces a “thick” cell membrane reaction (Fig. 12-48) because of the extensive microvillous surface of epithelial mesotheliomas. Henderson and coworkers410 demonstrated strong cell-surface EMA staining in epithelial mesotheliomas and found immunostaining in a surface distribution in some lymphoid cells. In our experience, most reactive (benign) epithelial mesothelial cells show no immunostaining for EMA, and we have found relatively intense EMA cell membrane staining in cases of nonmucinous bronchioloalveolar cell carcinomas and in pulmonary papillary adenocarcinomas.
455
Thrombomodulin is a plasma membrane–related glycoprotein that has anticoagulant activity. Thrombomodulin antigen is found in several cell types, including mesangial, synovial, mesothelial, and endothelial cells; megakaryocytes; and some squamous epithelial cells. Fink and colleagues411 demonstrated immunostaining for thrombomodulin in eight epithelial mesotheliomas and two mesothelial cell lines. The cell lines were shown by ISH to possess mRNA for thrombomodulin. In contrast, 93.3% (14/15) of adenocarcinomas were negative for thrombomodulin, and one showed focal positivity. Collins and associates412 found thrombomodulin expression in 31 of 31 epithelial mesotheliomas and in 8.3% (4/48) of pulmonary adenocarcinomas. In contrast, Brown and colleagues413 observed only 60% of epithelial and biphasic mesotheliomas to express thrombomodulin, whereas 58% of pulmonary adenocarcinomas were positive. Ascoli and coworkers414 identified thrombomodulin in 33 of 33 epithelial mesotheliomas, in reactive mesothelial cells in 35 effusions, and in 39.3% (57/145) of carcinomas in effusions. They reported a different IHC pattern of staining in benign reactive mesothelial cells, malignant epithelial mesotheliomas, and carcinomas. In benign reactive mesothelial cells, thin linear staining was observed. Thick membrane staining was seen in malignant epithelial mesotheliomas, and cytoplasmic staining was observed in most cases of carcinoma. Cadherins are a family of adhesion proteins important in sorting cells into specialized tissues during morphogenesis.415,416 Included in the cadherin family are epithelial (E) cadherin, nerve (N) cadherin, retina (R) cadherin, osteoblast (OB) cadherin, and placental (P) cadherin. N-cadherin is a 135,000-kD protein found in nerve cells, developing muscle cells, and mesothelial cells.417 Using 13A9 anti–N-cadherin monoclonal antibody on frozen sections, Peralta-Soler and coworkers418 observed strong immunoreactivity in 19 of 19 epithelial mesotheliomas and noted focal weak reactivity in 3 of 16 (18.8%) pulmonary adenocarcinomas. Using antigen
Figure 12-48 Most well to moderately differentiated epithelial mesotheliomas show “thick” cell membrane staining for epithelial membrane antigen (×400). CK, Cytokeratin; EMA, epithelial membrane antigen; p-CEA, polyclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor; WT1, Wilms tumor 1.
456
Immunohistology of Lung and Pleural Neoplasms
retrieval methodology on paraffin-embedded tissue sections, Han and colleagues419 reported 92.3% (12/13) of epithelial mesotheliomas to be positive for N-cadherin and 7.1% (1/14) of pulmonary adenocarcinomas to be positive. Ordonez420 evaluated 31 epithelioid mesotheliomas and 29 pulmonary adenocarcinomas for E-cadherin and N-cadherin expression using the 5H9, HECD-1, and clone 36 anti–E-cadherin antibodies and the 3B9 and clone 32 anti–N-cadherin antibodies. In this study, 68%, 52% and 19% of epithelial mesotheliomas reacted with anti–E-cadherin clone 36, clone HECD-1, and 5H9 respectively, and 74% and 71% of epithelial mesotheliomas reacted with anti–N-cadherin clone 3B9 and clone 32 respectively. In addition, 93%, 90%, and 90% of pulmonary adenocarcinomas reacted with anti–Ecadherin clones 36, HECD-1, and 5H9 respectively, and 45% and 34% of pulmonary adenocarcinomas reacted with anti–N-cadherin clones 32 and 3B9 respectively. Ordonez concluded that 5H9 anti–Ecadherin antibody had some utility in discriminating between pleural epithelioid mesothelioma and pulmonary adenocarcinoma. Wilms tumor suppressor gene 1 (WT1) resides on the 11p13 chromosome, the inactivation of which causes susceptibility to Wilms tumor. This gene is found predominantly in tissues of mesodermal origin. Using frozen tissue sections, Amin and colleagues421 observed nuclear immunostaining in 95.2% (20/21) of malignant mesotheliomas and in 0 of 26 nonmesothelioma tumors involving lung, including 20 primary NSCLCs. Using an antibody adaptable to FFPE tissue, Kumar-Singh and associates422 found positive staining of Wilms tumor suppressor gene (WT1) products in 92.9% (39/42) of mesotheliomas, 2 of 2 papillary carcinomas of ovary, and 1 of 1 renal cell carcinomas. Twelve adenocarcinomas of lung, 4 SCCs of lung, 8 metastatic breast adenocarcinomas, and 3 metastatic adenocarcinomas from colon did not express the WT1 suppressor gene products. Using molecular biology techniques, WT1 transcripts were found in 88.5% (23/26) of mesothelioma cell lines, in 62.5% (5/8) of human malignant mesotheliomas, but in no NSCLC cell lines and in only a few biopsy specimens.423 Mesothelin is a 40-kD glycoprotein of unknown function that is strongly expressed in mesothelial cells, ovarian serous cells, and pancreatic–bile duct cells. Using monoclonal antibody 5B2, Ordonez424 found it to immunostain normal mesothelial cells, mesotheliomas, nonmucinous ovarian carcinomas, and occasionally other neoplasms. Ordonez concluded that mesothelin staining could be useful in diagnosing mesotheliomas, although it was also expressed in 14 of 14 ovarian carcinomas, 12 of 14 pancreatic ductal adenocarcinomas, 7 of 12 DSRCTs, and 9 of 9 synovial sarcomas. This antibody should be interpreted carefully. D2-40, a clone of podoplanin, is a recently developed, commercially available antibody directed against the M2A antigen, a 40,000-kD sialoglycoprotein associated with germ cells and lymphatic endothelium. Chu and colleagues425 evaluated 53 cases of mesothelioma, 28 cases of reactive pleural tissue, 30 cases of pulmonary
adenocarcinoma, 35 cases of renal cell carcinoma, 26 cases of ovarian serous carcinoma, 16 cases of invasive breast carcinoma, 11 cases of prostatic adenocarcinoma, and 7 cases of urothelial carcinoma. D2-40 expression was found in 96% (51/53) of mesotheliomas, 96% (27/28) of reactive pleural tissues, and 65% (17/26) of ovarian serous carcinomas. D2-40 was not found in the other tumors examined, and the neoplastic cells immunostained in a cell membrane distribution. A cytoskeleton-associated protein, h-caldesmon is present in smooth and nonsmooth muscle cells. It combines with calmodulin, tropomyosin, and actin and is involved in the regulation of cellular contraction. Using IHC analysis with an antibody for h-caldesmon, a specific marker for smooth muscle tumors, Comin and colleagues426 examined 70 cases of epithelial mesothelioma and 70 cases of lung adenocarcinoma. In addition, IHC for muscle markers such as desmin, α-SMA, MSA, myoglobin, myogenin, myosin, and MyoD1 was performed on all mesothelioma cases. Reactivity for h-caldesmon was found in 97% (68/70) of epithelial mesotheliomas but in none of the adenocarcinoma cases. All mesotheliomas found were stated to be negative for other muscle markers. The authors426 concluded that h-caldesmon was a highly sensitive and specific marker and suggested its inclusion in the IHC panel for the differential diagnosis of epithelioid mesothelioma versus pulmonary adenocarcinoma. NEGATIVE MARKERS
Of all antibodies used as exclusionary antibodies for diagnosing epithelial mesothelioma, polyclonal CEA (pCEA) has been used most frequently. CEA is a glycoprotein of approximately 200 kD that contains approximately 50% carbohydrate.427-430 CEA is referred to as a family431 and is coded by 29 genes, 18 of which are expressed: 7 belong to the CEA subgroup, and 11 belong to the pregnancy-specific subgroup. Often referred to as an oncofetal antigen, CEA is expressed in normal adult tissues and in a variety of epithelial neoplasms. The CEA subgroup includes biliary glycoprotein and nonspecific cross-reacting substance. The antibody we use is polyclonal, and the immunogen is CEA isolated from hepatic metastases of a colonic adenocarcinoma. The antibody reacts with nonspecific cross-reacting substance and biliary glycoprotein. Nonspecific cross-reacting substance is present in granulocytes and monocytes, and biliary glycoprotein is expressed by a large number of normal epithelial cells and by granulocytes, lymphocytes, and possibly by endothelial cells. Therefore it is likely that in most tissue sections for pCEA, positive control staining is built in. In most epithelial mesotheliomas, pCEA shows no immunostaining.432-434 In contrast, it is found in a high percentage (85% to 100%) of pulmonary adenocarcinomas. Henderson and associates410 analyzed data from 21 separate reports in the evaluation of 598 cases of diffuse malignant mesothelioma and found 58 cases (9.7%) reported to express CEA. In the majority of cases in which a positive reaction was observed, it was usually focal and weak. In the same analysis, 88.9% (359/404) of
Pleural Neoplasms
pulmonary adenocarcinomas expressed CEA. We have observed CEA-positive staining in mesotheliomas only rarely, and it has occurred predominantly in those epithelial mesotheliomas that were mucin positive, which correlated with those that produce large quantities of hyaluronic acid or proteoglycan. In our experience, pCEA is the best negative marker of mesothelioma. LeuM1 is a monoclonal antibody against the membrane-related trisaccharide fucoysl-N-acetyllactosamine on myelomonocytic cells, in which the epitope is also known as CD15 or X-hapten. LeuM1 is found in Reed-Sternberg cells in most cases of Hodgkin lymphoma. In 1985 Sheibani and Battifora435 reported LeuM1 in a metastatic, poorly differentiated pulmonary adenocarcinoma. In 1986 Sheibani and colleagues436 performed IHC analyses on 400 malignant neoplasms and found LeuM1 immunostaining in 58.7% (105/179) of adenocarcinomas and 0 of 18 epithelial mesotheliomas. Sheibani and associates437 subsequently studied 50 primary pulmonary adenocarcinomas and 28 pleural epithelial mesotheliomas and found expression in 94% (47/50) of pulmonary adenocarcinomas and 0 of 28 epithelial mesotheliomas. In another study, Sheibani and associates438 reported no immunostaining for LeuM1 in 127 cases of malignant mesothelioma. Wick and colleagues409 identified LeuM1 in 52 of 52 pulmonary adenocarcinomas and 0 of 51 epithelial mesotheliomas. In contrast, Otis and coworkers439 observed LeuM1 immunostaining in only 50% of pulmonary adenocarcinomas and reported LeuM1 expression in epithelial mesotheliomas. Battifora and McCaughey355 observed focal LeuM1 expression in epithelial mesotheliomas. We have observed several LeuM1-positive epithelial mesotheliomas, and the staining has usually been focal. In contrast to what has been reported in the literature, and despite using heat-induced antigen retrieval, we have observed a positive reaction for LeuM1 (CD15) in only approximately 50% of primary pulmonary adenocarcinomas. B72.3 is an antibody that recognizes an HMW glycoprotein complex, tumor-associated glycoprotein 72 (TAG-72), derived from a membrane-enriched fraction of human metastatic breast carcinoma. Szpak and colleagues440 and Ordonez441 reported immunostaining in 84.4% (38/45) of pulmonary adenocarcinomas in comparison with 2.6% (1/38) of epithelial mesotheliomas. Wick and colleagues409 reported that 82.7% (43/52) of pulmonary adenocarcinomas expressed B72.3, whereas all 51 epithelial mesotheliomas they studied were negative for the marker. In an evaluation of peritoneal mesotheliomas and serous papillary adenocarcinomas of the peritoneum, Bollinger and colleagues442 reported 93.5% (43/46) of serous papillary carcinomas positive for B72.3, whereas eight epithelial mesotheliomas were negative. Ordonez370 tabulated the reported literature and found that 10.1% (69/684) of epithelial mesotheliomas show immunostaining for B72.3 (0% to 48% of cases), and 95.2% (578/607) of adenocarcinomas show positivity (47% to 100%). When positive for B72.3, epithelial mesotheliomas usually show a small percentage of positive cells. However, Henderson and associates369 reported more extensive expression of B72.3 in the cytoplasm of
457
epithelial mesotheliomas and described one case of intracytoplasmic crescentic staining that correlated ultrastructurally with intracytoplasmic glycogen. BerEP4 is a monoclonal antibody that recognizes the protein moiety of two, 34- and 39-kD glycopolypeptides on human epithelial cells. Latza and associates443 observed BerEP4 reactivity in 98.6% (142/144) of adenocarcinomas from various sites and in 0 of 14 epithelial mesotheliomas. Sheibani and coworkers444 observed BerEP4 immunoreactivity in 0.87% (1/115) of epithelial mesotheliomas and in 86.8% (72/83) of adenocarcinomas of various sites, and 8 of 25 breast carcinomas and 3 renal cell carcinomas studied were negative for BerEP4. Gaffey and associates445 found different results: they reported 86% (103/120) of adenocarcinomas positive; 20% (10/49) of epithelial mesotheliomas were also reactive, as were 22% (2/9) of adenomatoid tumors. Gaffey and associates445 reported one epithelial mesothelioma to be diffusely positive. Staining for BerEP4 was usually in a cell membrane distribution. A possible explanation for the difference in these results is that Sheibani and colleagues444 used protease type 14 predigestion before BerEP4 staining, whereas Gaffey and coworkers445 used 0.4% pepsin predigestion for 30 minutes before staining. We currently use heatinduced epitope retrieval (HIER) and observe approximately 20% of epithelial mesotheliomas to show predominantly low-intensity cell membrane staining for BerEP4. Occasional epithelial mesotheliomas show intense cell membrane immunostaining. Ordonez370 reviewed published studies of BerEP4 reactivity in epithelial mesotheliomas and adenocarcinomas of various types and found that 12.4% (76/611) of epithelial mesotheliomas (0% to 88%) and 67.2% (940/1399) of adenocarcinomas (35% to 100%) immunostained for BerEP4. MOC-31 is a monoclonal antibody that reacts with a 38-kD epithelial-associated transmembrane glycoprotein of SCLC, epithelial glycoprotein 2.446,447 MOC-31 activity was reported by DeLeij and colleagues448 in all pulmonary carcinomas, including 28 of 28 adenocarcinomas. Normal mesothelial cells and neoplastic epithelial mesothelial cells did not react with the antibody. In 1991 Delahaye and coworkers449 studied cytologic preparations of serous fluid and found positive MOC-31 staining in 8.3% (2/24) of epithelial mesotheliomas and in 58.1% (18/31) of adenocarcinomas of various origins. Riutenbeek and associates447 found MOC-31 reactivity in 98.4% (62/63) of adenocarcinomas and in 0 of 5 epithelial mesotheliomas. Ordonez450 reported intense MOC-31 reactivity in 40 of 40 (100%) pulmonary adenocarcinomas, 11 of 11 colon adenocarcinomas, 20 of 21 ovarian adenocarcinomas (95.2%), 9 of 10 breast adenocarcinomas (90%), and 5 of 13 kidney adenocarcinomas (38.5%). MOC-31 reactivity was seen in 2 of 38 epithelial mesotheliomas (5.3%), although the degree of staining was usually focal and involved less than 10% of cells. Ordonez370 reviewed published studies of MOC-31 reactivity in epithelial mesotheliomas and various types of adenocarcinomas. Immunostaining was found in 307 of 333 (92.2%) adenocarcinomas and 23 of 158 (14.6%) epithelial mesotheliomas.
458
Immunohistology of Lung and Pleural Neoplasms
Monoclonal antibody Bg8 reacts with SK-LU-3 lung cancer cells that recognize the blood group antigen Lewisy. Jordon and colleagues451 reported reactivity in 18 of 18 pulmonary adenocarcinomas and in 23.3% (7/30) of epithelial mesotheliomas. Reactivity in mesotheliomas was usually focal and limited to a few cells, whereas reactivity in pulmonary adenocarcinomas was usually strong and diffuse. Riera and associates452 evaluated Bg8 antibody and found expression in 92.7% (114/123) pulmonary adenocarcinomas and in 8.8% (5/57) of epithelial mesotheliomas. The staining in epithelial mesotheliomas was usually focal and weak. E-cadherin (epithelial cadherin) is a 120-kD cell adhesion molecule expressed in epithelial cells.415,453 Loss of E-cadherin expression is associated with a higher degree of invasiveness and increased malignant potential in several carcinomas, including lung cancers.454 Peralta-Soler and coworkers418 reported intense immunostaining for E-cadherin in 16 of 16 pulmonary adenocarcinomas and 8 of 19 (42.1%) epithelial mesotheliomas, with the staining usually involving only a few cells. With formalin-fixed HIER, 92.9% (13/14) of pulmonary carcinomas were positive for E-cadherin and 0 of 13 epithelial mesotheliomas were reactive. Leers and coworkers399 reported positive reactivity in 95.2% (20/21) of adenocarcinomas of various origin and in 15% (3/20) of epithelial mesotheliomas. Using the commercially available 5H9 anti–E-cadherin antibody on sections of FFPE tissue, Ordonez370 found reactivity in 83.3% (15/18) of pulmonary adenocarcinomas and 0 of 17 epithelial mesotheliomas. When Ordonez used anti– E-cadherin monoclonal antibody clone 36, strong reactivity was seen in 6 of 6 epithelial mesotheliomas. It would therefore appear that caution must be exercised using this antibody, and that 5H9 anti–E-cadherin antibody would be the appropriate choice. As discussed previously,Ordonez420 concluded that only 5H9 clone anti–Ecadherin antibody had utility in differentiating epithelial mesothelioma from pulmonary adenocarcinoma. Blood group–related antigen expression has been used to evaluate epithelial mesotheliomas and pulmonary adenocarcinomas. Wick and others409 found ABH isoantigen expression in 67.3% (35/52) of adenocarcinomas and in 0 of 51 epithelial mesotheliomas. Kawai and colleagues455 evaluated 20 epithelial, 3 biphasic, and 6 sarcomatous mesotheliomas, along with 5 reactive mesothelial cell lesions and 38 well-differentiated pulmonary adenocarcinomas using ABH blood group– related antigen (BGRA-g) antibody and Helix pomotia agglutinin (HPAgg). The reactive mesothelial lesions and the mesotheliomas showed no expression for BGRA-g or HPAgg, irrespective of the blood group type. In pulmonary adenocarcinoma, the 6GRAg antibody showed a high positive rate with the compatible blood group type, especially in type O cases (83%). A positive reaction for HPAgg was seen in 94.1% (16/17) of patients of blood type A and in all blood type AB patients. Positive staining for HPAgg was observed in 80% (4/5) of blood type B patients and in 33.3% (4/12) with blood type O. Riera and associates452 used HIER to study 268 FFPE tumors, including 57 epithelial mesotheliomas and 211
adenocarcinomas of various origin. After statistical analysis, they found CEA, BerEP4, and Bg8 to be the best discriminators between adenocarcinoma and epithelial mesothelioma within the entire panel; the mesotheliomaassociated antibodies HBME-1, calretinin, and thrombomodulin were less sensitive and less specific, although they were found to be useful in certain cases. All adenocarcinomas and mesotheliomas showed intense IHC staining for keratin, with no discernible difference in staining pattern between adenocarcinoma and mesothelioma, and 80.7% (46/57) of mesotheliomas and 31.3% (66/211) of adenocarcinomas expressed vimentin. The intensity and distribution of vimentin staining was stated to be greater in mesotheliomas than in adenocarcinomas. CEA immunoreactivity was observed in 82.9% (175/211) of adenocarcinomas and in no mesotheliomas. Ovarian and breast adenocarcinomas showed 44.8% and 79.3% CEA-positive staining, respectively. Focal immunostaining for BerEP4 was observed in 63.7% of adenocarcinomas and in no mesotheliomas; Bg8 reactivity was observed in 88.6% of adenocarcinomas and 8.8% (5/57) of mesotheliomas, and the neoplastic cells in mesotheliomas stained only focally positive and with less intensity. B72.3 immunostaining was observed in 80.6% (170/211) of adenocarcinomas and 3.5% (2/57) of mesotheliomas, in which immunostaining for B72.3 was focal but intense. Granular cytoplasmic staining for LeuM1 was found in 75.4% (159/211) of adenocarcinomas and in 84.6% (104/123) of pulmonary adenocarcinomas, and 3.5% (2/57) of mesotheliomas showed predominantly focal membrane staining for LeuM1. Diffuse, moderately intense cytoplasmic staining was observed for BerEP4 in 85.3% (180/211) of adenocarcinomas and in 28.1% (16/57) of mesotheliomas. Most adenocarcinomas were stated to show cytoplasmic reactivity. In contrast, 57.9% (33/57) of epithelial mesotheliomas showed cell membrane staining, usually without cytoplasmic staining. In several cases, a thick pattern of membrane staining was observed similar to that found with HBME-1. Concerning mesothelial-related antigens, 49.1% (28/57) of epithelial mesotheliomas immunostained for thrombomodulin compared with 6.2% (13/211) of adenocarcinomas, of which 7 were pulmonary adenocarcinomas. HBME-1 immunoreactivity was observed in 79% (45/57) of epithelial mesotheliomas, usually in a circumferentially thick or moderately thick distribution, and in 39.3% (83/211) of adenocarcinomas, usually in a thin pattern restricted to the apical region. A thick membrane pattern was observed in 9% (19/211) of adenocarcinomas, usually in an apical distribution. In addition, 42.1% (24/57) of epithelial mesotheliomas showed immunostaining for calretinin that was cytoplasmic, finely granular, and diffuse in 22 cases and focal in 2 cases, whereas 6.2% (13/211) of adenocarcinomas showed weak or moderate staining. The sensitivity and specificity of the adenocarcinoma and mesothelioma markers observed by Riera and colleagues452 are shown in Tables 12-30 and 12-31, respectively, and their study provides a great deal of practical information. Of particular interest was that the HBME-1 antibody was used in a dilution of 1 : 40, which is in
Pleural Neoplasms
459
TABLE 12-30 Immunohistochemical Tests Used to Evaluate Adenocarcinomas 1 Scoring
Sensitivity (%)
CEA-I
2 Specificity (%)
83
Sensitivity (%)
Specificity (%)
Sensitivity (%)
Specificity (%)
79
100
75
100
75
100
64
100
82
91
50
96
75
100
42
100
64
100
39
100
55
100
29
100
77
96
68
96
50
98
19
98
71
96
55
96
55
100
33
100
72
64
79
19
89
72
73
77
40
89
100
CEA-D Bg8-I
89
91
Bg8-D BerEP4-I
64
100
BerEP4-D B72.3-I
81
96
75
96
B72.3-D LeuM1-I LeuM1-D HMFG-2-CI
85
HMFG-2-CD
3
From Riera Jr, Astengo-Osuna C, Longmate JA, et al: The immunohistochemical diagnostic panel for epithelial mesothelioma: a re-evaluation after heat-induced epitope retrieval. Am J Surg Pathol 1997;21:1409-1419. Heat-induced epitope retrieval (HIER) was used when appropriate. CEA, Carcinoembryonic antigen; I, intensity of staining; D, distribution of staining; HMFG, human milk fat globule.
contrast to what we use (1 : 7500) and what Dr. Henderson369 uses (1 : 5000 to 1 : 15,000). In addition, we have found a much higher positive staining reaction for calretinin (~95%) in epithelial mesothelioma than that reported by Riera and colleagues. MISCELLANEOUS ANTIBODIES
Expression of p53 tumor suppressor gene products has been evaluated as a method of discriminating between mesothelioma and reactive mesothelial hyperplasia.456,457 Ramael and coworkers457 evaluated 40 cases of nonneoplastic reactive pleural mesothelial proliferative lesions and 36 epithelial mesotheliomas for p53 tumor
suppressor gene product. With DO-7 and CM-1 antibodies, nuclear immunolabeling for p53 was observed in 25% of mesotheliomas. No reactivity was observed using antibody Pab240, and no significant difference in reactivity was observed for the p53 tumor suppressor gene product among histologic subtypes of mesothelioma. Mayall and associates456 evaluated p53 gene product expression using DO-7 and CM-1 antibodies in pepsin predigested tissue sections and found positive reactions in 62.5% (10/16) of epithelial mesotheliomas, 47.4% (9/19) of biphasic mesotheliomas, 16.7% (2/12) of sarcomatoid mesotheliomas, and 0 of 20 reactive mesothelial cell proliferations. Mayall and others458 found no difference in p53 gene product expression in
TABLE 12-31 Immunohistochemical Tests Used to Evaluate Mesotheliomas 1 Scoring
2
3
Sensitivity
Specificity
Sensitivity
Specificity
Sensitivity
Specificity
Calretinin-I
42
94
31
96
8
100
Calretinin-D
49
94
39
97
16
98
Thrombomodulin-I
49
94
46
95
35
97
Thrombomodulin-D
49
94
32
96
32
96
HBME-1-I*
79
61
74
72
53
91
HBME-2-D*
79
61
65
73
39
89
From Riera JR, Astengo-Osuna C, Longmate JA, et al: The immunohistochemical diagnostic panel for epithelial mesothelioma: a re-evaluation after heat-induced epitope retrieval. Am J Surg Pathol 1997;21:1409-1419. Heat-induced epitope retrieval (HIER) was used when appropriate. *Only membrane staining was interpreted. I, intensity of staining D, distribution of staining.
460
Immunohistology of Lung and Pleural Neoplasms
asbestos-induced mesotheliomas versus those not caused by asbestos, suggesting that asbestos was not the cause of p53 gene mutation. Using FFPE tissue sections, Hurlimann459 reported IHC desmin expression in 56.3% (9/16) of mesotheliomas (8 epithelial, 1 biphasic). Staining was described as focal or was found only in rare tumor cells. Cases showed positive reactivity more frequently with HIER. In this study, 25% (4/16) of mesotheliomas (3 epithelial, 1 biphasic) expressed NSE, 31.3% (5/16) immunostained for chromogranin (4 epithelial, 1 biphasic), and 31.3% (5/16) of mesotheliomas, all epithelial, were positive for S-100 protein. Only rare tumor cells expressed these neuroepithelial markers. Azumi and associates460 used IHC techniques to study 33 mesotheliomas (32 pleural, 1 peritoneal; 18 epithelial, 10 biphasic, 4 sarcomatoid, 1 desmoplastic) and 37 adenocarcinomas for hyaluronate, and 8.1% (3/37) of adenocarcinomas and all mesotheliomas immunostained for hyaluronate. The location of the staining reaction in the mesotheliomas was membranous in 30 cases, cytoplasmic in 21 cases, and membranous and cytoplasmic in 19 cases. The staining reaction in mesotheliomas was classified as moderate or greater in 81.8% (27/33) of cases. The authors concluded that demonstration of hyaluronate should be considered an important adjunct to be used with other IHC tests and electron microscopy in diagnosing epithelial mesotheliomas. Hyaluronan detection in pleural fluid has been advocated as a method of diagnosing mesothelioma.461 Among 13 patients with pleural fluid hyaluronan concentrations greater than 225 mg/L, no other diagnosis but mesothelioma was identified. The specificity for mesothelioma was 96% with a cutoff level of 75 mg/L hyaluronan and 100% with a cutoff level of 225 mg/L hyaluronan. Martensson and colleagues462 evaluated hyaluronan in pleural fluid from 19 men with mesothelioma. Tumor volume was estimated on transilluminated CT scans with a digital planimeter. An elevated concentration of hyaluronan (7100 mg/L) was found in the pleural fluid in 68.4% (13/19) of patients. A positive correlation was found between the initial concentration of hyaluronan in the serum and the concentration of hyaluronan in the pleural fluid. Increasing concentration of circulating hyaluronan correlated positively with an increasing tumor volume in the hyaluronan-producing mesotheliomas but not in the nonhyaluronan-producing mesotheliomas. Thylen and colleagues463 evaluated the IHC differences between hyaluronan-producing and nonhyaluronan-producing malignant mesothelioma and found a significantly higher reactivity to EMA and CAM5.2 keratin and a lower reactivity to vimentin in the hyaluronan-producing epithelial mesotheliomas. All tumors were stated to be negative for CEA. Our experience is different from that of these authors; we have found that the mesotheliomas that produce excess amounts of hyaluronic acid and/or proteoglycans are more likely to be mucin positive and to express the negative markers of epithelial mesothelioma, such as CEA, LeuM1, and B72.3.
CA-125 is a glycoprotein identified in the cell membrane in celomic epithelium during embryogenesis and in neoplasms of the female genital tract.464-467 The antibody to detect CA-125 in histologic sections, OC-125, was initially thought to work only in frozen tissue sections, but it was adapted to work in FFPE sections by using enzymatic digestion468 or HIER. It soon became apparent that OC-125 reactivity was not restricted to neoplasms of the gynecologic tract, rather OC-125 reactivity was also observed in breast469 and lung neoplasms470,471 and in neoplasms of the pleura and peritoneal linings.472,473 The second-generation antibody against CA-125, M-11, was stated to show greater intensity staining than OC-125.474 M-11 reactivity was demonstrated in mesothelial linings of spontaneous abortion specimens of 6 to 14 weeks gestation.475 In rare instances, unusual substances have been demonstrated immunohistochemically in mesotheliomas. Okamoto and associates476 reported two neoplasms consistent with primary pleural mesotheliomas that contained anaplastic tumor giant cells that contained hCG as demonstrated by IHC. McAuley and coworkers477 evaluated a patient with malignant mesothelioma who had hypercalcemia and an elevated serum concentration of parathyroid-like hormone. They evaluated nine epithelial mesotheliomas for parathyroid-like peptide and found abundant immunopositive cells in eight of nine cases. They also observed parathyroid-like peptide immunoreactivity in normal and reactive epithelial mesothelial cells. Tateyama and colleagues478 reported CD5 expression in thymic carcinoma and atypical thymoma and in 69.2% (9/13) of mesotheliomas (5 epithelial, 3 biphasic, 1 sarcomatoid). All CD5-positive mesotheliomas showed intense intracytoplasmic staining, and 61.5% (8/13) of pulmonary adenocarcinomas showed low to moderately intense, predominantly cell membrane staining for CD5. A significant number of mesotheliomas show immunostaining for various CD antigens such as CD30, CD56, and CD99. Desmin and neuromarker expression in mesothelial cells and mesotheliomas can occur. Mesotheliomas show a variety of histologic patterns, including some that have an NE appearance, such as small cell mesotheliomas, which can occasionally express NE markers such as NSE, synaptophysin, and CD56.
Diagnostic Considerations SARCOMATOID MESOTHELIOMA
According to the Guidelines for Pathologic Diagnosis of Malignant Mesothelioma: Consensus from the International Mesothelioma Interest Group,479 an IHC panel that can be useful for the evaluation of sarcomatoid tumors involving the pleura should include cytokeratins, calretinin, and D2-40. As stated above, multiple cytokeratin antibodies, including AE1/AE3 and CAM5.2 (CK18) keratins and CK7, usually are expressed in sarcomatoid mesotheliomas. D2-40 and calretinin are the two positive markers most frequently expressed
Pleural Neoplasms
in sarcomatoid mesotheliomas in a variable percentage of cases.425,480-482 Most sarcomatoid mesotheliomas express keratin, usually LMW keratin, and also vimentin and frequently muscle-specific actin (MSA). In our experience, a relatively small percentage (10% to 20%) of sarcomatoid mesotheliomas express calretinin, and most sarcomatoid mesotheliomas do not express CK5/6. Most sarcomatoid mesotheliomas express CK7, often intensely. In our experience, sarcomatoid mesotheliomas, and tumors they may be confused with, do not express the negative markers used to evaluate potential epithelial mesotheliomas, including CEA, LeuM1, B72.3, BerEP4, Bg8, and TTF-1. Therefore we would not include those antibodies in a screening of a malignant spindle cell proliferative lesion of the pleura. Marchevsky368 evaluated sarcomatoid mesotheliomas and pointed out that the diagnosis of sarcomatoid mesothelioma was particularly difficult, because the sensitivity and specificity of mesothelial markers was considerably lower in these lesions than in malignant epithelial mesothelioma. Marchevsky found that immunostains for calretinin, thrombomodulin, and WT1 were positive in fewer than 20% of sarcomatoid mesotheliomas, but AE1/AE3 keratin was the most helpful marker, in that it showed staining of almost all cases of sarcomatoid mesothelioma. Klebe and associates483 reported 27 cases of malignant mesothelioma that had heterologous elements. Of these 27 cases, 16 (59%) were sarcomatoid, 10 (37%) were biphasic, and 1 was epithelioid. Forty-one percent (11 cases) showed osteosarcomatous elements only, 19% showed areas of rhabdomyosarcoma only, 19% showed areas of chondrosarcomatous differentiation only, and 22% showed osteochondromatous elements. The authors found labeling for cytokeratins in the majority of cases. These tumors had a very poor prognosis, and median survival was only 6 months. The authors also found that lack of labeling for cytokeratins in a spindle cell/sarcomatoid tumor did not exclude the diagnosis of mesothelioma irrespective of the presence of heterologous elements. The authors suggested that if the anatomic distribution of the tumor conformed to that of a mesothelioma, a diagnosis of heterologous mesothelioma should be made in preference to a diagnosis of a primary pleural osteosarcoma or chondrosarcoma, regardless of cytokeratin positivity (as for conventional nonheterologous sarcomatoid mesothelioma). In 2010, Klebe and colleagues484 studied 326 cases of sarcomatoid mesotheliomas among 2000 consecutive malignant mesothelioma cases received in consultation. Patients included 312 men (96%) and 14 women (4%) with a median age of 70 years (range 41 to 94 years). Most sarcomatoid mesotheliomas were pleural (319; 98%), and 7 were peritoneal (2%). Some desmoplastic features were identified in 110 cases (34%), and 70 (22%) were classified as desmoplastic mesotheliomas, characterized by being relatively hypocellular and showing slitlike spaces between the tumor cells; they can resemble hyaline pleural plaques. The difference between pleural plaques and desmoplastic
461
Figure 12-49 This neoplasm was in a diffuse pleural distribution and had the consistency of fat.
mesothelioma is that desmoplastic mesotheliomas are more cellular and often show areas of necrosis. Klebe and coworkers484 reported that pleural plaques were present in 79% of cases for which information was available, and asbestosis was diagnosed in 27% (34/127). Rare subtypes included two cases with a lymphohistiocytoid pattern (<1%) and eight heterologous mesotheliomas (2%). Cytokeratins were observed in 93% (261/280), calretinin in 31%, and vimentin in 91%. This study represents the largest series of sarcomatoid and desmoplastic mesotheliomas to date and confirms the diagnostic usefulness of cytokeratin IHC. Almost 100% of sarcomatoid mesotheliomas express vimentin, whereas only a relatively small percentage (~20%) express calretinin. In addition, rare sarcomatoid mesotheliomas show heterologous differentiation composed of fat (Figs. 12-49 and 12-50).485 The immunohistogram of a sarcomatoid mesothelioma is shown in Figure 12-51. The most important IHC finding in sarcomatoid mesothelioma is coexpression of keratin and vimentin.
Figure 12-50 Portions of tumor have morphology consistent with fat.
Immunohistology of Lung and Pleural Neoplasms
-4 0 D2
Ca
W T1
-2 lre tin in
A EM
FG HM
in pr ot ei n
sm
S10
0
tin Ac
De
tin
7
en
CK
Vi m
12 E
H 35
1/ AE AE
34
11
100 90 80 70 60 50 40 30 20 10 0 3
Percent positive
462
Figure 12-51 Immunohistogram of a sarcomatoid mesothelioma. CK, Cytokeratin; HFMG, human milk fat globule; WT1, Wilms tumor 1.
TRANSITIONAL MESOTHELIOMA
Transitional mesothelioma refers to a histologic type of mesothelioma that has epithelioid and sarcomatoid features. These were described by Bolen353 in 1986. Dardick and colleagues486 described features of what we referred to as transitional mesothelioma that were composed of large, polygonal to plump and occasionally spindleshaped cells present in nests or in no specific pattern. These transitional mesotheliomas typically express broad-spectrum keratin (AE1/AE3), CK8/18, and vimentin. Not infrequently, they express SMA. However, only approximately 15% to 20% express calretinin. METASTATIC MESOTHELIOMA
Metastatic tumors to the pleura are more common than primary pleural neoplasms. It is imperative that metastases be considered a possibility for any tumor involving the pleura that is being evaluated by IHC. In this context, it is important to know clinical information concerning the patient, because these may have an influence on the IHC tests performed. However, the pathologic diagnosis is based on objective findings and not on the clinical history. Metastatic tumors of unknown primary origin are discussed in Chapter 8 and may involve the pleura. It has been shown that the most cost-effective way of evaluating a suspected metastatic tumor of unknown primary origin is by pathologic techniques, including IHC and electron microscopy.487,488 The histologic appearance of the tumor dictates the antibody selection in such neoplasms. Pathologists should remember the variable histologic spectrum of mesotheliomas so that uncommon histologic types of mesothelioma are not misdiagnosed as metastatic neoplasms. In cases of suspected pleural mesothelioma, clinical information may be helpful by demonstrating radiographically that a tumor is encasing the lung and also by providing evidence that a neoplasm is not identified in other organs or tissues. LOCALIZED MALIGNANT MESOTHELIOMA
Localized malignant mesotheliomas do occur,489,490 and they show the same histologic and IHC features as diffuse malignant mesotheliomas. However,
mesotheliomas may be diffuse and beyond the resolution of current radiographic techniques, such as CT and magnetic resonance imaging (MRI), and may initially appear to be localized. DECIDUOID MESOTHELIOMA
Deciduoid mesothelioma was first described in a 13-year-old girl491 and was subsequently found in other young women.492,493 These neoplasms are composed of large round cells that have abundant eosinophilic cytoplasm and large nuclei. Deciduoid mesotheliomas were first thought to occur only in women and were thought to represent some type of gynecologic neoplasm. In 2012, Ordonez494 reported 21 cases of deciduoid mesothelioma, which he investigated using a large panel of IHC markers, and nine of these cases were studied by electron microscopy. As pointed out by Ordonez, deciduoid mesothelioma is a rare variant of epithelioid mesothelioma that was initially considered to occur exclusively in the peritoneal cavity of young women who had no history of asbestos exposure. Deciduoid mesotheliomas were described as having an aggressive clinical course. Additional cases of deciduoid mesothelioma were observed in older women and men, and a significant number had exposure to asbestos. Some studies reported the clinical course to be no different than the typical conventional course of an epithelioid mesothelioma. In the study by Ordonez,494 15 patients were men, and 6 were women; the mean age was 60 years. Seventeen cases originated in the pleura, and four began in the peritoneum. Histologically, all cases were composed of large, polygonal, or ovoid cells of welldefined cell borders with dense eosinophilic cytoplasm and single or multiple nucleoli. In some cases, the cells exhibited a wide variation in size and shape. IHC evaluation of available cases showed positive staining for calretinin in 21 of 21 cases, pancytokeratin in 16 of 16, CK7 in 15 of 15, CK5/6 in 14 of 14, podoplanin in 13 of 14, WT1 in 11 of 13, and mesothelin in 8 of 8 cases. There was no immunostaining of the neoplastic cells for MOC-31, CEA, TAG-72 (B72.3), CD15 (LeuM1), TTF-1, or hCG. Ordonez concluded that the differences in prognosis reported in deciduoid mesotheliomas were due to the existence of a high-grade subgroup that presented with a highly aggressive clinical behavior.
Pleural Neoplasms
PLEOMORPHIC MESOTHELIOMA
Pleomorphic mesothelioma can cause difficulties in diagnosis.145 These neoplasms are composed of pleomorphic epithelioid and sarcomatoid cells, although occasional multinucleated macrophage giant cells are associated with the tumor cells. Pleomorphic mesotheliomas must be differentiated from pleomorphic carcinomas of the lung. In most instances, pleomorphic mesotheliomas express pancytokeratin and vimentin. Approximately 10% to 20% of cases express CK5/6, CK7, and calretinin. Rarely, they express mesothelin, WT1, and D2-40. A significant number of different types of pleomorphic mesotheliomas exist, and they can show a variety of patterns. Photographs of these can be seen on pages 614 and 615 of Dail and Hammar’s Pulmonary Pathology, third edition.145 In 2012, Ordonez495 reported 10 cases of pleomorphic mesothelioma that were investigated using a large panel of IHC markers. Four cases were also evaluated by electron microscopy. All patients within this study were men, and seven had a history of asbestos exposure; 90% of cases (9/10) originated in the pleura, and one originated in the peritoneum. This type of neoplasm shows a variety of sizes and shapes and contains abundant eosinophilic cytoplasm with single or multiple irregular nuclei with large, sometimes irregular nucleoli. Mitotic activity is frequent, and atypical mitoses have been observed. Ordonez found immunoreactivity for pankeratin and CK7 in all cases. Expression of calretinin, WT1, podoplanin (D2-40), mesothelin, and CK5/6 was frequent but variable. All cases were negative for MOC-31, CEA, CD15, TAG-72, and TTF-1, and patients who had pleomorphic mesotheliomas and underwent extrapleural pneumonectomy had extensive lymph node metastases. The median survival was only 8.2 months, indicating that mesothelioma with pleomorphic features are very aggressive tumors. SMALL CELL MESOTHELIOMA
In 1992, Mayall and Gibbs496 drew attention to a small cell variant of malignant mesothelioma, likely to be confused with SCLC. Falconieri and associates497 reported four cases of small cell carcinoma of lung with spread into the pleura that simulated pleural malignant mesothelioma. Most of the cases reported by Mayall and Gibbs496 represented autopsy cases with the potential for the small cell features to be explicable, in part, by postmortem artifact. Krismann and colleagues498 expressed doubt about the existence of small cell mesothelioma, because the German Mesothelioma Registry, which contained more than 6000 mesothelioma cases at that time (2004), did not contain a single example of small cell mesothelioma. In cases of small cell mesothelioma, it is important to realize that the tumor can often show transition from small cell areas to other areas, where the appearance is more characteristic of a typical epithelioid mesothelioma. The nucleocytoplasmic features of small cell mesothelioma differ subtly from those of small cell carcinoma, and the mesothelioma cells often possess greater amounts of cytoplasm.
463
The nuclei are often more open and vesicular with finely divided chromatin in comparison to the “salt-and-pepper” nuclear chromatin pattern characteristic of small cell carcinomas with nuclear molding. IHC studies of small cell mesotheliomas showed features characteristic of mesothelial differentiation with no evidence of neuroendocrine differentiation, such as that shown by immunostaining for synaptophysin or chromogranin. We have encountered extremely rare cases of mesothelioma in which some focal evidence of NE differentiation was apparent, but such cases do not appear to have been described in detail in the literature. In 2012, Ordonez499 reported eight cases of epithelioid mesothelioma with small cell features (7 men, 1 woman), all of which originated in the pleura. Four patients had a history of asbestos exposure; histologically, four mesotheliomas were epithelioid, and four were biphasic. The proportion of small cells seen in these cases constituted 80% to 100% of the tumor included in the biopsy material and 15% to 20% of the tumor in the pneumonectomy specimen. Immunoreactivity was identified for pankeratin, CK5/6, CK7, calretinin, WT1, podoplanin, and mesothelin. All cases were negative for MOC-31, BerEP4, CEA, CD15, TAG-72, TTF-1, chromogranin A, synaptophysin, CD99, and desmin. This type of mesothelioma was associated with poor survival; the mean survival of the six patients for whom information was available was 8.2 months. It appears that the best way to differentiate pseudomesotheliomatous SCLC from small cell mesothelioma is with TTF-1 (positive in 90% of SCLC, negative in mesothelioma), CEA (positive in ~30% to 50% of SCLC, negative in small cell mesothelioma), and synaptophysin (positive in 90% of SCLC, negative in most small cell mesotheliomas). ROUND CELL MESOTHELIOMA
Most pathologists recognize that epithelioid mesothelioma can show a variety of histologic patterns, such as histiocytoid and deciduoid. However, they might not recognize that epithelial mesotheliomas can be composed almost entirely of round cells that are smaller than histiocytic and deciduoid mesotheliomas, and smaller than small cell mesotheliomas, that look like small cell NE lung cancers.
Rare Primary Pleural Neoplasms Certain rare mesotheliomas can cause diagnostic difficulties if the pathologist is not aware of their existence. These are discussed below. MESOTHELIOMA WITH RHABDOID FEATURES
Rhabdoid tumors were first identified in young children as a primary kidney cancer. It then became apparent that rhabdoid tumors could occur in other parts of the body and could occur in older individuals as well as in infants and young children. This type of tumor can also be identified in the lung, and it can be difficult to make
464
Immunohistology of Lung and Pleural Neoplasms
the distinction among deciduoid, rhabdoid, and pleomorphic mesotheliomas. Typically, rhabdoid tumors have characteristic nuclear features: the nucleus is composed of generally large cells that have large nuclei and abundant nucleoli; it is usually pushed to one side, and an inclusion in the cytoplasm composed of intermediate filaments is evident. These paranuclear collections of intermediate filaments can occupy most of the cytoplasm of the tumor. Ordonez500 published 10 cases of mesothelioma with rhabdoid features, nine of which originated in the pleura, and one that originated in the peritoneum. Eight of the patients were men, and two were women; six patients had a history of asbestos exposure. Histologically, seven of the mesotheliomas were epithelioid, two were sarcomatoid, and one was biphasic. The proportion of rhabdoid cells seen in these cases varied significantly, from 15% to 75% of the individual tumors. Cytoplasmic staining in the rhabdoid cells was seen for pankeratin and vimentin in 10 of 10 cases, CK5/6 in 7 of 10 (70%), CK7 in 8 of 10 (80%), and calretinin in 9 of 10 cases (90%). Nuclear staining of WT1 was seen in 4 of 7 cases (57%), and only one case was desmin positive in sparse cells in the nonrhabdoid component of the tumor. Immunostaining was negative for CEA, MOC-31, TAG-72 (B72.3), CD15 (LeuM1), CD34, Bcl-2, MSA, and TTF-1. Median survival for five of six patients for whom information was available was 3.8 months, and one patient survived 1 year, making this a very aggressive tumor.
Epithelioid Mesothelioma Expressing Mucin and Showing Crystalloid Structures In 1996, I (S.P.H.) worked with Bockus and others501 to evaluate mucin-positive epithelial mesotheliomas that frequently expressed PAS-diastase, mucicarmine, and Alcian blue with colloidal iron. We subsequently studied this type of neoplasm by electron microscopy and IHC and found that these tumors typically express broadspectrum keratin, CK5/6, WT1, and calretinin. Ultrastructurally, these tumors show crystalloid structures that, in our experience, are relatively unique, although Henderson and colleagues referred to them in 1992.410 In 2012, Ordonez502 reported nine cases of epithelioid mesothelioma, out of a total of 59 cases examined, that contained crystalloid structures. One case had oncocytic features. Unlike our experience, the tumors did not have features of mucin reported. SOLITARY FIBROUS TUMORS OF THE PLEURA
Localized solitary fibrous tumors (SFTs) of the pleura are uncommon neoplasms thought to arise from subpleural connective tissue cells.503-506 They occur as neoplasms in the subvisceral-subpleural zone of the lung, within the pleural space attached to the pleura (usually the visceral pleura) by a small pedicle, or in a subparietal pleural location. Such neoplasms may become extremely large and may be associated with unusual clinical
situations such as hypoglycemia. Histologically, these neoplasms are composed of spindle cells with varying degrees of cellularity and various amounts of extracellular collagen. Identifying these neoplasms as malignant can be difficult; malignant criteria include 1) more than 4 mitoses/10 hpf, 2) hemorrhage, 3) necrosis, and 4) invasion into lung and chest wall.505 One hundred percent of localized fibrous tumors of the pleura express vimentin, and they are uniformly negative for keratin. Approximately 75% to 80% express CD34,506,507 and a slightly greater percentage express the antiapoptotic substance Bcl-2.508 Rare localized fibrous tumors of the pleura express actin.505 Schirosi and colleagues509 performed a clinicopathologic, IHC, and molecular study of 88 cases of pleuropulmonary SFTs to confirm the prognostic value of the de Perrot staging system510 and expression of p53, c-Kit, BRAF, PDGFR-α, PDGFR-β, c-Met, and EGFR. The de Perrot staging system is shown in Box 12-3. The authors found that 52 cases (59%) had at least one clinicopathologic feature related to malignancy, whereas mortality and recurrences occurred in 10.2% and 18.2% of cases respectively. Staging and high p53 expression were significantly related to conventional clinicopathologic prognostic features and to overall survival and diseasefree survival. With respect to multivariate analysis, high p53 expression and tumor necrosis were the only parameters associated with overall survival and diseasefree survival (P = .017 and .012, respectively). IHC expression was frequently detected for PDGFR-α (97.7%), PDGFR-β (86.5%), and hepatocyte growth factor receptor (96.6%). Missense mutations were only identified in two cases, and both involved PDGFR-β (exons 18 and 20). The de Perrot stratification of SFTs was a reliable prognostic indicator and merited consideration in view of its suggestions for the management of these tumors in daily practice, and p53 represented a valid and easy method to test prognosis. Although mutations of the corresponding genes were rare events in SFTs, PDGFR-α, PDGFR-β, and hepatocyte growth
Box 12-3 DE PERROT STAGING SYSTEM OF PLEUROPULMONARY SOLITARY FIBROUS TUMOR Stage 0: Tumor with peduncle without features of malignancy at histology Stage 1: Tumor with sessile or “inverted” appearance without features of malignancy at histology Stage 2: Tumor with peduncle with features of malignancy at histology Stage 3: Tumor with sessile or “inverted” appearance with features of malignancy at histology Stage 4: Multiple metastatic tumor Modified from Schirosi L, Lantuejoul S, Cavazza A, et al: Pleuropulmonary solitary fibrous tumors: a clinicopathologic, immunohistochemical, and molecular study of 88 cases confirming the prognostic value of de Perrot staging system and p53 expression, and evaluating the role of c-kit, BRAF, PDGFRs (alpha/ beta), c-met, and EGFR. Am J Surg Pathol 2008;32:1627-1642.
Pleural Neoplasms
465
TABLE 12-32 Comparison of Immunohistochemical Features of Localized Fibrous Tumors of the Pleura, Sarcomatoid Mesothelioma, and Soft Tissue Sarcomas
Sarcomatoid Mesothelioma
Localized Fibrous Tumor of Pleura
Soft Tissue Sarcoma
AE1/AE3 keratin
S
−
R
LMW keratin
S
−
R
HMW keratin
S
−
R
CK7
S
−
R
Antibody Directed Against
Vimentin
+
+
+
Actin
S
S
S*
Desmin
R
−
S*
S-100 Protein
R
−
S*
CD34
R
+
R*
Bcl-2
R
+
R
*Positivity depends on the type of soft tissue sarcoma. Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive −, almost always negative. CK, Cytokeratin; HMW, high molecular weight; LMW, low molecular weight.
factor receptor tyrosine kinases should be further investigated, given the availability of specific inhibitory molecules that might provide useful and novel therapeutic approaches for patients with SFTs. Localized fibrous tumors of the pleura are contrasted to sarcomatoid mesotheliomas and sarcomas in Table 12-32. PSEUDOMESOTHELIOMATOUS CARCINOMAS OF THE LUNG
Rare primary neoplasms of lung grow in a distribution characteristic of mesothelioma. Babolini and Blasi511 described five such neoplasms in 1956. In 1976, Harwood and associates,512 who introduced the term pseudomesotheliomatous carcinoma, reported six examples. In 1992, Koss and colleagues513 reported on 30 cases of pseudomesotheliomatous adenocarcinoma of lung, 15 from a review of the published literature and 15 from the files of the Armed Forces Institute of Pathology. Hartman and Schutz514 reported on 72 cases and designated these neoplasms as mesothelioma-like tumors of the pleura. In 1998, Koss and others515 described 29 cases of pseudomesotheliomatous adenocarcinoma of lung. Robb et al516 reported 17 cases of pseudomesotheliomatous carcinoma of lung in abstract form, and at this writing, we were preparing a report on more than 160 cases of pseudomesotheliomatous lung cancer, including rare varieties. Attanoos and Gibbs517 reported an additional 53 cases of pseudomesotheliomatous carcinoma in 2003.
Figure 12-52 Macroscopically, pseudomesotheliomatous adenocarcinomas of lung look like diffuse malignant mesotheliomas.
Macroscopically these neoplasms look almost identical to pleural mesothelioma (Fig. 12-52). As reported by Koss and colleagues513,515 and in my experience (S.P.H),516 the majority of pseudomesotheliomatous lung neoplasms are adenocarcinomas, and the most common histologic subtype is what we refer to as a tubulodesmoplastic pseudomesotheliomatous adenocarcinoma (Fig. 12-53). Pseudomesotheliomatous pulmonary adenocarcinomas are usually mucin positive and express the IHC markers of a pulmonary adenocarcinoma. Occasionally, these neoplasms show squamous and small cell NE differentiation. Some are large cell undifferentiated carcinomas, and some may be poorly
Figure 12-53 The most common histologic appearance of pseudomesotheliomatous adenocarcinoma is a tubulodesmoplastic pattern (×400).
466
Immunohistology of Lung and Pleural Neoplasms
differentiated and challenging to distinguish from a poorly differentiated mesothelioma. The poorly differentiated pseudomesotheliomatous carcinomas usually coexpress keratin and vimentin and may not express specific carcinoma or mesothelioma markers; in fact, they may be impossible to differentiate from mesothelioma. Koss and colleagues513,515 reported two biphasic variants and Robb et al516 observed one. The IHC profile of a pseudomesotheliomatous adenocarcinoma of the lung is the same as for the typical primary pulmonary adenocarcinoma. As Robb et al516 reported and as reported by Koss and associates,513,515 a significant percentage of these neoplasms occurs in individuals who were exposed to asbestos and who have elevated concentrations of asbestos in their lung tissue. PSEUDOMESOTHELIOMATOUS EPITHELIOID HEMANGIOENDOTHELIOMA
Rare neoplasms composed of endothelial cells that resemble epithelial mesotheliomas are referred to as pseudomesotheliomatous epithelioid hemangioendothelioma or as epithelioid hemangioendothelioma mimicking mesothelioma.518,519 One of the authors (S.P.H.) contributed a case to a series by Lin and colleagues,519 which involved a 50-year-old man with a history of potential exposure to asbestos while working at a hardware store
at age 20. He came to medical attention with a right pleural effusion and a tumor that encased the right lung. The initial biopsy was diagnosed by the treating pathologist as adenocarcinoma and by another pathologist as an epithelial mesothelioma. The pattern of immunoreactivity was confusing in that the neoplastic cells expressed LMW (35βH11) and HMW (34βE12) keratins, vimentin, CD31, FVIII antigen (Fig. 12-54), and CD34. Ultrastructurally, the neoplastic cells resembled endothelial cells and contained Weibel-Palade bodies in their cytoplasm (Fig. 12-55). Of note, keratin expression has been reported in normal endothelial cells and in vascular neoplasms.520 CALCIFYING FIBROUS PSEUDOTUMOR OF THE PLEURA
Calcifying fibrous pseudotumor of the pleura is a newly recognized fibrous soft tissue tumor of the pleura that occurs predominantly in younger individuals and presents as a pleural mass radiographically.521 Patients with this neoplasm usually present with chest pain and/or cough and vague chest discomfort. The tumor consists of circumscribed but unencapsulated masses of dense, hyalinized collagenous tissue interspersed with a lymphoplasmacytic infiltrate and calcium deposits, many of which have the appearance of psammoma bodies. The
A
B
C
D
Figure 12-54 This pseudomesotheliomatous epithelioid hemangioendothelioma was initially diagnosed as an adenocarcinoma and then as an epithelial mesothelioma. In this case, the neoplastic cells expressed keratin and endothelial cell markers CD31, factor VIII antigen, and vimentin (×400). CK, Cytokeratin; EMA, epithelial membrane antigen; WT1, Wilms tumor 1.
Pleural Neoplasms
467
mesothelioma. The cases presented by Moran and associates524 showed no radiographic evidence of a mediastinal tumor, and six cases showed histologic features of a mixed (lymphocytic-epithelial) thymoma. The neoplastic thymic epithelial cells express keratin and CD5. SYNOVIAL SARCOMAS
Figure 12-55 Ultrastructurally, this pseudomesotheliomatous epithelioid hemangioendothelioma contains Weibel-Palade bodies in many of the neoplastic cells (×42,000).
spindle cells show immunostaining for vimentin and no immunostaining for keratin, alpha actin, desmin, S-100 protein, CD34, Bcl-2, or CD117. PRIMARY DESMOID TUMORS OF THE PLEURA
These tumors resemble desmoid tumors in other locations and show infiltration of adjacent fat and skeletal muscle by plump spindle cells.522 Immunohistochemically, the neoplastic spindle cells show immunostaining for vimentin, desmin, SMA, and MSA. They are negative for S-100 protein and keratin. Ultrastructurally, the neoplastic cells resemble myofibroblasts. Andino and colleagues523 evaluated the expression of β-catenin and cyclin D1 in desmoid tumors and SFTs and compared the utility of these for distinguishing between these entities with that of other more commonly used stains. Four desmoid tumors (1 pulmonary, 1 pleural, and 2 pleural/chest wall) and five benign and six malignant SFTs of the pleura were studied with β-catenin, cyclin D1, ALK-1, CD34, vimentin, desmin, SMA, MSA, S-100 protein, and pancytokeratin. Diffuse moderate or strong nuclear staining was observed for β-catenin in all desmoid tumors, four of five benign SFTs, and two of six malignant SFTs. All cases except one benign SFT showed concurrent cytoplasmic staining. Nuclear and cytoplasmic cyclin D1 staining was observed in all groups. The best distinction between desmoid tumors and SFTs was provided by CD34; none of the desmoid tumors stained, and 8 of 11 SFTs (73%) expressed CD34. MSA expression was observed in four desmoid tumors but in none of the 11 SFTs. The authors concluded that alterations in the adenomatous polyposis coli/β-catenin pathway and cyclin D1 dysregulation may have contributed to the pathogenesis of pleuropulmonary desmoid tumors and SFTs. PRIMARY PLEURAL THYMOMAS
Thymomas may occur in the pleura and can be confused with mesothelioma.524-527 These neoplasms may cause confusion with sarcomatoid mesothelioma with a heavy lymphoid infiltrate or with a lymphohistiocytoid
Synovial sarcomas may occur as primary neoplasms in the pleura and may be confused histologically with biphasic and sarcomatoid mesotheliomas.528-530 The neoplastic epithelial cells express keratin and show glandular differentiation. Cells that form the glandular structures frequently express CEA and BerEP4 and are positive for neutral mucins, findings absent in most epithelial mesotheliomas. Ultrastructurally, the neoplastic epithelial cells in a synovial sarcoma are different than those of epithelial mesothelioma, having short microvilli and showing glycocalyceal bodies.531 Monophasic synovial sarcomas are difficult to differentiate from sarcomatoid mesotheliomas, although sarcomatoid synovial sarcoma tumor cells usually show Bcl-2 expression and may express CD99. Colwell and colleagues530 and Yano and coworkers532 reported SYT and SYX fusion genes in synovial sarcomas that can be identified by FISH. A variety of other sarcomas can occur in the lung and pleura and are beautifully described and illustrated by Litzky.533 PLEUROPULMONARY BLASTOMA
Pleuropulmonary blastomas are rare neoplasms that occur predominantly in infants and children and involve the lung and/or pleura,534,535 although rare cases occur in adults.536 The neoplasm is frequently cystic, and the cysts are lined by benign metaplastic epithelium that may be ciliated. The malignant component is composed of differentiated and/or anaplastic sarcomatous elements that may include fibrosarcoma, chondrosarcoma, embryonal rhabdomyosarcoma, and mixtures of these elements. The immunohistologic findings are dependent on the type of sarcomatous differentiation that occurs. LYMPHOMAS INVOLVING THE PLEURA
Relatively little information exists concerning pleural involvement by lymphoma. In 1992, Celikoglu and associates537 reviewed involvement of the pleura by lymphomas. In 2006, Vega and colleagues538 reported on pleural involvement by lymphoma. Of the 34 cases, 22 were men and 12 were women, with an average age of 62 years and a range in age from 22 to 88 years. In that study, 26.5% of patients (9/34) had pleural involvement as the only site of disease, whereas 64.7% (22/34) had other sites of involvement, and 8.8% (3/34) had inadequate staging data. The authors found that 56.2% (18/32) of patients with adequate clinical data had a history of lymphoma, including three patients with pleural involvement as the only disease site. In 85.3% (29/34) of cases, a specific diagnosis according to the WHO classification could be made: 58.6% (17/29) diffuse large B-cell lymphoma; 17.2% (5/29) follicular
468
Immunohistology of Lung and Pleural Neoplasms
lymphoma, including a case with areas of diffuse large B-cell lymphoma; 6.9% (2/29) small lymphocytic lymphoma/chronic lymphocytic leukemia; 5.9% (2/29) precursor T-cell lymphoblastic lymphoma/leukemia; and 3.4% (1 case each) mantle cell lymphoma, posttransplant lymphoproliferative disorder, and classic Hodgkin lymphoma. Five cases were B-cell lymphomas that could not be further classified; diffuse large B-cell lymphoma was the most frequent type found, followed by follicular lymphoma, approximately 60% and 20% respectively. Obviously, IHC is very important in classifying these lymphomas. As stated in the section on lung cancer, primary effusion lymphomas and pyogenic lymphomas occur.
Diagnostic Pitfalls of Pleural Neoplasms An important pitfall in accurately diagnosing mesothelioma is the failure of pathologists to recognize the many histologic patterns exhibited by epithelial and sarcomatoid mesotheliomas. Most IHC literature that discusses mesothelioma concerns epithelial mesothelioma, specifically well-differentiated and moderately well-differentiated epithelial mesotheliomas. Most
antibodies used to differentiate mesothelioma from adenocarcinoma include tumors that are well or moderately well differentiated. When mesotheliomas become more poorly differentiated, many relatively specific positive markers—such as HBME-1, calretinin, and CK5/6—fail to stain. Poorly differentiated mesotheliomas characteristically express LMW keratin and occasionally HMW keratin and vimentin. An absolute diagnosis of such mesotheliomas may be difficult, and the diagnosis may have to state that the histologic and immunohistologic findings are consistent with a poorly differentiated mesothelioma. The “clinical” (diffuse pleural) distribution of a pleural neoplasm can help support the diagnosis of mesothelioma. Mesotheliomas are not necessarily composed of a uniform population of cells that look the same and can show a variety of histologic types within the same mesothelioma.145 Several published papers discuss the IHC tests used to distinguish epithelial mesotheliomas from pulmonary adenocarcinomas and other carcinomas.402-404,539-541 These have been extensively reviewed by Ordonez,541-543 who in 2003 evaluated 60 unequivocal epithelial mesotheliomas and 50 lung adenocarcinomas with a large panel of antibodies (Table 12-33). The results of his evaluation are shown in Table 12-34. The results of published IHC studies concerning the various IHC tests541
TABLE 12-33 Antibodies Used to Distinguish Epithelial Mesothelioma From Pulmonary Adenocarcinoma and Other Carcinomas Marker
Source
Type
Dilution
Antigen Retrieval
B72.3
BioGenox
B72.3
1 : 300
None
BerEP4
Dako
MAb
1 : 30
Enzymatic digestion
Bg8 (Lewis y)
Signet
Bg8 MAb
1 : 50
Citrate
CA 19-9
Dako
MAb
1 : 50
Citrate
Calretinin
Zymed
PAb (rabbit)
1 : 20
Citrate
CD44S
Vector Laboratories
F10-44.2 MAb
1 : 75
Citrate
CEA
NeoMarkers
PAb (rabbit)
1 : 175
None
CK5/6
Boehringer-Mannheim
D5/16B4 MAb
1 : 25
Citrate
E-cadherin
Zymed
HECD-1 MAb
1 : 20
Citrate
EMA
Dako
E29 MAb
1 : 20
Citrate
HBME-1
Dako
MAb
1 : 50
Citrate
Leu-M1 (CD15)
Becton-Dickinson
Leu-M1 (MAb)
1 : 40
Tris-EDTA
Mesothelin
Novocastra
5B2 MAb
1 : 30
Tris-EDTA
MOC-31
Dako
MAb
1 : 50
Citrate
N-cadherin
Zymed
3B9 MAb
1 : 20
Tris-EDTA
Thrombomodulin
Dako
1005 MAb
1 : 50
Citrate
TTF-1
Dako
BG7G3/1 MAb
1 : 25
Citrate
Vimentin
Dako
V9 MAb
1 : 500
Citrate
WT1
Dako
6F-H2 MAb
1 : 40
Tris-EDTA
CEA, Carcinoembryonic antigen; CK, cytokeratin; EMA, epithelial membrane antigen; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor 1.
Diagnostic Pitfalls of Pleural Neoplasms
469
TABLE 12-34 Immunohistochemical Results of Epithelial Mesotheliomas and Pulmonary Adenocarcinomas Epithelial Mesotheliomas
Marker
Pulmonary Adenocarcinomas Grade of Reactivity
(n = 60) + cases (%)
Trace
0 (0)
Grade of Reactivity
(n = 50) + cases (%)
Trace
1+
2+
3+
4+
1+
2+
3+
4+
0
0
0
0
0
42 (84)
0
7
12
15
8
11 (18)
2
9
0
0
0
50 (100)
0
0
0
13
37
Bg8 (Lewis y)
4 (7)
2
2
0
0
0
48 (96)
1
6
7
15
21
CA 19-9
0 (0)
0
0
0
0
0
24 (48)
0
6
10
6
2
Calretinin
60 (100)
0
0
0
15
45
4 (8)
2
2
0
0
0
0 (0)
0
0
0
0
0
44 (88)
0
3
8
17
16
CD44S
44 (73)
5
7
4
11
17
24 (48)
0
14
7
2
1
CK5/6
60 (100)
E-cadherin
24 (40)
2
3
7
16
32
1 (2)
0
1
0
0
0
0
14
5
2
3
44 (88)
0
9
8
20
7
EMA
56 (93)
0
7
9
19
21
50 (100)
0
3
12
10
25
HBME-1
51 (85)
0
7
11
14
28
34 (68)
0
4
5
9
16
0 (0)
0
0
0
0
0
36 (72)
0
4
9
13
7
60 (100)
0
11
4
17
28
19 (38)
0
8
5
5
1
5 (8)
2
3
0
0
0
50 (100)
0
3
7
19
21
N-cadherin
44 (73)
1
5
13
14
11
15 (30)
2
4
3
6
0
Thrombomodulin
46 (77)
0
10
16
16
3
7 (14)
2
5
0
0
0
0 (0)
0
0
0
0
0
37 (74)
0
4
10
15
8
Vimentin
33 (55)
1
28
4
0
0
19 (38)
1
16
2
0
0
WT1
56 (93)
0
4
9
16
27
0 (0)
0
0
0
0
0
B72.3 (TAG-72) BerEP4
CEA
LeuM1 (CD15) Mesothelin MOC-31
TTF-1
CEA, Carcinoembryonic antigen; CK, cytokeratin; EMA, epithelial membrane antigen; TTF-1, thyroid transcription factor 1.
are summarized in Table 12-35. Based on their sensitivity and specificity, Ordonez concluded that calretinin, CK5/6, and WT1 were the best positive markers, although calretinin and CK5/6 were stated to be more sensitive than WT1, and thrombomodulin was found to be less sensitive and less specific than calretinin, CK5/6, and WT1. Mesothelin was found to be a highly sensitive marker for epithelial mesothelioma, although it was less specific. HBME-1, N-cadherin, and CD44S were stated to not be helpful. CEA, MOC-31, BerEP4, Bg8, and B72.3 were found to be the most specific and sensitive negative epithelial mesothelioma markers. TTF-1, LeuM1 (CD15), and CA 19-9 were stated to be highly specific but less sensitive. Ordonez543 concluded that from a practical viewpoint, a panel of four markers—two positive and two negative—allowed for the distinction between epithelial mesothelioma and pulmonary adenocarcinoma. Ordonez’s543 recommendation was to use calretinin and CK5/6 (or WT1) for the positive markers and CEA and MOC-31 (or B72.3, BerEP4, or Bg8) for the negative markers. Others544 have come to a slightly different selection of positive and negative markers. Calretinin expression has been reported in a fairly diverse group of nonmesothelial neoplasms but is often identified only in the cytoplasm.545-548 WT1 expression has also been identified in a variety of other neoplasms,549-552 and
nerve growth factor receptors TrkA and p75 have been reported.553 As mentioned earlier, a consensus statement from the International Mesothelioma Interest Group was published in 2009 to set the guidelines for pathologic diagnosis of malignant mesothelioma.479 These included a list of IHC markers used to differentiate epithelial pleural mesothelioma from squamous carcinoma of the lung (Table 12-36); epithelioid pleural mesothelioma from renal cell carcinoma; and peritoneal malignant mesothelioma from papillary serous carcinoma and nongynecologic adenocarcinoma. KEY DIAGNOSTIC POINTS Immunohistochemistry of Mesothelioma • Calretinin and CK 5/6 or WT1 are used for the positive markers, and CEA and either MOC-31 or B72.3, BerEP4, or Bg8 are used for the negative markers.
Mucin-Positive Epithelial Mesothelioma Unusual and uncommon types of mesothelioma should be mentioned here, because they can cause diagnostic confusion. Mucin-positive epithelial mesotheliomas are
613
HBME-1
637
715
298
Thrombomodulin
Vimentin
WT1
231
418
501 43-95
16-100
30-100
0-88
0-33
173
34
57-100
0-100
64-100
0-45
47-100
50-100
6-24
0-88
0-48
516
101
286
68
336
648
34
139
68
Positive Cases
77.7
60.3
51.4
19.5
5.0
86.2
51.9
90.1
5.0
72
86.2
13.2
10.5
8.0
Average Positive Cases (%)
154
354
430
173
693
249
123
183
1023
159
238
108
222
490
No. of Cases
8
73
109
167
521
175
116
16
840
100
74
105
210
412
Positive Cases
0-20
0-50
5-77
90-100
44-100
55-100
84-100
0-19
25-100
15-57
0-70
96-100
57-100
35-100
Range of Positive Cases (%)
10
19.5
21.9
97.5
84.6
71.7
96.2
6.2
81.6
54
9.5
98
93.8
82.1
Average Positive Cases (%)
Primary Pulmonary Adenocarcinoma
Not given
6
Not given
Not given
Not given
5
3
Not given
Not given
Not given
16
Not given
Not given
Not given
Useful
9
9
6
8
Not Useful
1
2
Sometimes Useful
1
Not Applicable
Conclusion of Study
CK, Cytokeratin; CEA, carcinoembryonic antigen; WT1, Wilms tumor 1. Ordonez NG: Role of immunohistochemistry in differentiating epithelial mesothelioma from adenocarcinoma: review and update. Am J Clin Pathol. 112:75–89, 1999.
265
MOC-31
1423
224
E-cadherin
LeuM1 (CD15)
309
CK5/6
495
CD44S
1392
805
Calretinin
CEA
316
Bg8 (Lewis )
1055
BerEP4
y
797
No. of Cases
B72.3
Antigen Tested for
Range of Positive Cases (%)
Epithelial Mesothelioma
TABLE 12-35 Summary of Immunohistochemical Tests Referred to by Ordonez
2
1
No Conclusion
470 Immunohistology of Lung and Pleural Neoplasms
Diagnostic Pitfalls of Pleural Neoplasms
471
TABLE 12-36 Immunohistochemical Markers Used to Differentiate Epithelioid Pleural Mesothelioma from Primary Pulmonary Adenocarcinoma Antigen
Epithelioid Pleural Mesothelioma
Primary Pulmonary Adenocarcinoma
CK5/6
Positive staining in 75%-100%
Focal positive staining in 5%-10%
Calretinin
Virtually all positive both nuclear and cytoplasmic staining required
Focally positive in 2%-19%
CEA
Focal positive staining in <5%
50%-90% positive
CD15 (LeuM1)
Rare focal positive staining
50%-70% positive
MOC-31
Focal positive staining in 2%-10%
90%-95% positive
BerEP4
Up to 20% are focally positive
95%-100% strongly positive
B72.3 (TAG-72)
Very few positive
75%-85% positive
Wilms tumor 1 (WT1)
Positive nuclear staining in 43%-93%
Negative
TTF-1
Negative
Nuclear positivity in 75%-85%
Cell membrane staining in 86%-100%
Focally positive in up to 7%
Focal reactivity in 3%-7%
89%-100% positive
D2-40 (podoplanin) y
Bg8 (Lewis )
Two positive markers (e.g., CK5/6 and calretinin) and two negative markers (e.g., CEA and TTF-1) should be used to discriminate an epithelioid malignant mesothelioma from a primary pulmonary adenocarcinoma. CEA, Carcinoembryonic antigen; CK, cytokeratin; TTF-1, thyroid transcription factor 1.
not uncommon, and patterns of mucin staining have been studied.501 Between 1% and 5% of all moderately differentiated to well-differentiated epithelial mesotheliomas show mucicarmine and/or PAS-diastase staining (Fig. 12-56). Mucin-positive epithelial mesotheliomas are the ones that most frequently express IHC markers that are negative in most epithelial mesotheliomas and that are usually positive in pulmonary adenocarcinomas (CEA, LeuM1, B72.3, BerEP4). Mucin-positive epithelial mesotheliomas express calretinin and HBME-1 and show cell membrane staining for EMA and HMFG-2. Ultrastructurally, mucin-positive epithelial mesotheliomas frequently contain crystalloid material (Fig. 12-57) in intracellular neolumens, in glandular spaces formed by neoplastic cells, or in the extracellular space. In our experience, this crystalloid material is unique to epithelial mesotheliomas.
Figure 12-56 This mucin-positive epithelial mesothelioma shows intracellular mucicarmine staining. This reaction can result in an incorrect diagnosis of a mucin-producing adenocarcinoma (×400).
Atypical Carcinoid Presenting as Mesothelioma Two cases of atypical carcinoid presenting as mesothelioma were reported by van Hengel.554 In both cases, the individuals had been exposed to asbestos. A recently observed case of a neoplasm composed of nodules of fleshy tumor involving the visceral and parietal pleura was also reported (Fig. 12-58). In this case, the tumor
Figure 12-57 In this mucin-positive epithelial mesothelioma, crystalloid material is located in glandular spaces formed by the neoplastic cells and in intracellular neolumens (×20,000).
472
Immunohistology of Lung and Pleural Neoplasms
Figure 12-58 This neoplasm consists of fleshy nodules attached to the visceral and parietal pleura.
was composed of neoplastic cells that showed carcinoidlike features, and the individual cells showed a very high mitotic rate (up to 10/hpf) and focal areas of necrosis (Fig. 12-59). These neoplastic cells showed immunostaining for synaptophysin, chromogranin-A, and TTF-1 (Figs. 12-60 through 12-62).
Lymphohistiocytoid Mesothelioma Lymphohistiocytoid mesothelioma555 is a rare form of sarcomatoid mesothelioma that was initially diagnosed histologically as a lymphoma. Lymphohistiocytoid mesothelioma is composed of large, mostly round cells admixed with numerous inflammatory cells (Fig. 12-63).
Figure 12-59 Histologically, this neoplasm has the features of an atypical carcinoid.
This neoplasm can be misdiagnosed as a large cell lymphocytic lymphoma. The neoplastic cells characteristically express LMW and HMW keratins and vimentin. Most lymphohistiocytoid mesotheliomas are negative for calretinin, HBME-1, and EMA. Ultrastructurally, the neoplastic cells show intercellular junctions and intracellular tonofilaments.
Well-Differentiated Papillary Epithelial Mesothelioma Well-differentiated papillary epithelial mesothelioma can cause diagnostic confusion, often because pathologists are not familiar with this neoplasm.355-357,556 Most
Figure 12-60 These neoplastic cells express synaptophysin.
Diagnostic Pitfalls of Pleural Neoplasms
473
Figure 12-61 These neoplastic cells express chromogranin A.
cases occur in the peritoneal cavity of young women (20- to 30-year age group), although these now have been reported in the pleura.357,557 Macroscopically, they present as multiple serosal surface nodules that typically involve the omentum, mesentery, pelvic cavity, and pleura. The nodules vary from a few millimeters to several centimeters in maximum dimension. Histologically, they show tubulopapillary differentiation and are composed of well-differentiated, relatively uniform cuboidal cells (Fig. 12-64). The IHC pattern is generally that of a “typical” well-differentiated epithelial mesothelioma and, in most cases we have seen, they show cell membrane immunostaining for EMA and no immunostaining for desmin. These tumors differ from “typical” epithelial mesotheliomas in that they usually have a good prognosis and do not rapidly progress. However, cases also occur that progress, invade, and cause death.
Cystic Mesothelioma Some epithelial mesotheliomas are composed of small cysts formed by uniform cuboidal mesothelial cells and associated numerous blood vessels (Fig 12-65). This type of mesothelioma may be difficult to differentiate from a vascular neoplasm. The epithelial mesothelial
Figure 12-62 The nuclei of these neoplastic cells show immunostaining for thyroid transcription 1 (TTF-1).
Figure 12-63 This mesothelioma is composed of large, round cells admixed with numerous lymphoid cells. Histologically, this tumor resembles a lymphoma.
cells may contain intracytoplasmic hemosiderin (Fig. 12-66). The immunophenotype of such neoplasms is identical to that of other epithelial mesotheliomas, and the vascular proliferation may be related to an endothelial growth factor produced by neoplastic mesothelial cells.558
Adenomatoid Tumors Adenomatoid tumors are localized benign mesothelial proliferations that most frequently occur in the
Figure 12-64 This well-differentiated papillary epithelial mesothelioma involving pleura shows papillary differentiation and is composed of relatively small, uniform cuboidal cells. This type of mesothelioma usually pursues a benign clinical course (×200).
474
Immunohistology of Lung and Pleural Neoplasms
aware, however, that epithelial mesotheliomas may present in lymph nodes as metastatic tumors of unknown primary origin.565
Primary Papillary Serous Carcinoma of the Serosa
Figure 12-65 This epithelial mesothelioma is composed of small cysts associated with numerous blood vessels (×200).
epididymis and cornua of the uterus.559 Adenomatoid tumors have also been identified in the adrenal gland560 and pancreas.561 These tumors are formed by uniform small cuboidal cells and can appear as invasive neoplasms. They express keratin and other markers of mesothelial cells and have the characteristic ultrastructural features of mesothelial cells. Adenomatoid tumors have also been reported in the pleura.145
Mesothelial Cells in Lymph Nodes Hyperplastic mesothelial cells may be found in mediastinal lymph nodes and may simulate metastatic epithelial mesothelioma or carcinoma.562,563 These atypical mesothelial cells usually occur in nodal sinuses and are most prominent in the subcapsular sinuses. They may be confused with macrophages and, in fact, Ordonez and associates564 reported lesions composed of cells thought to represent reactive mesothelial cells that were, in fact, macrophages. The mesothelial cells express the usual “positive” epithelial-mesothelial cell markers and are negative for the antigens that are characteristically negative in epithelial mesothelioma. One must be
Figure 12-66 Neoplastic mesothelial cells show abundant intracytoplasmic hemosiderin as demonstrated in this Prussian-Blue ironstained section (×200).
Well-differentiated epithelial mesotheliomas and welldifferentiated papillary epithelial mesotheliomas must be differentiated from primary papillary serous carcinomas of the serosa, nearly all of which occur in the peritoneal cavity of women.442,566-576 One case was reported in a man,574 and rare cases involve the pleural cavity.369 Histologically, these neoplasms are well differentiated and exhibit a papillary morphology. Psammoma bodies are more commonly seen in primary papillary carcinomas than in papillary epithelial mesotheliomas, although psammoma bodies are seen in papillary mesotheliomas. Primary serosal papillary carcinomas are usually mucicarmine and PAS-diastase negative. By IHC, the staining pattern can be similar to epithelial mesotheliomas, although Khoury and colleagues576 found serous papillary tumors to express one or more of the antigens CEA, LeuM1 (CD15), and TAG-72 (B72.3). In 17 of 20 cases, 7 expressed monoclonal CEA, 6 expressed LeuM1, and 13 were positive for B72.3 when tissue sections were predigested with pepsin. Ultrastructurally, primary papillary serous carcinomas show occasional cilia and outpouchings of straight and relatively short microvilli covered by a fuzzy glycocalyx, and sometimes the microvilli are longer and are branched.
Sarcomatoid Renal Cell Carcinomas Sarcomatoid renal cell carcinoma (RCC) can metastasize to the pleura in a macroscopic distribution identical to mesothelioma and can very closely simulate a sarcomatoid mesothelioma.577 Sarcomatoid RCC is considered to be a pseudomesotheliomatous carcinoma but not a primary pulmonary pseudomesotheliomatous carcinoma. The immunostaining pattern of such neoplasms can be identical to a sarcomatoid mesothelioma, and the situation can be complicated further, because sarcomatoid mesotheliomas can metastasize to the kidney. If an individual is known to have a sarcomatoid RCC and also has a spindle cell neoplasm of the pleura, the pathologist must consider the possibility of a metastatic sarcomatoid RCC. If the tumor shows a clear cell and a sarcomatoid pattern, metastatic RCC is most likely, although mesotheliomas may show a clear cell pattern.578 Pathologists should be aware that RCC-associated markers, such as erythropoietin and CD10, can be identified in diffuse malignant mesotheliomas and in metastatic RCCs. Butnor and colleagues579 evaluated 100 diffuse malignant mesotheliomas and 20 metastatic RCCs for immunoexpression of erythropoietin. These same cases and an additional 45 diffuse malignant mesotheliomas were evaluated for CD10 and the RCC marker RCCma. Erythropoietin was expressed in 100% of diffuse malignant mesotheliomas and metastatic
Diagnostic Pitfalls of Pleural Neoplasms
RCCs. Staining for CD10 was observed in 54% of diffuse malignant mesotheliomas and in 100% of metastatic RCCs. RCC marker stained 26% of diffuse malignant mesotheliomas and 55% of metastatic RCCs. Given the overlap in the expression of RCC markers in metastatic RCC and diffuse malignant mesothelioma, these markers must be interpreted cautiously and should be used in conjunction with mesothelial-associated markers. The authors also concluded that differences in expression may potentially help distinguish metastatic RCC from diffuse malignant mesothelioma, inasmuch as strong and diffuse expression of RCC marker and CD10 supports a diagnosis of metastatic RCC over diffuse malignant mesothelioma. Husain and associates479 listed a panel of IHC markers to aid in the differential diagnosis between epithelioid pleural mesothelioma and RCC; these are listed in Table 12-37.
Separation of Benign and Malignant Mesothelial Cell Proliferations As reported by Henderson and coworkers,358 nonneoplastic reactive proliferative pleural lesions may very
TABLE 12-37 Immunohistochemical Markers to Differentiate Epithelioid Pleural Mesothelioma From Renal Cell Carcinoma Antigen
Pleural Epithelioid Mesothelioma
Renal Cell Carcinoma
CK5/6
75%-100% positive
Negative
Calretinin
Virtually all positive both nuclear and cytoplasmic staining required
4%-10% focally positive
CD15 (LeuM1)
Rare focal positivity can stain any necrotic tissue
63% positive
MOC-31
2%-10% show focal staining
50% positive
BerEP4
Up to 20% are focally positive
42% positive
Mesothelin
100% positive
Negative
D2-40 (podoplanin)
86%-100% positive cell membrane distribution
Negative
WT1
43%-93% positive nuclear staining
4% positive
RCC marker
8% focally positive
50%-70% positive
Bg8 (Lewisy)
3%-7% positive
4% positive
BerEP4 and Bg8 were not considered useful in differentiating between epithelioid pleural mesothelioma and renal cell carcinoma. MOC-31 was considered to be of limited utility. CK, Cytokeratin; WT1, Wilms tumor 1.
475
closely simulate mesothelioma and may cause the most difficult diagnostic challenge in pleural pathology. Several publications have evaluated the issue of benign/reactive versus malignant mesothelial proliferations.361,362,479 In 2012, Churg and Galateau-Salle361 discussed the issues involved in the separation of benign and malignant mesothelial proliferations. The IHC markers that have been claimed in the literature to separate benign from malignant mesothelial proliferations include pancytokeratin, seen in benign and malignant mesothelial processes; desmin, claimed to be a marker of benign mesothelial cells; and EMA, p53, GLUT-1, X-linked inhibitor of apoptosis, and IMP3, all of which are claimed to be markers of malignancy. The authors concluded that the aforementioned markers have no diagnostic utility in an individual case for separating benign from malignant mesothelial cells. Kato and associates580 reported that in histologic sections, 40 of 40 mesotheliomas and 0 of 40 reactive mesotheliomas stained for glucose transporter 1 (GLUT-1) whereas Monaco and colleagues581 found positive staining for 27 of 41 mesotheliomas (66%), 5 of 70 benign mesothelial proliferations (7%), and 1 of 14 cases of atypical hyperplasia (7%). Wu and associates582 observed staining for X-linked inhibitor of apoptosis in 0 of 9 “normal” mesotheliomas, 1 of 13 hyperplasias (8%), and 25 of 31 mesotheliomas (81%). Shi and coworkers583 reported that of 64 reactive mesothelial proliferations, none stained for IMP3, whereas 33 of 45 mesotheliomas (73%) did stain for IMP3, in addition to two cases of atypical hyperplasia that later turned out to be mesothelioma. The authors opined that at this point in time, the literature on these three markers is too scanty to recommend them for general use, and the overall use of IHC staining has yet to provide markers that reliably separate benign from malignant mesothelial proliferations. In 2012 Lagana and associates584 studied the utility of GLUT-1 in the distinction between benign and malignant thoracic and abdominal mesothelial lesions. They studied 135 malignant peritoneal mesotheliomas and 30 malignant pleural mesotheliomas and compared them with 56 reactive mesothelial lesions stained with GLUT-1 monoclonal antibody; overall, the sensitivity for GLUT-1 was 53%, and the specificity was 98%. The sensitivity for epithelioid malignant mesothelioma was 49%, and the sensitivity in sarcomatoid/biphasic malignant mesothelioma was 66%. In the thorax, the sensitivity was 50%, and the sensitivity in peritoneum was 54%. The positive predictive value of GLUT-1 immunoreactivity was 98%, and the negative predictive value was 40%. The authors concluded that GLUT-1 staining of pleural mesotheliomas showed higher specificity but lower sensitivity than previously reported, and peritoneal mesotheliomas showed similar results. In both sites, thoracic and abdominal, the utility of GLUT-1 was limited by nonspecific staining (e.g., in necrotic areas) and by brightly staining erythrocytes and occasional lymphoid elements. Nonetheless, GLUT-1 could help differentiate malignant mesothelioma from reactive or otherwise benign mesothelium.
476
Immunohistology of Lung and Pleural Neoplasms
TABLE 12-38 Differentiating Reactive Mesothelial Hyperplasia From Mesothelioma
Characteristics
Reactive Mesothelial Hyperplasia
Mesothelioma
Stromal invasion
Absent (beware of entrapment and en face cuts)
Usually apparent (highlight with pancytokeratin staining)
Cellularity
May be prominent but within the mesothelial space and not the stroma
Dense, including cells surrounded by stroma
Papillae
Simple single cell layers
Complex tubules, cellular stratification
Necrosis
Rare
Occasionally present
Inflammation
Common
Usually minimal
Growth
Uniform (highlighted with cytokeratin staining)
Disorganized expansile nodules highlighted on cytokeratin staining
EMA, p53
Usually negative
Often positive
Desmin
Often positive
Often negative
Mitotic activity
Usually not useful
Cytologic atypia
Usually not useful
EMA, Epithelial membrane antigen.
MALIGNANT MESOTHELIOMA VS. REACTIVE MESOTHELIAL HYPERPLASIA
In 2009, the International Mesothelioma Interest Group479 contrasted the difference between reactive mesothelial hyperplasia and mesothelioma (Table 12-38). Comparison of the IHC features of reactive mesothelial hyperplasia versus epithelial mesothelioma is shown in Table 12-39. Attanoos and colleagues585 evaluated 60 cases of malignant pleural mesothelioma and 40 cases of reactive mesothelial hyperplasia with antibodies stained against desmin, EMA, p53, Bcl-2, P-glycoprotein, and platelet-derived growth factor receptor (PDGF-R) beta chain by the avidin-biotin complex method. The cohort of malignant pleural mesotheliomas was immunoreactive to desmin, EMA, and p53 in 6 of 60 (10%), 48 of 60 (80%), and 27 of 60 cases (45%), respectively. In contrast, the cohort of reactive mesothelial hyperplasia was immunoreactive to desmin, EMA, and p53 in 34 of 40 (85%), 8 of 40 (20%), and 0 of 40 cases, respectively. In a smaller cohort (n = 15) of malignant pleural mesotheliomas, Bcl-2, P-glycoprotein, and PDGF-R beta were expressed in 0 of 15, 2 of 15 (13%),
and 15 of 15 cases, respectively. In a small cohort of reactive mesothelial hyperplasias (n = 15), Bcl-2, P-glycoprotein, and PDGF-R beta were immunoreactive in 0 of 15, 0 of 15, and 6 of 15 (40%) cases respectively. Desmin and EMA appeared to be the most useful markers in distinguishing benign from malignant mesothelial cell proliferation; desmin appeared to be preferentially expressed in reactive mesothelium, and EMA was preferentially expressed in neoplastic mesothelium. IHC detection of mutated p53 oncoprotein appeared to be of less utility in this study on account of the low marker sensitivity for malignant mesothelioma. Bcl-2, P-glycoprotein, and PDGF-R beta chain appeared to be of no use in distinguishing reactive from neoplastic mesothelium. Wu and associates582 evaluated benign and malignant mesothelial tissue samples for the presence of X-linked inhibitor of apoptosis protein (XIAP), a potent constituent of the inhibitor of apoptosis family of caspase inhibitors. The authors studied 31 malignant mesotheliomas, 2 well-differentiated peritoneal mesotheliomas, 13 pleural mesothelial hyperplasias, and 9 benign mesothelial tissues from archival FFPE surgical tissue blocks with citrate-based antigen retrieval. All nine normal mesothelial samples were negative for XIAP. Of the 13 mesothelial hyperplasias, one was weakly positive in fewer than 10% of cells, as was one of the two welldifferentiated papillary peritoneal mesotheliomas. Of the 31 malignant mesotheliomas, 25 (81%) expressed XIAP positivity. The authors concluded that XIAP immunostaining, when strong, allows for distinction of malignant from benign and hyperplastic mesothelial cell populations and is a potentially useful immunodiagnostic marker in small samples and morphologically controversial cases. Kato and colleagues580 evaluated whether GLUT-1 has diagnostic utility for the differential diagnosis
TABLE 12-39 Comparison of the Immunohistochemical Features of Reactive Mesothelial Hyperplasia vs. Epithelial Mesothelioma Mesothelial Hyperplasia
Epithelial Mesothelioma
Desmin
S
R
Epithelial membrane antigen
R
S
Glucose transporter 1 (GLUT-1)
−
+
XIAP
R
S
Bcl-2
−
−
p-Glycoprotein
−
R
PDGF-R
S
+
Antibody
Reactivity: +, almost always diffuse, strong positivity; S, sometimes positive; R, rare cells positive; −, almost always negative. GLUT-1, Glucose transporter 1; PDGF-R, platelet-derived growth factor receptor; XIAP, X-linked inhibitor of apoptosis protein.
Molecular Biology and Theranostic Features in Diffuse Malignant Mesothelioma
477
TABLE 12-40 Differentiating Fibrosing Pleuritis From Desmoplastic Mesothelioma Characteristics
Fibrous Pleurisy
Desmoplastic Mesothelioma
Storiform pattern
Not prominent
Often prominent
Stromal invasion
Absent
Present (use pancytokeratin to confirm)
Necrosis
If present, at surface and often associated with acute inflammation
Bland, paucicellular collagenized tissue
Thickness
Uniform
Uneven with disorganized growth, expansile nodules, and abrupt changes in cellularity
Maturation
Hypercellularity at the surface and deep, decreased cellularity (so-called zonation)
Lack of maturation from the surface to depths of the process
Orientation
Perpendicularly oriented
Without paucity of vessels
Cellularity
Usually not useful
Atypia (unless severe)
Usually not useful
Mitotic activity (unless numerous atypical mitotic figures)
Usually not useful
between reactive mesothelium and malignant pleural mesotheliomas. They found IHC GLUT-1 expression in 40 of 40 malignant pleural mesotheliomas, and in all cases, the expression was demonstrated by linear plasma membrane staining, sometimes with cytoplasmic staining in addition. GLUT-1 expression was also observed in 96.6% (56/58) of lung carcinomas. No reactive mesothelium cases were stated to have expressed GLUT-1, therefore the authors concluded that GLUT-1 was a sensitive and specific IHC marker that enables differential diagnosis of reactive mesothelioma from malignant pleural mesothelioma, although it is not useful in discriminating malignant pleural epithelial mesothelioma from lung carcinoma. Acurio and associates586 evaluated IHC profiles of 85 mesothelial tissues, including 20 normal, 20 hyperplastic, and 45 malignant mesotheliomas using desmin, EMA, p53 protein, and GLUT-1. The p53 failed to distinguish between benign and malignant mesothelial lesions, but desmin was stated to have identified benign mesothelium and distinguished it from malignant mesothelioma. EMA and GLUT-1 were stated to have been positive in the majority of malignant mesotheliomas and negative or only weakly positive in benign mesothelial tissues. Because some malignant lesions were negative for EMA and GLUT-1, diagnosis should not be based exclusively on immunoreactivity. Instead, EMA and GLUT-1 could be used as part of an IHC profile and could be used as adjuncts to histomorphology in the diagnosis of malignant mesothelioma. DESMOPLASTIC MESOTHELIOMA VERSUS REACTIVE FIBROSING PLEURITIS
Another area of difficulty in pleural pathology is the differentiation between fibrosing pleuritis and desmoplastic mesothelioma.361,479 IHC is not particularly useful in differentiating fibrosing pleuritis from desmoplastic mesothelioma, because the spindle cells of both conditions characteristically express keratin and
vimentin. A comparison for differentiating fibrosing pleuritis from desmoplastic mesothelioma is shown in Table 12-40.
Molecular Biology and Theranostic Features in Diffuse Malignant Mesothelioma One of the most common genetic alterations in mesothelioma is the homozygous deletion of the 9p21 locus within a cluster of genes that includes CDKN2A, CDKN2B, and MTAP.587,588 Several cytogenetic and molecular studies have reported CDKN2A (p16) deletions in as many as 72% of primary mesotheliomas.589,590 Recent studies demonstrated that this alteration, detected by FISH, may be useful for differentiating benign from malignant mesothelial proliferations in surgical and cytology specimens.591,592 Homozygous deletion of 9p21 is an adverse prognostic factor in malignant mesotheliomas.588,593 Allen and colleagues594 constructed a tissue microarray of 19 cases of diffuse malignant mesothelioma, which included 19 cases of diffuse malignant mesothelioma with rare long survival (>3 years) and 21 cases that were typical of diffuse malignant mesothelioma with short survival. The authors found that osteopontin and hypoxia-inducible factor 1 (HIF-1) were commonly overexpressed in diffuse malignant mesothelioma, including both long- and short-survival varieties. The lack of osteopontin and HIF-1 were observed only in the long-survival diffuse malignant mesothelioma cases, suggesting the lack of osteopontin and HIF-1 expression may have had some role in prolonging survival. Chung and coworkers595 studied dual-color FISH for CDKN2A and chromosome 9 on paraffin-embedded sections in 56 biopsy or resected malignant mesothelioma cases (43 epithelioid, 1 sarcomatoid, 12 biphasic), 11 reactive mesothelial proliferations, and 8 equivocal
478
Immunohistology of Lung and Pleural Neoplasms
biopsy cases for which a histopathologic distinction between benign and malignant was uncertain. Others found a high prevalence of CDKN2A deletion in malignant mesothelioma, which was consistent with the findings. The findings of CDKN2A homozygous or hemizygous deletion by FISH added further support for a malignant diagnosis. Xu and colleagues596 studied IHC KOC/IMP3 in 53 malignant pleural mesotheliomas and 12 reactive mesothelial hyperplasias. The K homology domain contained proteins overexpressed in cancer (KOC), also known as IMP3 and L523S, and were members of the insulin-like growth factor family. KOC/IMP3 was strongly and diffusely expressed in a large proportion of malignant pleural mesotheliomas and was only occasionally expressed in reactive mesothelial hyperplasias, suggesting this marker’s usefulness in a diagnostic setting in distinguishing malignant mesothelioma from benign mesothelial lesions. The high frequency of expression in sarcomatoid and biphasic malignant pleural mesotheliomas suggested KOC expression could be associated with an aggressive biologic behavior. Gardner and colleagues597 studied the expression of matrix metalloproteinase-7 (MMP-7) in 45 diffuse malignant mesotheliomas using a tissue microarray and concluded that strong expression of MMP-7 in their series indicated MMP-7 was likely to play a role in the progression of malignant mesothelioma. Moore and associates598 studied topoisomerase II-α, minichromosome maintenance protein 2 (MCM2), and XIAP expression in diffuse pleural malignant mesotheliomas using tissue microarrays immunostained for these substances. The authors found increased expression of ProExC and XIAP in a substantial percentage of diffuse pleural malignant mesotheliomas with a higher positive rate in epithelioid versus sarcomatoid subtypes. Westerhoff and coworkers599 studied c-Met receptor tyrosine kinase (c-Met) by IHC in 24 malignant mesotheliomas (18 epithelioid, 3 sarcomatoid, and 3 biphasic). Phosphorylated protein kinase C (p-PKC) β2 expression was an adverse prognostic factor in malignant mesothelioma. They also found that c-Met expression was an adverse prognostic factor, and the expression of p-Met correlated with the downstream target of KDR (formerly VEGFR2) p-PKC β2. Their study suggested that dual targeting of c-Met and p-PKC β2 could be an important therapeutic strategy in malignant mesothelioma.
Prognosis of Mesothelioma Based on Morphology and Immunohistochemical Analysis Most pathologists realize that sarcomatoid mesotheliomas are much more aggressive neoplasms than
epithelioid mesotheliomas and, in general, patients with sarcomatoid mesothelioma have a much shorter life expectancy of 3 months. In contrast, some mesotheliomas—such as papillary, tubulopapillary, and those made up of relatively uniform small cells—are not as aggressive, although it is very difficult to predict what an individual patient’s survival rate would be. Kadota and colleagues600 studied a nuclear grading system of diffuse pleural epithelioid mesothelioma and discovered that the larger the nucleus and nucleolus, the worse the prognosis. The authors studied 232 epithelioid, diffuse, malignant pleural mesotheliomas (stage I, 14 cases; stage II, 54 cases; stage III, 130 cases; stage IV, 34 cases) and evaluated them for 1) nuclear atypia, 2) nuclear/cytoplasmic ratio, 3) chromatin pattern, 4) intranuclear inclusions, 5) prominence of nucleoli, 6) mitotic count, and 7) atypical mitoses. Median overall survival of all patients was 16 months and correlated with nuclear atypia (P < .001), chromatin pattern (P = .031), prominence of nucleoli (P < .001), mitotic count (P < .001), and atypical mitosis (P < .001) by univariate analysis. Multivariate analysis revealed nuclear atypia (P = .012) and mitotic count (P < .001) as independent prognostic factors. These two factors were used to create a three-tier nuclear grade score as follows: • Grade I (n = 107, median overall survival, 28 months) • Grade II (n = 91, median survival, 14 months) • Grade III (n = 34, median survival, 5 months) The authors found nuclear grade to be an independent predictor of overall survival, but it was also a stronger discriminator of survival than all currently available factors. Furthermore, nuclear grade was associated with time to recurrence (P = .004) in patients who underwent complete surgical resection (n = 159). MIB-1 labeling index correlated with mitotic count (P < .001), nuclear atypia (P = .037), stratified overall survival (P < .001), and time of recurrence (P = −0.048), confirming the prognostic value of the nuclear grade. Whether this grading system is of value or use to the clinician/oncologist is worthy of consideration.
Summary IHC is a valuable technique in accurately diagnosing primary and metastatic tumors of the lung and pleura. As with all adjuvant diagnostic pathologic techniques, the results of IHC tests have to be correlated with histologic, histochemical, and ultrastructural observations and with clinical observations. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Han H, Landreneaux RJ, Santucci TS, et al: Prognostic value of immunohistochemical expression of p53, HER-2-neu and bcl-2 in stage I non-small cell lung cancer. Hum Pathol. 33:105–110, 2002. 2. Minami K, Saito Y, Imamura H, et al: Prognostic significance of p53, Ki67, VEGF and GLUT-1 in resected stage I adenocarcinoma of the lung. Lung Cancer. 38:51–57, 2002. 3. Nakanishi K, Kawai T, Kumaki F, et al: Survivin expression in atypical adenomatous hyperplasia of the lung. Am J Clin Pathol. 120:712–719, 2003. 4. Yamaguchi NH, Lichetenfels AJ, Demarchi LMM, et al: Cox-2, MMP-9 and Noguchi classification provide additional prognostic information about adenocarcinomas of the lung: a study of 117 patients from Brazil. Am J Clin Pathol. 121:78–86, 2004. 5. Saad RS, Liu Y, Han H, et al: Prognostic significance of HER2/ neu, p53 and vascular endothelial growth factor expression in early stage conventional adenocarcinoma and bronchioloalveolar carcinoma of the lung. Mod Pathol. 17:1234–1242, 2004. 6. Pelosi G, Pasini F, Fraggetta F, et al: Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids. Lung Cancer. 42:203– 213, 2003. 7. Pelosi G, Pastorini O, Pasini F, et al: Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer. Br J Cancer. 88:537–547, 2003. 8. Jagirdar J: Immunohistochemistry: Then and Now. Arch Pathol Lab Med. 132:323–325, 2008. 9. Dubinski W, Leighl NB, Tsao M-S, et al: Ancillary testing in lung cancer diagnosis. Pulm Med. 2012:249082, 2012. 10. Travis WD, Brambilla E, Noguchi M, et al: International Association for the Study of Lung Cancer/American Thoracic Society/ European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma. J Thorac Oncol. 6:244– 285, 2011. 11. Yoshizawa A, Motoi N, Riely GJ, et al: Prognostic significance of the proposed IASLC/ATS/ERS revised classification of lung adenocarcinoma in 514 stage 1 lung adenocarcinomas (ADC). USCAP 2010 Annual Meeting Abstract, Publication 1859. 12. Moll R, Franke WW, Schiller DL, et al: The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell. 31:11–24, 1982. 13. Moll R, Lowe A, Laufer J, et al: Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Surg Pathol. 140:427–447, 1992. 14. Ramaekers F, Huysmans A, Schaart G, et al: Tissue distribution of keratin 7 as monitored by a monoclonal antibody. Exp Cell Res. 170:235–249, 1987. 15. Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases cases. Mod Pathol. 13:962–972, 2000. 16. Sano H, Tanaka T, Fukuoka J: Central and peripheral squamous cell carcinoma of the lung. Are they different? USCAP 2012 Annual Meeting Abstract; Publication 2041. 17. Hammar SP, Hallman KO: Unusual primary lung neoplasms: spindle cell and undifferentiated carcinomas expressing only vimentin. Ultrastruct Pathol. 14:407–422, 1990. 18. Upton MP, Hirohashi S, Tome Y, et al: Expression of vimentin in surgically resected adenocarcinomas and large cell carcinomas of lung. Am J Surg Pathol. 10:560–567, 1986. 19. Guazzi S, Price M, DeFelice M, et al: Thyroid nuclear factor-1 (TTF-1) contains a homeodomain and displays a novel DNAbinding specificity. EMBO J. 9:3631–3639, 1990. 20. Lazzaro D, Price M, DeFelice M, et al: The transcription factor TTF-1 expressed at the onset of thyroid and lung morphogenesis and in the restricted regions of the foetal brain. Development. 113:1093–1094, 1991. 21. Bohinski RJ, DiLauro R, Whitsett JA: The lung specific surfactant protein B promoter is a target for thyroid transcription factor-1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the Foregut axis. Mol Cell Biol. 14:5671–5678, 1994. 22. Bohinski RJ, Huffman JA, Whitsett JA, et al: Cis-active elements controlling lung cell-specific expression of human pulmonary surfactant B gene. J Biol Chem. 268:11160–11166, 1993.
478.e1
23. Bejarano PA, Baughman RP, Biddinger PW, et al: Surfactant proteins and thyroid transcription factor-1 in pulmonary and breast carcinomas. Mod Pathol. 9:445–452, 1996. 24. Fabbro D, DiLoreto C, Stamerra O, et al: TTF-1 gene expression in human lung tumors. Eur J Cancer. 32A:512–573, 1996. 25. DiLoreto C, DiLauro V, Puglisi F, et al: Immunohistochemical expression of tissue specific transcription factor-1 in lung carcinoma. J Clin Pathol. 50:30–32, 1997. 26. Holzinger A, Dingle S, Bejarano PA, et al: Monoclonal antibody to thyroid transcription factor-1: Production, characterization and usefulness in tumor diagnosis. Hybridoma. 15:49–53, 1996. 27. Folpe AL, Gown AM, Lamps LW, et al: Thyroid transcription factor-1: Immunohistochemical evaluation in pulmonary neuroendocrine tumors. Mod Pathol. 12:5–8, 1999. 28. Au NHC, Gown AM, Cheang M, et al: p63 expression in lung carcinomas: A tissue microarray study of 408 cases. Appl Immunohistochem Mol Morphol. 12:240–247, 2004. 29. Bishop JA, Teruya-Feldstein J, Westra WH, et al: p40 (?Np63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 25:405–415, 2012. 30. Savci-Heijink CD, Kosari F, Aubry M-C, et al: The role of desmoglein-3 in the diagnosis of squamous cell carcinoma of the lung. Am J Pathol. 174:1629–1637, 2009. 31. Fukuoka J, Dracheva T, Shih JH, et al: Desmoglein 3 as a prognostic factor in lung cancer. Hum Pathol. 38:276–283, 2007. 32. de Peralta-Venturina M, Phan D, Parakh R, et al: Clinical utility of immunohistochemistry for the diagnosis of lung squamous cell carcinoma in biopsy and cytologic specimens. USCAP 2010 Annual Meeting Abstracts; Publication 1789. 33. Ordonez NG: Thyroid transcription factor 1 is expressed in squamous cell carciomas of the lung: an immunohistochemical study with review of the literature. Appl Immunohistochem Mol Morphol. 20:525–535, 2012. 34. Findeis-Hosey JJ, Yang Q, McMahon LA, et al: IMP3: an immunohistochemical marker for invasive squamous cell carcinoma and its high-grade precursor lesions of the lung. USCAP 2010 Annual Meeting Abstracts; Publication 1795. 35. Sheikh HA, Fuhrer K, Cieply K, et al: p63 expression in assessment of bronchioloalveolar proliferations of the lung. Mod Pathol. 17:1134–1140, 2004. 36. Hammar SP: Metastatic adenocarcinoma of unknown primary origin. Hum Pathol. 29:1393–1402, 1998. 37. Amin MB, Tamboli P, Merchant SH: Micropapillary component in lung adenocarcinoma: a distinctive histologic feature with possible prognostic significance. Am J Surg Pathol. 26:358–364, 2002. 38. Nakamura N, Miyagi E, Murata S, et al: Expression of thyroid transcription factor-1 in normal and neoplastic lung tissues. Mod Pathol. 15:1058–1067, 2002. 39. Lau SK, Desrochers MJ, Luthringer DJ: Expression of thyroid transcription factor-1, cytokeratin 7 and cytokeratin 20 in bronchioloalveolar carcinomas: an immunohistochemical evaluation of 67 cases. Mod Pathol. 15:538–542, 2002. 40. Simsir A, Wei XJ, Yee H, et al: Differential expression of cytokeratins 7 and 20 and thyroid transcription factor-1 in bronchioloalveolar carcinoma: an immunohistochemical study in fine needle aspiration biopsy specimens. Am J Clin Pathol. 11:350– 357, 2004. 41. Johansson L: Histopathologic classification of lung cancer: relevance of cytokeratin and TTF-1 immunophenotyping. Ann Diagn Pathol. 8:259–267, 2004. 42. Yatabe Y, Mitsudomi T, Takahashi T: TTF-1 expression in pulmonary adenocarcinomas. Am J Surg Pathol. 26:767–773, 2002. 43. Chang Y, Lee Y, Lia W, et al: The utility and limitation of thyroid transcription factor-1 protein in primary and metastatic pulmonary neoplasms. Lung Cancer. 44:149–157, 2004. 44. Saad RS, Liu YL, Han H, et al: Prognostic significance of thyroid transcription factor-1 expression in both early-stage conventional adenocarcinoma and bronchioloalveolar carcinoma of the lung. Hum Pathol. 35:3–7, 2004. 45. Jagirdar J: Application of immunohistochemistry to the diagnosis of primary and metastatic carcinoma to the lung. Arch Pathol Lab Med. 132:384–396, 2008.
478.e2
Immunohistology of Lung and Pleural Neoplasms
46. Sainz IM, Fukuoka J, Cagle PT, et al: Napsin A: a new marker for lung adenocarcinoma is complementary and more sensitive than TTF-1 (thyroid transcription factor-1): evaluation of 967 cases by tissue microarray. Mod Pathol. 21:349A, 2008. 47. Turner BM, Cagle PT, Sainz IM, et al: Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma. Arch Pathol Lab Med. 136:163–171, 2012. 48. Whithaus K, Fukuoka J, Prihoda TJ, et al: Evaluation of Napsin A, cytokeratin 5/6, p63, and thyroid transcription factor 1 in adenocarcinoma versus squamous cell carcinoma of the lung. Arch Pathol Lab Med. 136:155–162, 2012. 49. Ordonez NG: Value of thyroid transcription factor-1 immunostaining in tumor diagnosis: a review and update. Appl Immunohistochem Mol Morphol. 20:429–444, 2012. 50. Kadota K, Nitadori J-I, Suzuki K, et al: Thyroid transcription factor-1 expression correlates with predominant histologic subtypes and recurrence in stage I lung adenocarcinoma patients. USCAP 2012 Annual Meeting Abstracts; Publication 1996. 51. Ang DC, Ghaffar H, Zakowski MF, et al: Expression of squamous markers in lung adenocarcinoma (AD): clinicopathologic and molecular correlates, and implications for differentiation of squamous cell carcinoma (SqCC). USCAP 2010 Annual Meeting Abstracts; Publication 1770. 52. Zenali M, Kalhor N, Soto LMS, et al: Mucinous carcinoma (colloid carcinoma) of the lung, an immunohistochemical and molecular analysis. USCAP 2010 Annual Meeting Abstracts; Publication 1860. 53. Yesner R: Small cell tumors of the lung. Am J Surg Pathol. 7:775–785, 1983. 54. Carter D: Small-cell carcinoma of the lung. Am J Surg Pathol. 7:787–795, 1983. 55. Yesner R: Classification of lung cancer histology. N Engl J Med. 312:652–653, 1985. 56. Arrigoni MG, Woolner LB, Berantz PE: Atypical carcinoid tumors of the lung. J Thorac Cardiovasc Surg. 64:413–421, 1972. 57. Leschke H: Über nur regionär bösartige und über krebsig entartete Bronchusadenome bzw. Carcinoide. Virch Arch [Pathol Anat]. 328:635–657, 1956. 58. Warren WH, Memoli VA, Gould VE: Immunohistochemical and ultrastructural analysis of bronchopulmonary neuroendocrine neoplasms: II. Well-differentiated neuroendocrine carcinomas. Ultrastruct Pathol. 7:185–199, 1984. 59. Mark EJ, Ramirerz JF: Peripheral small-cell carcinoma of the lung resembling carcinoid tumor: a clinical and pathologic study of 14 cases. Arch Pathol Lab Med. 109:263–269, 1985. 60. Paladugu RR, Benfield JR, Pak HY, et al: Bronchopulmonary Kulchitzky cell carcinomas. Cancer. 55:1303–1311, 1985. 61. Travis WD, Colby TV, Corrin B, et al: Histological Typing of Lung and Pleural Tumors, New York, 1999, Springer-Verlag. 62. Thomas CF, Tazelaar HD, Jett JR: Typical and atypical pulmonary carcinoids: outcome in patients presenting with regional lymph node involvement. Chest. 119:1143–1150, 2001. 63. Slodkowska J, Langfort R, Rudzinski P, et al: Typical and atypical pulmonary carcinoids–pathologic and clinical analysis of 77 cases. Pneumonol Alergol Pol. 66:297–303, 1998. 64. Gould VE, Chejfec G: Ultrastructural and biochemical analysis of pulmonary “undifferentiated” carcinomas. Hum Pathol. 9:377– 384, 1978. 65. Hammond ME, Sause WT: Large cell neuroendocrine tumors of the lung: Clinical significance and histological definition. Cancer. 56:1624–1629, 1985. 66. Neal MH, Kosinki R, Cohen P, et al: Atypical endocrine tumors of the lung: A histologic, ultrastructural and clinical study of 19 cases. Hum Pathol. 17:1264–1277, 1986. 67. Barbareschi M, Mariscotti C, Barberis M, et al: Large cell neuroendocrine of the lung. Tumor. 75:583–588, 1989. 68. McDowell EM, Wilson TS, Trump BF: Atypical endocrine tumors of the lung. Arch Pathol Lab Med. 105:20–28, 1981. 69. Travis WD, Linnoila I, Tsokos MG, et al: Neuroendocrine tumors of the lung with proposed criteria for large cell neuroendocrine carcinoma: an ultrastructural, immunohistochemical and flow cytometric study of 35 cases. Am J Surg Pathol. 15:529–533, 1991.
70. Hammar S, Bockus D, Remington F, et al: The unusual spectrum of neuroendocrine lung neoplasms. Ultra Pathol. 13:515–560, 1989. 71. Jiang SX, Kameya T, Shoji M, et al: Large cell neuroendocrine carcinoma of the lung: a histologic and immunohistochemical study of 22 cases. Am J Surg Pathol. 22:526–537, 1998. 72. Sturm N, Lantuejoul S, Laverriere MH, et al: Thyroid transcription factor-1 and cytokeratins 1, 5, 10, 14 (34βE12) expression in basaloid and large cell neuroendocrine carcinomas of the lung. Hum Pathol. 32:918–925, 2001. 73. Jung KJ, Lee KS, Han J, et al: Large cell neuroendocrine carcinoma of the lung: clinical, CT and pathologic findings in 11 patients. J Thorac Imaging. 16:156–162, 2001. 74. Mazieres J, Daste G, Molinier L, et al: Large cell neuroendocrine carcinoma of the lung: pathological study and clinical outcome of 18 resected cases. Lung Cancer. 37:287–292, 2002. 75. Paci M, Cavazza A, Annessi V, et al: Large cell neuroendocrine carcinoma of the lung: a 10 year clinicopathologic retrospective study. Ann Thorac Surg. 77:1163–1167, 2004. 76. Doddoli C, Barlesi F, Chetaille B, et al: Large cell neuroendocrine carcinoma of the lung: an aggressive disease potentially treatable with surgery. Ann Thorac Surg. 77:1168–1172, 2004. 77. Peng WX, Sano T, Oyama T, et al: Large cell neuroendocrine carcinoma of the lung: a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma. Lung Cancer. 47:225–233, 2005. 78. Lim E, Goldstraw P, Nicholson AG, et al: Proceedings of the IASLC International Workshop on Advances in Pulmonary Neuroendocrine Tumors 2007. J Thorac Oncol. 3:1194–1201, 2008. 79. Rossi G, Marchioni A, Milani M, et al: TTF-1, cytokeratin 7, 34betaE12, and CD56/NCAM immunostaining in the subclassification of large cell carcinomas of the lung. Am J Clin Pathol. 122:884–893, 2004. 80. Pearse AGE: The diffuse neuroendocrine system: an extension of the APUD concept. In Taylor S, editor: Endocrinology, London, 1972, Heinemann, pp 145–222. 81. Gould VE, DeLellis RA: The neuroendocrine cell system: its tumors, hyperplasias and dysplasias. In Silverberg SG, editor: Principles and practice of surgical pathology, New York, 1983, John Wiley; pp 1488–1501. 82. Hammar SP, Gould VE: Neuroendocrine neoplasms. In Azar HA, editor: Pathology of human neoplasms: an atlas of diagnostic electron microscopy and immunohistochemistry, New York, 1988, Raven Press, pp 333–404. 83. Jahn B, Schibler W, Ouimet C, et al: A 38,000 dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci USA. 82:4137–4141, 1985. 84. Wiedenmann B, Franke WW: Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell. 41:1017–1028, 1985. 85. Carmichael SW, Winkler H: The adrenal chromaffin cell. Sci Am. 253:40–49, 1985. 86. O’Connor DT, Frigon RP: Chromogranin A, the major catecholamine storage vesicle protein. J Biol Chem. 259:3237–3247, 1984. 87. Banks P, Helle K: The release of protein from the stimulated adrenal medulla. Biochem J. 97:C40–C41, 1965. 88. Haimoto H, Takahashi V, Koshikawa T, et al: Immunohistochemical localization of gamma-enolase in normal human tissues other than nervous and neuroendocrine tissues. Lab Invest. 52:257–263, 1985. 89. Pahlman S, Esscher T, Nilsson K: Expression of gamma-subunit of enolase, NSE in human non-neuroendocrine tumors and derived cell lines. Lab Invest. 54:554–560, 1986. 90. Leoncini P, DeMarco EB, Bognoli M, et al: Expression of phosphorylated and non-phosphorylated neurofilament subunits and cytokeratins in neuroendocrine lung tumors. Pathol Res Pract. 185:848–855, 1989. 91. Komminoth P, Roth J, Lackie PM, et al: Polysialic acid of the neural cell adhesion molecule distinguishes small cell carcinoma from carcinoids. Am J Pathol. 139:297–304, 1991. 92. Kibbelaar RE, Moolenaar CEC, Michalides RJAM, et al: Expression of the embryonal neural cell adhesion molecule N-CAM in lung carcinoma: diagnostic usefulness of monoclonal antibody 735 for the distinction between small cell lung cancer and nonsmall cell lung cancer. J Pathol. 159:23–28, 1989.
References 93. Bunn P, Linnoila I, Minna J, et al: Small-cell lung cancer, endocrine cells of the fetal bronchus and other neuroendocrine cells express Leu-7 antigenic determinant present on natural killer cells. Blood. 65:764–768, 1985. 94. Matsuki Y, Tanimoto A, Hamada T, et al: Histidine decarboxylase expression as a new sensitive and specific marker for small cell lung carcinoma. Mod Pathol. 16:72–78, 2003. 95. Gibbs AR, Whimster WF: Tumors of the lung and pleura. In Fletcher CDM, editor: Diagnostic Histopathology of Tumors, Edinburg, 1995, Churchill Livingstone, pp 127–150. 96. Findeis-Hosey JJ, Yang Q, McMahon LA, et al: Napsin A is frequently expressed in pulmonary adenocarcinoma but rarely detected in neuroendocrine tumors of the lung. USCAP 2010 Annual Meeting Abstract; Publication No. 1794. 97. Shibani A, Panjawani N, Arab A, et al: Usefulness of Ki-67 for predicting metastatic potential of carcinoid tumour of the lung: a study of 48 cases. USCAP 2010 Annual Meeting Abstract; Publication 1845. 98. Kirby S, Frankel W, March W, et al: PAX2, PAX5, and PAX8 expression in pulmonary neuroendocrine tumors. USCAP 2012 Annual Meeting Abstract; Publication 1999. 99. Ko H-B M, Hechtman JF, Kinoshita Y, et al: Histone 1.5 (H1.5) staining directly correlates with high grade in pulmonary neuroendocrine tumors. USCAP 2012 Annual Meeting Abstract; Publication 2000. 100. Dail DH: Uncommon neoplasms. In Dail DH, Hammar SP, editors: Pulmonary Pathology, ed 2, New York, 1994, SpringerVerlag, pp 1279–1461. 101. Wick MR, Swanson PE: Carcinosarcomas: current perspective and a historical review of nosologic concepts. Semin Diag Pathol. 10:118–127, 1993. 102. Addis BJ, Corrin B: Pulmonary blastoma, carcinosarcoma and spindle cell carcinoma: an immunohistochemical study of keratin intermediate filaments. J Pathol. 147:291–301, 1985. 103. Zarbo RJ, Crissman JD, Venkat H, et al: Spindle cell carcinoma of the upper aerodigestive tract mucosa: an immunohistologic and ultrastructural study of 18 biphasic tumors and comparison with seven monophasic spindle cell tumors. Am J Surg Pathol. 10:741–753, 1986. 104. Humphrey PA, Scroggs MW, Roggli VL, et al: Pulmonary carcinomas with a sarcomatoid element: an immunohistochemical and ultrastructural analysis. Hum Pathol. 19:155–165, 1988. 105. Colby TV, Bilbao JE, Battifora H, et al: Primary osteosarcoma of the lung: a re-appraisal following immunohistologic study. Arch Pathol Lab Med. 113:1147–1150, 1989. 106. Nappi O, Glasner SD, Swanson PE, et al: Biphasic and monophasic sarcomatoid carcinomas of the lung: a re-appraisal of “carcinosarcomas” and “spindle cell carcinomas”. Am J Clin Pathol. 102:331–340, 1994. 107. Fishback NF, Travis WD, Moran CA, et al: Pleomorphic (spindle/ giant cell) carcinoma of the lung. Cancer. 73:2936–2945, 1994. 108. Pelosi G, Fraggetta F, Nappi O, et al: Pleomorphic carcinomas of the lung show a selective distribution of gene products involved in cell differentiation, cell cycle control, tumor growth and tumor cell motility: a clinicopathologic and immunohistochemical study of 31 cases. Am J Surg Pathol. 27:1203–1215, 2003. 109. Koss MN, Hochholzer L, O’Leary T: Pulmonary blastomas. Cancer. 73:265–294, 1983. 110. Yousem SA, Wick MR, Randhawa P, et al: Pulmonary blastoma: an immunohistochemical analysis and comparison with fetal lung in its pseudoglandular stage. Am J Clin Pathol. 93:167–175, 1990. 111. Etienne-Mastroianni B, Falchero L, Chalabreysse L, et al: Primary sarcomas of the lung: a clinicopathologic study of 12 cases. Lung Cancer. 38:283–290, 2002. 112. Ognibene FP, Steis RG, Macher AM, et al: Kaposi’s sarcoma causing pulmonary infiltrates and respiratory failure in the acquired immunodeficiency syndrome. Ann Intern Med. 102:471–475, 1985. 113. Garay SM, Belenko M, Fazzini E, et al: Pulmonary manifestations of Kaposi’s sarcoma. Chest. 91:39–43, 1987. 114. White DA, Matthay RA: Noninfectious pulmonary complications of infection with the human immunodeficiency virus. Am Rev Respir Dis. 140:1763–1787, 1989. 115. McLoud TC, Naidich DP: Thoracic disease in the immunocompromised patient. Radiol Clin North Am. 30:525–554, 1992.
478.e3
116. Ognibene FP, Shelhamer JH: Kaposi’s sarcoma. Clin Chest Med. 9:459–465, 1988. 117. Heitzman ER: Pulmonary neoplastic lymphoproliferative disease in AIDS: a review. Radiology. 177:347–351, 1990. 118. Purdy LJ, Colby TV, Yousem SA, et al: Pulmonary Kaposi’s sarcoma. Premortem histologic diagnosis. Am J Surg Pathol. 10:301–311, 1986. 119. Hymes KB, Cheung T, Greene JB, et al: Kaposi’s sarcoma in homosexual men: a report of eight cases. Lancet. 2:598–600, 1981. 120. Kaposi’s sarcoma and Pneumocystis pneumonia in homosexual men—New York City and California. MMWR. 30:305–308, 1981. 121. Gottlieb GJ, Ackerman AB: Kaposi’s sarcoma: an extensively disseminated form in young homosexual men. Hum Pathol. 13:882–892, 1982. 122. Adem C, Aubry MC, Tazelaar HD, et al: Metastatic angiosarcoma masquerading as diffuse pulmonary hemorrhage: clinicopathologic analysis of 7 new patients. Arch Pathol Lab Med. 125:1562–1565, 2001. 123. Tschernig T, Pabst R: Bronchus-associated lymphoid tissue (BALT) is not present in the normal adult lung but in different disease. Pathobiology. 68:1–8, 2000. 124. Richmond I, Pritchard GE, Ashcroft T, et al: Bronchus associated lymphoid tissue (BALT) in human lungs: Its distribution in smokers and nonsmokers. Thorax. 48:1130–1134, 1993. 125. Swigris JJ, Berry GJ, Raffin TA, et al: Lymphoid interstitial pneumonia. A narrative review. Chest. 122:2150–2164, 2002. 126. Bienenstock J, Johnston N, Perey DYE: Bronchial lymphoid tissue. I. Morphologic characteristics. Lab Invest. 28:686–692, 1973. 127. Bienenstock J, Johnston N, Perey DYE: Bronchial lymphoid tissue II. Functional characteristics. Lab Invest. 28:693–698, 1973. 128. Pisani RJ, Witzit TE, Li CY, et al: Confirmation of lymphomatous pulmonary involvement by immunophenotypic and gene rearrangement analysis of broncho-alveolar lavage fluid. Mayo Clin Proc. 65:651–656, 1990. 129. Kuroso K, Yumo T, Rom WN, et al: Oligoclonal T cell expansions in pulmonary lymphoproliferative disorders: Demonstration of the frequent occurrence of oligoclonal T cells in human Immunodeficiency virus-related lymphoid interstitial pneumonia. Am J Respir Crit Care Med. 165:254–259, 2002. 130. Colby TV: Lymphoproliferative diseases. In Dail DH, Hammar SP, Colby TV, editors: Pulmonary Pathology Tumors, New York, 1995, Springer Verlag, pp 343–368. 131. Lymphoproliferative Disorders and Leukemia. In Fraser RS, Pare PD, Muller NL, et al, editors: Diagnosis of Diseases of the Chest, ed 4, Philadelphia, 1999, WB Saunders Company, pp 1269–1330. 132. Jaffe ES, Harris NL, Stein H, et al: Genetics of tumours of hematopoietic and lymphoid tissues, Lyon, 2001, IARC Press. 133. Nicholson AG: Lymphoproliferative lung disease. In Corrin B, editor: Pathology of Lung Tumors, New York, 1997, Churchill Livingstone, pp 213–223. 134. Yousem SA: Lung tumors in the immunocompromised host. In Corrin B, editor: Pathology of Lung Tumors, New York, 1997, Churchill Livingstone, pp 189–212. 135. Koss MN: Pulmonary lymphoid disorders. Sem Diag Pathol. 158–171, 1995. 136. Kradin RL, Mark EG: Benign lymphoid disorders of the lung with a theory regarding their development. Hum Pathol. 14:857– 867, 1983. 137. Colby TV: Lymphoproliferative diseases. In Dail DH, Hammar SP, editors: Pulmonary Pathology, ed 2, New York, 1994, SpringerVerlag, pp 1097–1122. 138. Saldana MJ, Patchfsky AS, Israel HL, et al: Pulmonary angiitis and granulomatosis. The relationship between histologic features, organ involvement and response to treatment. Hum Pathol. 8:391–409, 1977. 139. Israel HL, Patchfsky AS, Saldana MJ: Wegener’s granulomatosis, lymphomatoid granulomatosis, and benign lymphocytic angiitis and granulomatosis of lung. Recognition and treatment. Ann Intern Med. 87:691–699, 1977. 140. Jaffe ES, Banks PM, Natwhani B, et al: Recommendations for reporting of lymphoid neoplasms: A report from the association
478.e4
Immunohistology of Lung and Pleural Neoplasms
of directors of anatomic and surgical pathology. The ad hoc committee of reporting of lymphoid neoplasms. Hum Pathol. 33:1064–1068, 2002. 141. His ED, Yegaptan S: Lymphoma Immunophenotyping: A new era in paraffin-sectioned immunohistochemistry. Adv Anat Pathol. 8:218–239, 2001. 142. Rosenow C, Wilson WR, Cockerill FR: Pulmonary disease in the immunocompromised host. Mayo Clin Proc. 60:473–487, 1985. 143. Lin Y, Rodriguez GD, Turner JF, et al: Plasmablastic lymphoma of the lung: Report of a unique case and review of the literature. Arch Pathol Lab Med. 125:282–285, 2001. 144. L’Hoste RJ, Filippa DA, Lieberman PH, et al: Primary pulmonary lymphomas: a clinicopathologic analysis of 36 cases. Cancer. 54:1397–1406, 1984. 145. Hammar SP, Henderson DW, Klebe S, et al: Chapter 43: Neoplasms of the Pleura. In Tomashefski JF, editor: Dail and Hammar’s Pulmonary Pathology, ed 3, New York, 2008, Springer. 146. Kagan E, Jacobson RJ: Lymphoid and plasma cell malignancies: asbestos-related disorders of long latency. Am J Clin Pathol. 80:14–20, 1983. 147. Radin AI: Primary pulmonary Hodgkin’s disease. Cancer. 65:550–563, 1990. 148. Liebow AA, Carrington CB, Friedman PJ: Lymphomatoid granulomatosis. Hum Pathol. 3:457–558, 1972. 149. Katzenstein AL, Carrington CB, Liebow AA: Lymphomatoid granulomatosis: a clinicopathologic study of 152 cases. Cancer. 43:360–373, 1979. 150. Guinee D, Kingma D, Fishback N, et al: Pulmonary lesions with features of lymphomatoid granulomatosis/angiocentric immunoproliferative lesion (LYT/AIL); evidence for Epstein-Barr virus within B lymphocytes. USCAP Abstract #881, 1994. 151. Fauci AS, Haynes BF, Costa J, et al: Lymphomatoid granulomatosis: prospective, clinical and therapeutic experience over 10 years. N Engl J Med. 306:68–74, 1982. 152. Colby TV: Current histological diagnosis of lymphomatoid granulomatosis. Modern Pathol. 25 Suppl 1:S39–S42, 2012. 153. Katzenstein AL, Doxtader E, Narendra S: Lymphomatoid granulomatosis: insights gained over 4 decades. Am J Surg Pathol. 34:e35–e48, 2010. 154. Demirer T, Dail DH, Aboulafia DM: Four varied cases of intravascular lymphomatosis and a literature review. Cancer. 73:1738–1745, 1994. 155. Tan TB, Spaander PJ, Blaisse M, et al: Angiotropic large cell lymphoma presenting as interstitial lung disease. Thorax. 43:578–579, 1988. 156. Snyder LS, Harmon KR, Estensen RD: Intravascular lymphomatosis (malignant angioendotheliomatosis) presenting as pulmonary hypertension. Chest. 96:1199–1200, 1989. 157. Yousem SA, Colby TB: Intravascular lymphomatosis presenting in the lung. Cancer. 65:349–353, 1990. 158. Pellicone JT, Goldstein HB: Pulmonary malignant angioendotheliomatosis: presentation with fever and syndrome of inappropriate anti-diuretic hormone. Chest. 98:1292–1294, 1990. 159. Takamura K, Nasuhara Y, Mishina T, et al: Intravascular lymphomatosis diagnosed by transbronchial lung biopsy. Eur Respir J. 10:955–957, 1997. 160. Walls JG, Hong YG, Cox JE, et al: Pulmonary intravascular lymphomatosis: presentation with dyspnea and air trapping. Chest. 115:1207–1210, 1999. 161. Ansari MQ, Dawson DB, Nador R, et al: Primary body cavitybased AIDS-related lymphomas. Am J Clin Pathol. 105:221–229, 1996. 162. Banks PM, Warnke RA: Primary effusion lymphoma. In WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, Lyon, 2001, IARC Press, pp 179–180. 163. Banks PM, Harris NL, Warnke RA: Primary effusion lymphoma. In Travis WD, Brambilla E, editors: WHO Classification of Tumors of the Lung, Pleura and Mediastinum, Lyon, 2004, IARC Press. 164. Cesarman E, Chang Y, Moore PS, et al: Kaposi’s sarcomaassociated herpes virus-like DNA sequencing in age-related body cavity lymphoma. N Engl J Med. 332:1186–1191, 1995. 165. Aozasa K, Ohsaw AM, Kanno H: Pyothorax-associated lymphoma: a distinctive type of lymphoma strongly associated with Epstein-Barr virus. Adv Anat Pathol. 4:58–63, 1997.
166. Gaulard P, Harris NL: Pyothorax-associated lymphoma. In Travis WD, Brambilla E, editors: WHO Classification of Tumors of the Lung, Pleura and Mediastinum, Lyon, 2004, IARC Press. 167. Ibuka T, Fukayama M, Hayashi Y, et al: Pyothorax-associated pleural lymphoma. Cancer. 73:738–744, 1994. 168. Allen TC, Barrios-Chevez P, Shetlar DJ, et al: Pulmonary and opthalmic involvement with Erdheim-Chester disease; a case report and review of the literature. Arch Pathol Lab Med. 128:1428–1431, 2004. 169. Hammar SP: Pulmonary histiocytosis-X (Langerhans cell granulomatosis). In Dail DH, Hammar SP, editors: Pulmonary Pathology, ed 2, New York, 1994, Springer-Verlag, pp 567–596. 170. Carter D, Patchefsky AS, Mountain CF: Clear cell lesions. In Carter D, Patchefsky AS, editors: Tumors and Tumor-like Lesions of the Lung, Philadelphia, 1998, WB Saunders, pp 271–274. 171. Bonetti F, Pea M, Martigoni G, et al: Clear cell (“sugar tumor”) of the lung is a lesion strictly related to angiomyolipoma—the concept of a family of lesions characterized by the presence of the perivascular epithelioid cells (PEC). Pathology. 26:230–236, 1994. 172. Panizo-Santos A, Sola I, de Alava E, et al: Angiomyolipoma and PEComa are immunoreactive for MyoD1 in cell cytoplasmic staining pattern. Appl Immunohistochem Mol Morphol. 11:156– 160, 2003. 173. Gaffey M, Mills S, Zarbo R, et al: Clear cell tumor of the lung: Immunohistochemical and ultrastructural evidence of melanogenesis. Am J Surg Pathol. 15:644–653, 1991. 174. Sullivan EJ: Lymphangioleiomyomatosis: a review. Chest. 114:1689–1703, 1998. 175. Johnson S: Lymphangioleiomyomatosis: clinical features, management and basic mechanism. Thorax. 54:254–264, 1999. 176. Mandal S, Hyjek E, Husain A, et al: Placental alkaline phosphatase (PLAP) and podoplanin (D2-40) define two subtypes of cells in lymphangioleiomyomatosis. USCAP 2010 Annual Meeting Abstracts; Publication 1824. 177. Liebow AA, Hubbell DS: Sclerosing hemangioma (histocytoma, xanthoma) of the lung. Cancer. 9:53–75, 1956. 178. Sogio K, Yokoyama H, Kanedo S, et al: Sclerosing hemangioma of the lung: radiographic and pathologic study. Ann Thorac Surg. 53:295–300, 1992. 179. Katzenstein A-LA, Gmelich JT, Carrington CB: Sclerosing hemangioma of the lung: a clinicopathologic study of 51 cases. Am J Surg Pathol. 4:343–356, 1982. 180. Devouassoux-Shisheboran M, Hayashi T, Linnoila RI, et al: A clinicopathologic study of 100 cases of pulmonary sclerosing hemangioma with immunohistochemical studies: TTF-1 is expressed in both round and surface cells, suggesting an origin from primitive respiratory epithelium. Am J Surg Pathol. 24:906–916, 2000. 181. Devouassoux-Shisheboran M, de la Fouchardiere A, ThivoletBejui F, et al: Endobronchial variant of sclerosing hemangioma of the lung: histological and cytological features on endobronchial material. Mod Pathol. 17:252–257, 2004. 182. Miyagawa-Hayashino A, Tazelaar HD, Langel DJ, et al: Pulmonary sclerosing hemangioma with lymph node metastases: report of 4 cases. Arch Pathol Lab Med. 127:321–325, 2003. 183. Beckwith JB, Palmer NF: Histopathology and prognosis of Wilms’ tumor: results from the first national Wilms’ tumor study. Cancer. 55:2850–2853, 1978. 184. Small EJ, Gordon GJ, Dahms BB: Malignant rhabdoid tumor of the heart in an infant. Cancer. 55:2850–2853, 1985. 185. Balaton AJ, Vaury P, Videgrain M: Paravertebral malignant rhabdoid tumor in an adult: a case report of immunocytochemical study. Pathol Res Pract. 182:713–718, 1987. 186. Harris M, Eyden BP, Joglekar VM: Rhabdoid tumour of the bladder: a histological, ultrastructural and immunohistochemical study. Histopathology. 11:1083–1089, 1987. 187. Biggs PJ, Garren PD, Posers JM, et al: Malignant rhabdoid tumor of the central nervous system. Hum Pathol. 18:332–337, 1987. 188. Dervan PA, Cahalane SF, Kneafsey P, et al: Malignant rhabdoid tumor of soft tissue: an ultrastructural and immunohistological study of a pelvic tumour. Histopathol. 11:183–190, 1987. 189. Parham DM, Peiper S, Robicheaux G, et al: Malignant rhabdoid tumor of the liver: evidence for epithelial differentiation. Arch Pathol Lab Med. 112:61–64, 1988.
References 190. Jakate SM, Mardsen HB, Ingram L: Primary rhabdoid tumour of the brain. Virch Arch [A]. 412:393–397, 1988. 191. Uchida H, Yokoyama S, Nakayama I, et al: An autopsy case of malignant rhabdoid tumor arising from soft parts in the left inguinal region. Acta Pathol Jpn. 38:1087–1096, 1988. 192. Patron M, Palacious J, Rodriguez-Peralto JL, et al: Malignant rhabdoid tumor of the tongue: a case report with immunohistochemical and ultrastructural findings. Oral Surg Oral Med Oral Path. 65:67–70, 1988. 193. Carter RL, McCarthy KP, al-Sam SZ, et al: Malignant rhabdoid tumour of the bladder with immunohistochemical and ultrastructural evidence suggesting histiocytic origin. Histopathology. 14:179–190, 1989. 194. Jsujimura T, Wasa A, Kawano K, et al: A case of malignant rhabdoid tumor arising from soft parts of the prepubic region. Acta Pathol Jpn. 37:677–682, 1989. 195. Cho KR, Rosenshein NB, Epstein JI: Malignant rhabdoid tumor of the kidney and soft tissues: evidence for a diverse morphological and immunocytochemical phenotype. Arch Pathol Lab Med. 113:115–120, 1989. 196. Tsokos M, Kouraklis G, Chandra RS, et al: Malignant rhabdoid tumor of the kidney and soft tissues: evidence for a diverse morphological and immunocytochemical phenotype. Arch Pathol Lab Med. 113:115–120, 1989. 197. Molenaar WM, DeJong B, Dam-Meiring A, et al: Epithelioid sarcoma or malignant rhabdoid tumor of soft tissue. Epithelioid immunophenotype and rhabdoid karyotype. Hum Pathol. 20:347–351, 1989. 198. Cavazza A, Colby TV, Tsokos M, et al: Lung tumors with a rhabdoid phenotype. Am J Clin Pathol. 105:182–188, 1996. 199. Miyagi J, Tsuhako K, Kinjo T, et al: Rhabdoid tumor of the lung is a dedifferentiated phenotype of pulmonary adenocarcinoma. Histopathology. 37:37–44, 2000. 200. Bahadori H, Liebow AA: Plasma cell granuloma of the lung. Cancer. 31:191–208, 1973. 201. Lane JD, Krohn S, Kolozzi W, et al: Plasma cell granuloma of the lung. Dis Chest. 27:216–221, 1955. 202. Berardi RS, Lee SS, Chen HP, et al: Inflammatory pseudotumors of the lung. Surg Gynecol Obstet. 156:89–96, 1983. 203. Tang TT, Segura AD, Oechler HW, et al: Inflammatory myofibrohistiocytic proliferation simulating sarcoma in children. Cancer. 65:1626–1634, 1990. 204. Tan-Liu NS, Matsubara MD, Grillo HC, et al: Invasive fibrous tumor of the tracheobronchial tree: clinical and pathological study of seven cases. Hum Pathol. 20:180–184, 1989. 205. Ledet SC, Brown RW, Cagle PT: p53 immunostaining of the differentiation of inflammatory pseudotumor from sarcoma involving the lung. Mod Pathol. 8:282–286, 1995. 206. Snyder CS, Dell-Aquila M, Haghighi P, et al: Clonal changes in inflammatory pseudotumor of the lung. Cancer. 76:1545–1549, 1995. 207. Yousem SA, Shaw H, Cieply K: Involvement of 2p23 pulmonary inflammatory pseudotumors. Hum Pathol. 32:428–433, 2001. 208. Freeman A, Geddes N, Munson P, et al: Anaplastic lymphoma kinase (ALK 1) staining and molecular analysis in inflammatory myofibroblastic tumors of the bladder: a preliminary clinicopathologic study of nine cases and review of the literature. Mod Pathol. 17:765–771, 2004. 209. Lae ME, Roche PC, Jin L, et al: Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical and molecular study of 32 tumors. Am J Surg Pathol. 26:823–835, 2002. 210. Syed S, Hague AK, Hawkins HK, et al: Desmoplastic small round cell tumor of the lung. Arch Pathol Lab Med. 126:1226– 1228, 2002. 211. Ostoros G, Orosz Z, Kovacs G, et al: Desmoplastic small round cell tumor of the pleura: a case report with unusual follow-up. Lung Cancer. 36:333–336, 2002. 212. Tsuji N, Tateishi R, Ishigoro S, et al: Adenomyoepithelioma of the lung. Am J Surg Pathol. 19:956–962, 1995. 213. Veeramechaneni R, Gulic J, Halldorsson AO, et al: Benign myoepithelioma of the lung: a case report and review of the literature. Arch Pathol Lab Med. 125:1494–1496, 2001. 214. Pelosi G, Fraggetta F, Maffini F, et al: Pulmonary epithelialmyoepithelial tumor of unproven malignant potential: report of
478.e5
a case and review of the literature. Mod Pathol. 14:521–526, 2001. 215. Ro K, Srivastava A, Tischer AS: Bronchial epithelial-myoepithelial carcinoma. Arch Pathol Lab Med. 128:92–94, 2004. 216. Dearers M, Guinee D, Koss MN, et al: Granular cell tumors of the lung: clinicopathologic study of 20 cases. Am J Surg Pathol. 19:627–635, 1995. 217. de Araujo VC, de Sousa SOM, Carvalho YR, et al: Application of immunohistochemistry to the diagnosis of salivary gland tumors. Appl Immunohist Mol Morphol. 8:195–202, 2000. 218. Moran CA, Suster S, Fishback NF, et al: Primary intrapulmonary thymoma: a clinicopathologic and immunohistochemical study of eight cases. Am J Surg Pathol. 19:304–312, 1995. 219. Ionescu DN, Sasatomi E, Aldeeb D, et al: Pulmonary meningothelial-like nodules: a genotypic comparison with meningiomas. Am J Surg Pathol. 28:207–214, 2004. 220. Mukhopadhyay S, El-Zammar OA, Katzenstein A-LA: Pulmonary meningothelial-like nodules: new insights into a common but poorly understood entity. Mod Pathol. 21:348A, 2008. 221. Hochholzer L, Moran CA, Koss MN: Pulmonary lipomatosis: a variant of placental transmogrification. Mod Pathol. 10:846–849, 1997. 222. Fidler ME, Koomen M, Sebek B, et al: Placental transmogrification of the lung, a histologic variant of giant bullous emphysema. Am J Surg Pathol. 19:563–570, 1995. 223. Abrams HJ, Spiro R, Goldstein N: Metastases in carcinoma, analysis of 1000 autopsied cases. Cancer. 3:74–85, 1950. 224. Dail DH: Metastases to and from the lung. In Dail DH, Hammar SP, Colby TV, editors: Pulmonary Tumors, New York, 1995, Springer-Verlag, pp 369–403. 225. Wang NP, Zee S, Zarbo RJ, et al: Coordinate expression of cytokeratins 7 and 20 defines unique subsets of carcinomas. Appl Immunohist. 3:99–107, 1995. 226. Pelosi G, Sonzogni A, Papotti M, et al: Difference prevalence of transactivating (TA) p63 and non-TAp63 isoforms in pulmonary adenocarcinomas: a useful diagnostic tool. USCAP 2010 Annual Meeting Abstracts; Publication 1838. 227. Kargi A, Gurel D, Tuna B: The diagnostic value of TTF-1, CK5/6, and p63 immunostaining in classification of lung carcinomas. Appl Immunohistochem Mol Morphol. 15:415–420, 2007. 228. Nonaka D: A study of ?Np63 (p40) expression in non-small cell lung carcinomas. USCAP 2012 Annual Meeting Abstract; Publication 2022. 229. Brown AF, Sirohi D, Fukuoka J, et al: Evaluation of TTF-1/p40 cocktail in adenocarcinoma versus squamous cell carcinoma of the lung. Archol Pathol Lab Med Abstracts. 1111, Poster 9, 2012. 230. Amador-Ortiz C, Chernock RD, El-Mofty SK, et al: Distinguishing primary from metastatic squamous cell carcinoma of the lung in patients with known head and neck or gynecological tract carcinoma. USCAP 2010 Annual Meeting Abstract; Publication 1768. 231. Brambilla E: Basaloid carcinoma of the lung. In Corrin B, editor: Pathology of Lung Tumors, New York, 1997, Churchill Livingstone, pp 71–82. 232. Nonaka D, Chiriboga L: Basal cell differentiation in lung adenocarcinoma. Mod Pathol. 21:348A, 2008. 233. Crapanzano JP, Loukeris K, Borczuk AC, et al: Cytological, histological, and immunohistochemical findings of pulmonary carcinomas with basaloid features. USCAP 2010 Annual Meeting Abstracts; Publication 1788. 234. Dodds L, Davis S, Polissar L: A population-based study of lung cancer incidence by histological type. J Natl Cancer Inst. 76:21– 29, 1986. 235. Thun MJ, Lally CA, Flannery JT, et al: Cigarette smoking and changes in the histopathology of lung cancer. J Natl Cancer Inst. 89:1580–1586, 1997. 236. Amaro DE, Lin GY: Napsin A: utility in identifying primary mucinous lung adenocarcinomas versus mucinous metastasis. USCAP 2012 Meeting Abstracts; Publication 1960. 237. Striebel JM, Dacic S, Yousem SA: Gross Cystic Disease Fluid Protein (GCDFP-15): expression in primary lung adenocarcinoma. Am J Surg Pathol. 32(3):426–432, 2008. 238. Lin F, Zhu S, Deng H, et al: Identification of an effective immunohistochemical panel in distinction of breast carcinoma from
478.e6
Immunohistology of Lung and Pleural Neoplasms
lung adenocarcinoma. USCAP 2012 Annual Meetings Abstract; Publication 2012. 239. Pereira TC, Share SM, Conroy EP, et al: PAX-2 and Napsin A in the differential diagnosis of lung versus kidney primary site in patients with renal cell carcinoma and a new lung mass: a comparison with TTF-1 and RCC antigen. USCAP 2010 Annual Meeting Abstract; Publication 1839. 240. Trump BF, McDowell EM, Glavin F, et al: The respiratory epithelium. III. Histogenesis of epidermoid metaplasia and carcinoma in situ in the human. J Nat Cancer Inst. 61:563–575, 1978. 241. Arcila ME, Rekhtman N, Teruja-Feldstein J: Immunohistochemical analysis of Napsin A in 77 lung carcinomas and 509 nonpulmonary malignancies. USCAP Annual Meeting Abstracts Publication 1771, 2010. 242. Ye J, Findeis-Hosey JJ, McMahon LA, et al: Usefulness of Napsin A and TTF-1 in discriminating metastatic carcinoma from primary adenocarcinoma of the lung. USCAP 2010 Annual Meetings Abstract; Publication 1857. 243. Cooper WA, Thourani VH, Gal AA, et al: The surgical spectrum of pulmonary neuroendocrine neoplasms. Chest. 119:14–18, 2001. 244. Lyda MH, Weiss LM: Immunoreactivity for epithelial and neuroendocrine antibodies are useful in the differential diagnosis of lung carcinomas. Hum Pathol. 31:980–987, 2000. 245. Sturm N, Rossi G, Lantuejoul S, et al: Expression of thyroid transcription factor-1 in the spectrum of neuroendocrine cell lung proliferations with special interest in carcinoids. Hum Pathol. 33:175–182, 2002. 246. Barbareschi M, Roldo C, Zamboni G, et al: CDX-2 homeobox gene product expression in neuroendocrine tumors: its role as a marker of intestinal neuroendocrine tumors. Am J Surg Pathol. 28:1169–1176, 2004. 247. Lin X, Saad RS, Luckasevic TM, et al: Diagnostic value of CDX-2 and TTF-1 expressions in separating metastatic neuroendocrine neoplasms of unknown origin. Appl Immunohistochem Mol Morphol. 15:407–414, 2007. 248. Lin O, Olgac S, Green I, et al: Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms. Am J Clin Pathol. 120:209–216, 2003. 249. Pelosi G, Rodriguez J, Viale G, et al: Typical and atypical pulmonary carcinoid tumor overdiagnosed as small cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients. Am J Surg Pathol. 29:179–187, 2005. 250. Visscher DW, Zarbo RJ, Trojanowski JQ, et al: Neuroendocrine differentiation in poorly-differentiated lung carcinomas: a light microscopic and immunohistochemical study. Mod Pathol. 3:508–512, 1990. 251. Linnoila RI, Mulshine JL, Steinberg SM, et al: Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas. Am J Clin Pathol. 90:641–652, 1988. 252. Mooi WJ, Dewar A, Springall D, et al: Non-small cell lung carcinomas with neuroendocrine features: a light microscopic, immunohistochemical and ultrastructural study of 11 cases. Histopathology. 13:329–337, 1988. 253. Wick MR, Berg LC, Hertz MI: Large cell carcinoma of the lung with neuroendocrine differentiation: a comparison with large cell “undifferentiated” pulmonary tumors. Am J Clin Pathol. 97:796–805, 1992. 254. Loy TS, Darkow GVD, Quesenberry JT: Immunostaining in the diagnosis of pulmonary neuroendocrine carcinomas: an immunohistochemical study with ultrastructural correlations. Am J Surg Pathol. 19:173–182, 1995. 255. Xu H, Bourne PA, Spaulding BO, et al: High-grade neuroendocrine carcinomas of the lung express K homology domain containing protein overexpressed in cancer but carcinoid tumors do not. Hum Pathol. 38:555–563, 2007. 256. Schleusener JT, Tazelaar HD, Jung S, et al: Neuroendocrine differentiation is an independent prognostic factor in chemotherapytreated non-small cell lung carcinoma. Cancer. 77:1284–1291, 1996. 257. Ralston J, Chiriboga L, Nonaka D: MASH1: a useful marker in differentiating pulmonary small cell carcinoma from Merkel cell carcinoma. Mod Pathol. 21:1357–1362, 2008.
258. Rajagopalan A, Browning D, Salama S: CD99 expression in Merkel cell carcinoma: a case series with an unusual paranuclear dot-like staining pattern. J Cutan Pathol. 40:19–24, 2013. 259. Bobos M, Hytiroglou P, Kostopoulos I, et al: Immunohistochemical distinction between merkel cell carcinoma and small cell carcinoma of the lung. Am J Dermatopathol. 28:99–104, 2006. 260. Lau SK, Luthringer DJ, Eisen RN: Thyroid transcription factor-1: a review. Appl Immunohistochem Mol Morphol. 10:97–102, 2002. 261. Leech SN, Kolar AJ, Barrett PD, et al: Merkel cell carcinoma can be distinguished from metastatic small cell carcinoma using antibodies to cytokeratin 20 and thyroid transcription factor 1. J Clin Pathol. 54:727–729, 2001. 262. Butler AE, Colby TV, Weiss L, et al: Lymphoepithelioma-like carcinoma of lung. Am J Surg Pathol. 13:632–639, 1989. 263. Chen F, Yan J, Lai W, et al: Epstein-Barr virus-associated nonsmall cell lung carcinoma: undifferentiated “lymphoepitheliomalike” carcinomas as a distinct entity with better prognosis. Cancer. 82:2334–2342, 1998. 264. Ginsberg SS, Buzaid AC, Stern H, et al: Giant cell carcinoma of the lung. Cancer. 70:606–610, 1992. 265. Gustmann C, Altmannsberger M, Osborn M, et al: Cytokeratin expression and vimentin content in large cell anaplastic lymphoma and other non-Hodgkin’s lymphoma. Am J Pathol. 38:1413–1422, 1991. 266. Delsol G, AlSaati T, Gatter KC, et al: Coexpression of epithelial membrane antigen (EMA), Ki-1 and interleukin-2 receptor by anaplastic large cell lymphomas. Diagnostic value in so-called malignant histiocytosis. Am J Pathol. 130:59–70, 1988. 267. Osborne BM, Mockay B, Butler JJ, et al: Large cell lymphoma with microvillus-like projections: an ultrastructural study. Am J Clin Pathol. 79:433–450, 1983. 268. Chu PG, Arber DA, Weiss LM: Expression of T/NK-Cell and plasma cell antigens in nonhematopoietic epithelioid neoplasms. Am J Clin Pathol. 120:64–70, 2003. 269. Bishop PW, Menasce LP, Yates AJ, et al: An immunophenotypic survey of malignant melanomas. Histopathology. 23:159–166, 1993. 270. Kaimaktchiev V, Terracciano L, Tornillo L, et al: The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal carcinomas. Mod Pathol. 17:1392–1399, 2004. 271. Rossi G, Murer B, Cavazza A, et al: Primary mucinous (so-called colloid) carcinomas of the lung: a clinicopathologic and immunohistochemical study with special reference to CDX2 homeobox gene and MUC2 expression. Am J Surg Pathol. 28:442–452, 2004. 272. Mazziotta RM, Borczuk AC, Powell CA, et al: CDX2 immunostaining as a gastrointestinal marker: expression in lung carcinomas is a potential pitfall. Appl Immunohistochem Mol Morphol. 13:55–60, 2005. 273. Bejarano PA, Mousavi F: Incidence and significance of cytoplasmic thyroid transcription factor-1 immunoreactivity. Arch Pathol Lab Med. 127:193–195, 2003. 274. Herrera GA, Turbat-Herrera EA, Lott RL: S100 protein expression by primary and metastatic adenocarcinoma. Am J Clin Pathol. 89:168–176, 1988. 275. Hammar SP, Bockus D, Remington F, et al: Langerhans cells and serum precipitating antibodies against fungal antigens in bronchioloalveolar cell carcinomas: Possible association with eosinophilic granuloma. Ultrastruct Pathol. 1:19–37, 1980. 276. Colasante A, Castrilli G, Aiello FB, et al: Role of cytokines in the distribution of dendritic cells/Langerhans cell lineage in human primary carcinomas of the lung. Hum Pathol. 26:866–872, 1995. 277. Hammar SP, Bockus D, Remington F, et al: The widespread distribution of Langerhans cells in pathologic tissues: An ultrastructural and immunohistochemical study. Hum Pathol. 17:894–905, 1986. 278. Dorion P, Shi J, Zhang K, et al: Utility of S100 protein in differentiating adenocarcinoma of the lung from papillary carcinoma and follicular carcinoma of the thyroid. Modern Pathol. 21:340, 2008. 279. Torikata C, Ishiwata K: Intranuclear tubular structures observed in the cells of alveolar cell carcinomas of the lung. Cancer. 40:1194–1201, 1977.
References 280. Singh G, Katyal SL, Torikata C: Carcinoma of type II pneumocytes: Immunodiagnosis of a subtype of bronchioloalveolar carcinoma. Am J Pathol. 102:195–208, 1981. 281. Singh G, Katyal SL, Torikata C: Carcinoma of type II pneumocytes: PAS staining as a screening test for nuclear inclusions of surfactant-specific apoprotein. Cancer. 50:946–948, 1982. 282. Ghadially FN, Harawi S, Khan W: Diagnostic ultrastructural markers in alveolar cell carcinoma. J Submicrosc Cytol. 17:269– 278, 1985. 283. Tigrani DY, Weydert JA: Expression of inhibin-alpha in primary pulmonary non-small cell carcinomas by immunohistochemistry. Mod Pathol. 21:352A, 2008. 284. Zhang K, Shi J, Lin F: Expression of inhibin-alpha in non-small cell carcinoma of the lung with a focus on adenocarcinoma: a diagnostic pitfall in the evaluation of metastatic pulmonary carcinoma. Modern Pathol. 21:353A, 2008. 285. Ollayos CW, Riordan P, Rushin JM: Estrogen receptor detection in paraffin section of adenocarcinoma of colon, pancreas and lung. Arch Pathol Lab Med. 118:630–632, 1994. 286. Canver CC, Memoli VA, Vanderveer PL, et al: Sex hormones in non-small cell lung cancer in human beings. J Thorac Cardiovasc Surg. 108:153–157, 1994. 287. Bacchi CE, Garcia RL, Gown AM: Immunolocalization of estrogen and progesterone receptors in neuroendocrine tumors of lung, skin, gastrointestinal and female genital tracts. Appl Immunohist. 5:17–22, 1997. 288. DiNunno LD, Larsson LG, Rinehart JJ, et al: Estrogen and progesterone receptors in non-small cell lung cancer in 248 consecutive patients who underwent surgical resection. Arch Pathol Lab Med. 124:1467–1470, 2000. 289. Dabbs DJ, Landreneau RJ, Liu Y, et al: Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg. 73:403–406, 2002. 290. Selvaggi G, Scagliotti GV, Torri V, et al: HER-2/neu overexpression in patients with radically resected nonsmall cell lung carcinoma: impact on long-term survival. Cancer. 94:2669–2674, 2002. 291. Yoshimoto T, Higashino K, Hada T, et al: A primary lung carcinoma producing alpha-fetoprotein, carcinoembryonic antigen and human chorionic gonadotropin. Cancer. 60:2744–2750, 1987. 292. Kuida CA, Braunstein GD, Shintaku P, et al: Human chorionic gonadotropin expression in lung, breast and renal carcinomas. Arch Pathol Lab Med. 112:282–285, 1988. 293. Dirnhofer S, Freund M, Rogatsch H, et al: Selective expression of trophoblastic hormones by lung carcinoma: neuroendocrine tumors produce human chorionic gonadotropin alpha-subunit (hCGα). Hum Pathol. 31:966–972, 2000. 294. Nasu M, Soma T, Fukushima H, et al: Hepatoid carcinoma of the lung with production of alpha fetoprotein and abnormal prothrombin: an autopsy case report. Mod Pathol. 10:1054– 1058, 1997. 295. Asirvatham JR, Esposito MJ, Bhuiya TA: Utility of PAX8, CD117, and CD5 in distinguishing thymic carcinoma from poorly differentiated lung carcinomas. USCAP 2012 Annual Meeting Abstracts; Publication 1962. 296. Lee HJ, Xu X, Choe G, et al: CD24, a novel cancer biomarker predicting disease-free survival of nonsmall cell lung carcinomas: from the viewpoint of forthcoming (seventh) new TNM classification. USCAP 2010 Annual Meeting Abstract; Publication 1815. 297. Zhu CQ, Shih W, Ling CH, et al: Immunohistochemical markers of prognosis in non-small cell lung cancer: a review and proposal for a multiphase approach to marker evaluation. Am J Clin Pathol. 59:790–800, 2006. 298. Olaussen KA, Dunant A, Fouret P, et al: DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. [see comment]. NEJM. 355:983–991, 2006. 299. Simon G, Sharma A, Li X, et al: Feasibility and efficacy of molecular analysis-directed individualized therapy in advanced non-small-cell lung cancer. J Clin Oncol. 25:2741–2746, 2007. 300. Clark GM, Zborowski DM, Culbertson JL, et al: Clinical utility of epidermal growth factor receptor expression for selecting patients with advanced non-small cell lung cancer for treatment with erlotinib. J Thorac Oncol. 1:837–846, 2006.
478.e7
301. Jeon YK, Sung S-W, Chung J-H, et al: Clinicopathologic features and prognostic implications of epidermal growth factor receptor (EGFR) gene copy number and protein expression in non-small cell lung cancer. Lung Cancer. 54:387–398, 2006. 302. Tsao M-S, Sakurada A, Cutz J-C, et al: Erlotinib in lung cancer— molecular and clinical predictors of outcome. [see comment] [erratum appears in N Engl J Med 2006:355(16):1746]. NEJM. 353:133–144, 2005. 303. Hirsch FR, Dziadziuszko R, Thatcher N, et al: Epidermal growth factor receptor immunohistochemistry: comparison of antibodies and cutoff points to predict benefit from gefitinib in a phase 3 placebo-controlled study in advanced nonsmall-cell lung cancer. Cancer. 112:1114–1121, 2008. 304. Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. [see comment]. NEJM. 350:2129–2139, 2004. 305. Paez JG, Janne PA, Lee JC, et al: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. [see comment]. Science. 304:1497–1500, 2004. 306. Pao W, Miller V, Zakowski M, et al: EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 101:13306–13311, 2004. 307. Jackman DM, Yeap BY, Sequist LV, et al: Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib. Clin Cancer Res. 12:3908–3914, 2006. 308. Pao W, Ladanyi M: Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method.[comment]. Clin Cancer Res. 13:4954–4955, 2007. 309. Sequist LV, Joshi VA, Janne PA, et al: Epidermal growth factor receptor mutation testing in the care of lung cancer patients. Clin Cancer Res. 12:S4403–S4408, 2006. 310. Shih J-Y, Gow C-H, Yu C-J, et al: Epidermal growth factor receptor mutations in needle biopsy/aspiration samples predict response to gefitinib therapy and survival of patients with advanced nonsmall cell lung cancer. [see comment]. Int J Cancer. 118:963–969, 2006. 311. Shih J-Y, Gow C-H, Yang P-C: EGFR mutation conferring primary resistance to gefitinib in non-small-cell lung cancer. NEJM. 353:207–208, 2005. 312. Bean J, Brennan C, Shih J-Y, et al: MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A. 104:20932–20937, 2007. 313. Engelman JA, Zejnullahu K, Mitsudomi T, et al: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043, 2007. 314. Ahrendt SA, Decker PA, Alawi EA, et al: Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung. Cancer. 92:1525– 1530, 2001. 315. Eberhard DA, Johnson BE, Amler LC, et al: Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. [see comment]. J Clin Oncol. 23:5900–5909, 2005. 316. Shigematsu H, Gazdar AF: Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer. 118:257–262, 2006. 317. Cappuzzo F, Hirsch FR, Rossi E, et al: Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-smallcell lung cancer. [see comment][comment]. J Natl Cancer Inst. 97:643–655, 2005. 318. Daniele L, Macri L, Schena M, et al: Predicting gefitinib responsiveness in lung cancer by fluorescence in situ hybridization/ chromogenic in situ hybridization analysis of EGFR and HER2 in biopsy and cytology specimens. Mol Cancer Ther. 6:1223– 1229, 2007. 319. Gallegos Ruiz MI, Floor K, Vos W, et al: Epidermal growth factor receptor (EGFR) gene copy number detection in non-small-cell
478.e8
Immunohistology of Lung and Pleural Neoplasms
lung cancer; a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization. Histopathology. 51:631– 637, 2007. 320. Hirsch FR, Varella-Garcia M, McCoy J, et al: Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. [see comment]. J Clin Oncol. 23:6838–6845, 2005. 321. Johnson BE, Janne PA: Selecting patients for epidermal growth factor receptor inhibitor treatment: A FISH story or a tale of mutations? [see comment][comment]. J Clin Oncol. 23:6813– 6816, 2005. 322. Hirsch FR, Herbst RS, Olsen C, et al: Increased EGFR gene copy number detected by fluorescent in situ hybridization predicts outcome in non-small-cell lung cancer patients treated with cetuximab and chemotherapy. J Clin Oncol. 26:3351–3357, 2008. 323. Sholl LM, John Iafrate A, Chou Y-P, et al: Validation of chromogenic in situ hybridization for detection of EGFR copy number amplification in nonsmall cell lung carcinoma. Mod Pathol. 20:1028–1035, 2007. 324. Barletta JA, Weir BA, Perner S, et al: Clinical significance of TTF-1 protien expression and TTF-1 gene amplification in lung adenocarcinomas. Mod Pathol. 21:335A, 2008. 325. Beasley MB, Castro CY, Garza L, et al: Expression of HIF-1 alpha in pulmonary non-small cell carcinomas and neuroendocrine carcinomas—a tissue microarray analysis of 442 cases. Mod Pathol. 21:336A, 2008. 326. Beheshti J, Sabo E, Janne PA, et al: TTF-1 positivity is a sensitive predictor of EGFR mutation and treatment response in pulmonary adenocarcinomas, by pathologist interpretation and by image analysis. Mod Pathol. 21:336A, 2008. 327. Kundu UR, Li W, Tan D, et al: Alpha-methylacyl-co-a-racemase (AMACR) expression is a biomarker of poor prognosis in stage 1 and 2 large cell carcinomas of the lung. Mod Pathol. 21:345A, 2008. 328. Perner S, Demichelis F, Johnson L, et al: TTF1 amplification defines TTF1 overexpression in lung adenocarcinomas. Mod Pathol. 21:349A, 2008. 329. Vohra P, Cagle PT, Allen TC, et al: Tissue microarray study of RNA-binding protein IMP3 in primary non-small cell lung cancers: prediction of survival in early stage squamous cell carcinomas. Mod Pathol. 21:352A, 2008. 330. Cutz J-C, Tsakaridis TS, Wright J, et al: Immunohistochemistry for phosphorylated EGF receptor predicts survival and phyosphorylated ERK predicts tumor response to chemoradiotherapy in unresectable, locally advanced non-small cell lung cancer patients. Mod Pathol. 21:339A, 2008. 331. Li QK, Tully E, Askin FB, et al: Overexpression of NRF2 is correlated with elevated p53 levels in pulmonary papillary adenocarcinoma by tissue microarray analysis. Mod Pathol. 21:345A, 2008. 332. Luu M, Sabo E, Resnick M, et al: Prognostic value of aspartyl (asparaginyl) hydroxylase (AAH) in lung carcinomas: correlation with survival. Mod Pathol. 21:346A, 2008. 333. Shahjahan M, Zhai QJ, Allen TC, et al: ProEx C expression in non-small cell lung cancers (NSCLC) is associated with longer patient survival. Mod Pathol. 21:350A, 2008. 334. Sienko AE, Allen TC, Barrios R, et al: Expression of galectin-4 in non-small cell lung carcinoma (NSCLC) and correlation with survival. Mod Pathol. 21:351A, 2008. 335. Song J, Tretiakova MS, Salgia R, et al: c-Met expression in pulmonary neuroendocrine tumors. Mod Pathol. 21:351–352A, 2008. 336. Haque AK, Singhal N, Allen T, et al: Lack of E-cadherin and Pan-cadherin immunoexpression is associated with metastasis in small cell lung cancer. Mod Pathol. 21:342A, 2008. 337. Homan SM, Sheehan CE, Ross JS, et al: Downregulation of GADD45α protein expression is associated with advanced tumor stage and decreased survival in squamous cell carcinoma of the lung. Mod Pathol. 21:343A, 2008. 338. Malik A, Krishnaswamy S, Gong C, et al: RON overexpression and activation in primary and metastatic lung carcinoma. Mod Pathol. 21:346A, 2008.
339. Lonardo F, Pass HI, Lucas DR: Immunohistochemistry frequently detects c-Kit expression in pulmonary small cell carcinoma and may help select clinical subsets for a novel form of chemotherapy. Appl Immunohist Mol Morphol. 11:51–55, 2003. 340. Pelosi G, Barisella M, Pasini F, et al: CD117 immunoreactivity in stage I adenocarcinoma and squamous carcinoma of the lung: relevance to prognosis in a subset of adenocarcinoma patients. Mod Pathol. 17:711–721, 2004. 341. Casali C, Stefani A, Rossi G, et al: The prognostic role of c-Kit protein expression in resected large cell neuroendocrine carcinoma of the lung. Ann Thorac Surg. 77:252–253, 2004. 342. Butnor KJ, Burchette JL, Sporn TA, et al: The spectrum of Kit (CD117) immunoreactivity in lung and pleural tumors: a study of 96 cases using a single-source antibody with a review of the literature. Arch Pathol Lab Med. 128:538–543, 2004. 343. Maddau C, Confortini M, Bisanzi S, et al: Prognostic significance of p53 and Ki-67 antigen expression in surgically treated nonsmall cell lung cancer: immunocytochemical detection with imprint cytology. Am J Clin Pathol. 125:425–431, 2006. 344. Kobayashi S, Kubo H, Suzuki T, et al: Endogenous secretory receptor for advanced glycation end products in non-small cell lung carcinoma. Am J Respir Crit Care Med. 175:184–189, 2007. 345. Allen TC, Granville LA, Cagle PT, et al: Expression of glutathione S-transferase π and glutathione synthase correlates with survival in early stage non-small cell carcinomas of the lung. Hum Pathol. 38:220–227, 2007. 346. Lee Y-C, Wu C-T, Kuo S-W, et al: Significance of extranodal extension of regional lymph nodes in surgically resected nonsmall cell lung cancer. Chest. 131:993–999, 2007. 347. Aviel-Ronen S, Lau SK, Pintilie M, et al: Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma. Mod Pathol. 21:817–825, 2008. 348. LaPoint RJA, Bourne PA, Wang HL, et al: Coexpression of c-kit and bcl-2 in small cell carcinoma and large cell neuroendocrine carcinoma of the lung. Appl Immunohistochem Mol Morphol. 15:401–406, 2007. 349. Meyronet D, Massoma P, Thivolet F, et al: Extensive expression of collapsing response mediator protein 5 (CRMP5) is a specific marker of high-grade neuroendocrine carcinoma. Am J Surg Pathol. 32:1699–1708, 2008. 350. Yousem SA, Nikiforova M, Nikiforov Y: The histopathology of BRAF-V600E-mutated lung adenocarcinoma. Am J Surg Pathol. 32:1317–1321, 2008. 351. Hammar SP: Pleural diseases. In Dail DH, Hammar SP, Colby TV, editors: Pulmonary Pathology Tumors, New York, 1995, Springer-Verlag, pp 405–530. 352. Davies J: Human developmental anatomy, New York, 1963, Roland Press, pp 51–52. 353. Bolen JW, Hammar SP, McNutt MA: Reactive and neoplastic serosal tissue: a light-microscopic, ultrastructural and immunocytochemical study. Am J Surg Pathol. 10:34–47, 1986. 354. Selikoff IJ, Seidman H: Asbestos-associated deaths among insulation workers in the United States and Canada, 1967-1987. Annls NY Acad Sci. 643:1–14, 1991. 355. Battifora H, McCaughey WTE: Tumors of the serosal membranes, Washington DC, 1995, Armed Forces Institute of Pathology, pp 17–88. 356. Butnor KJ, Sporn TA, Hammar SP, et al: Well-differentiated papillary mesothelioma. Am J Surg Pathol. 25:1304–1309, 2001. 357. Galateau-Salle F, Vignaud J, Burke L, et al: Well-differentiated papillary mesothelioma of the pleura: a series of 24 cases. Am J Surg Pathol. 28:534–540, 2004. 358. Henderson DW, Shilkin KB, Whitaker D: Reative mesothelial hyperplasia vs. mesothelioma, including mesothelioma in situ: a brief review. Am J Clin Pathol. 110:397–404, 1998. 359. US-Canadian Mesothelioma Reference Panel; Churg A, Colby TV, Cagle P, et al: The separation of benign and malignant mesothelial proliferations. Am J Surg Pathol. 24:1183–1200, 2000. 360. Betta P-G, Magnani C, Bensi T, et al: Immunohistochemistry and molecular diagnosis of pleural malignant mesothelioma. Arch Pathol Lab Med. 136:253–261, 2012. 361. Churg A, Galateau-Salle F: The separation of benign and malignant mesothelial proliferations. Arch Pathol Lab Med. 136:1217– 1226, 2012.
References 362. Anttila S: Epithelioid lesions of the serosa. Arch Pathol Lab Med. 136:241–252, 2012. 363. Le Stang N, Karanian M, Paciencia M, et al: Diagnosis of pleural epithelioid malignant mesothelioma: a meta-analysis based on routinely used immunohistochemical markers. Presented at the 11th International Conference of the International Mesothelioma Interest Group, Boston, Massachusetts; September 2012. 364. King JE, Thatcher N, Pickering CA, et al: Sensitivity and specificity of immunohistochemical markers used in the diagnosis of epithelioid mesothelioma: a detailed systematic analysis using published data. Histopathology. 48:223–232, 2006. 365. Yaziji H, Battifora H, Barry TS, et al: Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: identification of a three-body immunohistochemical panel with maximal sensitivity and specificity. Mod Pathol. 19:514–523, 2006. 366. Kushitani K, Takeshima Y, Amatya VJ, et al: Immunohistochemical marker panels for distinguishing between epithelioid mesothelioma and lung adenocarcinoma. Pathol Int. 57:190–199, 2007. 367. Klebe S, Nurminen M, Leigh J, et al: Diagnosis of epithelial mesothelioma using tree-based regression analysis and a minimal panel of antibodies. Pathology. 41:140–148, 2009. 368. Marchevsky AM: Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Arch Pathol Lab Med. 132:397–401, 2008. 369. Henderson DW, Comin CE, Hammar SP, et al: Malignant mesothelioma of the pleura: current surgical pathology. In Corrin B, editor: Pathology of Lung Tumors, New York, 1997, Churchill Livingstone, pp 241–280. 370. Ordonez NG: The immunohistochemical diagnosis of epithelial mesothelioma. Hum Pathol. 30:313–323, 1999. 371. Blobel GA, Moll R, Franke WW, et al: The intermediate filament cytoskeleton of malignant mesothelioma and its diagnostic significance. Am J Pathol. 121:235–247, 1985. 372. Blobel GA, Moll R, Franke WW, et al: Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines. Virchows Arch Cell Pathol. 45:407–429, 1984. 373. Moll R, Dhovailly D, Sun T-T: Expression of keratin 5 as a distinctive feature of epithelial and biphasic mesotheliomas: an immunohistochemical study using monoclonal antibody AE14. Virchows Archiv B Cell Pathol. 58:129–145, 1989. 374. Clover J, Oates J, Edwards C: Anti-cytokeratin 5/6: a positive marker for epithelial mesothelioma. Histopathology. 31:140– 143, 1997. 375. Kahn HJ, Thorner PS, Yeager H, et al: Immunohistochemical localization of pre-keratin filaments in benign and malignant cells in effusions: comparison with intermediate filament distribution by electron microscopy. Am J Pathol. 109:206–214, 1982. 376. Kahn HJ, Thorner PS, Yeger H, et al: Distinct keratin patterns demonstrated by immunoperoxidase staining of adenocarcinomas, carcinoids and mesotheliomas using polyclonal and monoclonal keratin antibodies. Am J Clin Pathol. 86:566–574, 1986. 377. Montag AG, Pinkus GS, Corson JM: Keratin protein reactivity immunoreactivity of sarcomatoid and mixed types of diffuse malignant mesotheliomas: an immunoperoxidase study of 30 cases. Hum Pathol. 19:336–342, 1988. 378. Battifora H: The pleura. In Sternberg SS, editor: Diagnostic Surgical Pathology, Volume I, New York, 1989, Raven Press, pp 829–855. 379. Roggli VL, Kolbeck J, Sanfilippo F, et al: Pathology of human mesothelioma: etiologic and diagnostic considerations. Pathol Annu. 22:(pt 2):91–131, 1987. 380. Al-Izzi M, Thurlow NP, Corrin B: Pleural mesothelioma of connective tissue type, localized fibrous tumour of the pleura and reactive submesothelial hyperplasia: an immunohistochemical comparison. J Pathol. 157:41–44, 1989. 381. Mayall FG, Goddard H, Gibbs AR: Intermediate filament expression in mesotheliomas: leiomyoid mesotheliomas are not uncommon. Histopathol. 21:453–457, 1992. 382. Yousem SA, Hochholzer L: Malignant mesotheliomas with osseous and cartilaginous differentiation. Arch Pathol Lab Invest. 111:62–66, 1987.
478.e9
383. Wirth PR, Legler J, Wright GL: Immunohistochemical evaluation of seven monoclonal antibodies for differentiation of pleural mesothelioma from lung adenocarcinoma. Cancer. 67:655–662, 1991. 384. Carter D, Otis CN: Three types of spindle cell tumors of pleura: Fibroma, sarcoma and sarcomatoid mesothelioma. Am J Surg Pathol. 12:747–753, 1988. 385. Azumi N, Battifora H, Carlson G, et al: Sarcomatous (spindlecell) mesothelioma of pleura: Immunohistochemical study. Lab Invest. 60:4A, 1989. 386. Lucas DR, Pass HI, Madan SK, et al: Sarcomatoid mesothelioma and its histologic mimics. Histopathol. 42:270–279, 2003. 387. Attanoos RL, Dojcinov SD, Webb R, et al: Anti-mesothelial markers in sarcomatoid mesothelioma and other spindle cell neoplasms. Histopathology. 37:224–231, 2000. 388. McNutt MA, Bolen JW, Gown AM, et al: Coexpression of intermediate filaments in human epithelial neoplasms. Ultrastruct Pathol. 9:31–43, 1985. 389. Azumi N, Battifora H: The distribution of vimentin and keratin in epithelial and non-epithelial neoplasms. A comprehensive study on formalin and alcohol-fixed tumors. Am J Clin Pathol. 88:286–296, 1987. 390. Raymond WA, Leong AS-Y: Vimentin—a new prognostic marker in breast carcinoma. J Pathol. 158:107–114, 1989. 391. Churg A: Immunohistochemical staining for vimentin and keratin in malignant mesothelioma. Am J Surg Pathol. 9:360– 365, 1985. 392. Jasani B, Edwards RE, Thomas ND, et al: The use of vimentin antibodies in the diagnosis of malignant mesothelioma. Virch Arch A Pathol Anat. 406:441–448, 1985. 393. Mullink H, Henzen-Logmans SC, Alons-van Kordelaan JJM, et al: Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from pulmonary adenocarcinoma. Virch Arch B Pathol Anat. 42:55–65, 1986. 394. Andersen C, Blumcke I, Celio MR: Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res. 271:181–208, 1993. 395. Schwaller B, Buchwald P, Blucke I, et al: Characterization of a polyclonal antiserum against the purified human recombinant calcium binding protein calretinin. Cell Calcium. 14:639–648, 1993. 396. Gotzos V, Schwaller B, Hertzel N, et al: Expression of the calcium binding protein calretinin in Wi Dr cells and its correlation to their cell cycle. Exp Cell Res. 202:292–302, 1992. 397. Doglioni C, Dei Tos AP, Laurino L, et al: Calretinin: a novel immunocytochemical marker for mesothelioma. Am J Surg Pathol. 20:1037–1046, 1996. 398. Barberis MCP, Faleri M, Veronese S, et al: Calretinin: a selective marker of normal and neoplastic mesothelial cells in serous effusions. Acta Cytol. 41:1757–1761, 1997. 399. Leers MPG, Aarts MMJ, Theunissen PHMH: E-cadherin and calretinin: a useful combination of immunochemical markers for differentiation between mesothelioma and metastatic adenocarcinoma. Histopathol. 32:209–216, 1998. 400. Ordonez NG: Value of calretinin immunostaining in differentiating epithelial mesothelioma from lung adenocarcinoma. Mod Pathol. 10:929–933, 1998. 401. Chenard-Neu MP, Kabou A, Mechine A, et al: Immunohistochemistry in the differential diagnosis of mesothelioma and adenocarcinoma: evaluation of 5 new and 6 traditional antibodies. Ann Pathol. 18:460–465, 1998. 402. Curry PM, Butcher DN, Fisher C, et al: Value of mesotheliumassociated antibodies thrombomodulin, cytokeratin 5/6, calretinin and CD44H in distinguishing epithelioid mesothelioma from adenocarcinoma metastatic to the pleura. Mod Pathol. 13:107–112, 2000. 403. Oates J, Edwards C: HBME-1, MOC-31, WT1 and calretinin: an assessment of recently described markers for mesothelioma and adenocarcinoma. Histopathol. 36:341–347, 2000. 404. Chieng DC, Yee H, Schaefer D, et al: Calretinin staining pattern aids in the differentiation of mesothelioma from adenocarcinoma in serous effusions. Cancer. 25:194–200, 2000.
478.e10
Immunohistology of Lung and Pleural Neoplasms
405. O’Hara CJ, Corson JM, Pinkus GS, et al: ME1: a monoclonal antibody that distinguishes epithelial-type mesothelioma from pulmonary adenocarcinoma and extra-pulmonary malignancies. Am J Pathol. 136:421–428, 1990. 406. Sheibani K, Esteban JM, Bailey A, et al: Immunopathologic and molecular studies as an aid to the diagnosis of malignant mesothelioma. Hum Pathol. 23:107–116, 1992. 407. Hammar SP, Bolen JW, Bockus D, et al: Ultrastructural and immunohistochemical features of common lung tumors: an overview. Ultrastruct Pathol. 9:283–318, 1985. 408. Walz R, Koch HK: Malignant pleural mesotheliomas: some aspects of epidemiology, differential diagnosis and prognosis. Histological and immunohistochemical evaluation and follow-up of mesotheliomas diagnosed from 1964 to January 1985. Pathol Res Pract. 186:124–134, 1990. 409. Wick MR, Loy T, Mills SE, et al: Malignant epithelioid pleural mesothelioma versus peripheral pulmonary adenocarcinoma: a histochemical, ultrastructural and immunohistologic study of 103 cases. Hum Pathol. 21:759–766, 1990. 410. Henderson DW, Shilkin KB, Whitaker D, et al: The pathology of mesothelioma, including immunohistology and ultrastructure. In Henderson DW, Shilkin KB, Langlois SL, et al, editors: Malignant Mesothelioma, New York, 1992, Hemisphere, pp 69–139. 411. Fink L, Collins CL, Schaefer R, et al: Thrombomodulin expression can be used to differentiate between mesotheliomas and adenocarcinomas. Lab Invest. 66:113A, 1992. 412. Collins CL, Ordonez NG, Schaefer R, et al: Thrombomodulin expression and pulmonary adenocarcinoma. Am J Pathol. 141:827–833, 1992. 413. Brown RW, Clark GM, Tandon AK, et al: Multiple-marker immunohistochemical phenotypes distinguishing malignant pleural mesothelioma from pulmonary adenocarcinoma. Hum Pathol. 24:347–354, 1993. 414. Ascoli V, Scalzo CC, Taccogna S, et al: The diagnostic value of thrombomodulin immunolocalization in serous effusions. Arch Pathol Lab Med. 119:1136–1140, 1995. 415. Geiger B, Ayalon O: Cadherins. Annu Rev Cell Biol. 8:307–332, 1992. 416. Takeichi M: Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 251:1451–1455, 1991. 417. Hatta K, Takagi S, Fujisawa H, et al: Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogetic processes of chicken embryos. Dev Biol. 120:215–227, 1987. 418. Peralta-Soler A, Knudsen KA, Jaurand MC, et al: The differential expression of N-cadherin, E-cadherin distinguished pleural mesotheliomas from lung adenocarcinomas. Hum Pathol. 26:1363–1369, 1995. 419. Han AC, Peralta-Soler A, Knudsen KA, et al: Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues. Hum Pathol. 28:641–645, 1997. 420. Ordonez NG: Value of E-cadherin and N-cadherin immunostaining in the diagnosis of mesothelioma. Hum Pathol. 34:749– 755, 2003. 421. Amin KM, Litzky LA, Smythe WR, et al: Wilms’ tumor 1 susceptibility (WT1) gene products are selectively expressed in malignant mesothelioma. Am J Pathol. 146:344–356, 1995. 422. Kumar-Singh S, Segers K, Rodeck O, et al: WT1 mutation in malignant mesothelioma and WT1 immunoreactivity in relation to p53 growth factor expression, cell-type transition and prognosis. J Pathol. 181:67–74, 1997. 423. Walker C, Rutlen F, Yuan X, et al: Wilms’ tumor suppressor gene expression in rat and human mesothelioma. Cancer Res. 54:1301–1306, 1994. 424. Ordonez NG: Application of mesothelin immunostaining in tumor diagnosis. Am J Surg Pathol. 27:1418–1428, 2003. 425. Chu AY, Litzky LA, Pasha TL, et al: Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma. Mod Pathol. 18:105–110, 2005. 426. Comin CE, Dini S, Novelli L, et al: h-Caldesmon, a useful positive marker in the diagnosis of pleural malignant mesothelioma, epithelioid type. Am J Surg Pathol. 30:463–469, 2006.
427. Shivley JE, Beatty JD: CEA-related antigens: molecular biology and clinical significance. CRC Crit Rev Oncol Hematol. 2:355– 399, 1985. 428. Thompson J, Grunert F, Zimmermann W: Carcinoembryonic antigen gene family: molecular biology and clinical perspectives. J Clin Labor Anal. 5:344–366, 1991. 429. Hammarstrom S, Khan WN, Teglund S, et al: The carcinoembryonic antigen family. In Van Regenmortel MHV, editor: Structure of Antigens, Boca Raton, 1993, CRC Press, pp 341–376. 430. Hammarstrom S, Olsen A, Teglund S, et al: The nature and expression of the human CEA family. In Stanners C, editor: Cell Adhesion and Clinical Perspectives, Amsterdam, 1997, Harwood Academic Publishers, pp 1–30. 431. Hammarstrom S: The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Cancer Biol. 9:67–81, 1999. 432. Wang N-S, Huang S-N, Gold P: Absence of carcinoembryonic antigen-like material in mesothelioma: an immunohistochemical differentiation from other lung cancers. Cancer. 44:437–943, 1979. 433. Whitaker D, Shilkin KB: Carcinoembryonic antigen in the tissue diagnosis of malignant mesothelioma. Lancet. 1:1369–1370, 1981. 434. Whitaker D, Sterret GF, Shilkin KB: Detection of tissue CEAlike substance as an aid in the differential diagnosis of malignant mesothelioma. Pathology. 14:255–258, 1982. 435. Sheibani K, Battifora H: Leu-M1 positivity is not specific for Hodgkin’s disease. Am J Clin Pathol. 84:682, 1985. 436. Sheibani K, Battifora H, Burke JS, et al: Leu-M1 in human neoplasms: An immunohistologic study of 400 cases. Am J Surg Pathol. 10:227–236, 1986. 437. Sheibani K, Battifora H, Burke J: Antigenic phenotype of malignant mesotheliomas and pulmonary adenocarcinomas: an immunohistologic analysis demonstrating the value of Leu-M1 antigen. Am J Pathol. 123:212–219, 1986. 438. Sheibani K, Azumi N, Battifora H: Further evidence demonstrating the value of LeuM1 antigen in differential diagnosis of malignant mesothelioma and adenocarcinoma: an immunohistologic evaluation of 395 cases. Lab Invest. 58:84A, 1988. 439. Otis CN, Carter O, Cole S, et al: Immunohistochemical evaluation of pleural mesothelioma and pulmonary adenocarcinoma. Am J Surg Pathol. 11:445–456, 1987. 440. Szpak CA, Johnston WW, Roggli V, et al: The diagnostic distinction between malignant mesothelioma and adenocarcinoma of the lung as defined by a monoclonal antibody (B72.3). Am J Pathol. 122:252–260, 1986. 441. Ordonez NG: The immunohistochemical diagnosis of mesothelioma: Differentiation of mesothelioma and lung adenocarcinoma. Am J Surg Pathol. 13:276–291, 1989. 442. Bollinger DJ, Wick MR, Dehner LP, et al: Peritoneal malignant mesothelioma versus serous papillary adenocarcinoma: A histochemical and immunohistochemical comparison. Am J Surg Pathol. 13:659–670, 1989. 443. Latza V, Niedobitek G, Schwarting R, et al: Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelia. J Clin Pathol. 43:213–219, 1990. 444. Sheibani K, Shin SS, Kezirian J, et al: Ber-EP4 antibody as a discriminant in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. Am J Surg Pathol. 15:779–784, 1991. 445. Gaffey MJ, Mills SE, Swanson PE, et al: Immunoreactivity for Ber-EP4 in adenocarcinomas, adenomatoid tumors and malignant mesotheliomas. Am J Surg Pathol. 16:593–599, 1992. 446. Souhami RL, Beverly PCL, Bobrow LG: Antigens of small cell lung cancer. First International Workshop. Lancet. 2:325–326, 1987. 447. Riutenbeek T, Gouw ASH, Poppema S: Immunocytology of body cavity fluids: MOC-31, a monoclonal antibody discriminating between mesothelial and epithelial cells. Arch Pathol Lab Med. 118:265–269, 1994. 448. DeLeij L, Broers J, Ramaekers F, et al: Monoclonal antibodies in clinical and experimental pathology of lung cancer. In Roiter DJ, Fleuren GJ, Warner SO, editors: Applications of Monoclonal Antibodies in Tumor Pathology, Dordrecht, 1987, Martinus Nijhoff Publishers, pp 191–210.
References 449. Delahaye M, Hoogsteden HC, van der Kwast TH: Immunocytochemistry of malignant mesothelioma: OV 632 as a marker of malignant mesothelioma. J Pathol. 165:137–143, 1991. 450. Ordonez NG: Value of MOC-31 monoclonal antibody in differentiating epithelial pleural mesothelioma from lung adenocarcinoma. Hum Pathol. 29:166–169, 1998. 451. Jordon D, Jagirdar J, Kaneko M: Blood group antigens Lewis x and Lewis y in the diagnostic discrimination of malignant mesothelioma versus adenocarcinoma. Am J Pathol. 135:931–937, 1989. 452. Riera JR, Astengo-Osuna C, Longmate JA, et al: The immunohistochemical diagnostic panel for epithelial mesothelioma. A reevaluation after heat-induced epitope retrieval. Am J Surg Pathol. 21:1409–1419, 1997. 453. Kinsella AR, Green B, Lepts GC, et al: The role of cell-cell adhesion molecule E-cadherin in large bowel tumour cell invasion and metastasis. Br J Cancer. 67:904–909, 1993. 454. Williams CL, Hayes VY, Hummel AM, et al: Regulation of E-cadherin mediated adhesion by muscarinic actetylcholine receptor in small cell lung carcinoma. J Cell Biol. 121:643–654, 1993. 455. Kawai T, Suzuki M, Torikata C, et al: Expression of blood grouprelated antigens and Helix pomatia agglutinin in malignant pleural mesothelioma and pulmonary adenocarcinoma. Hum Pathol. 22:118–124, 1991. 456. Mayall FG, Goddard H, Gibbs AR: p53 immunostaining in the distinction between benign and malignant mesothelial proliferations using formalin-fixed paraffin sections. J Pathol. 168:377– 381, 1992. 457. Ramael M, Lemmens G, Eerdekens C, et al: Immunoreactivity for p53 protein in malignant mesothelioma and non-neoplastic mesothelium. J Pathol. 168:371–375, 1992. 458. Mayall FG, Goddard H, Gibbs AR: The frequency of p53 immunostaining in asbestos-associated mesotheliomas. Histopathology. 22:383–386, 1993. 459. Hurlimann J: Desmin and neural marker expression in mesothelial cells and mesotheliomas. Hum Pathol. 25:753–757, 1994. 460. Azumi N, Underhill CB, Kagan E, et al: A novel biotinylated probe specific for hyaluronate. Am J Surg Pathol. 16:116–121, 1992. 461. Thylen A, Wallin J, Martensson G: Hyaluronan in serum as an indicator of progressive disease in hyaluronan-producing malignant mesothelioma. Cancer. 86:2000–2005, 1999. 462. Martensson G, Thylen A, Lindquist U, et al: The sensitivity of hyaluronan analysis of pleural fluid from patients with malignant mesothelioma and a comparison of different methods. Cancer. 73:1406–1410, 1994. 463. Thylen A, Levin-Jacobsen AM, Hjerpe A, et al: Immunohistochemical differences between hyaluronan and non-hyaluronanproducing malignant mesothelioma. Eur Respir J. 10:404–408, 1997. 464. Kabawat SE, Bast RC, Bhan AK, et al: Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumors of serous, endometrioid and clear cell types. Am J Clin Pathol. 79:98–104, 1983. 465. Kabawat SE, Bast RC, Bhan AK, et al: Tissue distribution of celomic epithelium related antigen recognized by monoclonal antibody OC-125. Int J Gynecol Pathol. 2:275–285, 1983. 466. Dabbs DJ, Geisinger KR: Selective application of immunohistochemistry in gynecological neoplasms. Pathol Annu. 28:(pt 1):329–353, 1993. 467. Bast RC, Freeney M, Lazarus H, et al: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest. 68:1331– 1337, 1981. 468. Koelma IA, Nap M, Rodenburg CJ, et al: The value of tumor marker CA-125 in surgical pathology. Histopathology. 11:287– 294, 1987. 469. Nanbu Y, Fujii S, Konishi I, et al: Immunohistochemical localization of CA-130 in fetal tissue and in normal and neoplastic tissues of the female genital tract. Asia Oceania J Obstet Gynecol. 16:379–387, 1990. 470. Tamura S, Yamaguchi K, Terada M, et al: Immunohistochemical analysis of CA19-9, SLX and CA-125 in adenoidcystic carcinoma of trachea and bronchus. Nippon Kyobu Shikkan Gakkai Zasshi. 3:407–411, 1992.
478.e11
471. Zhou J, Iwasa Y, Konishi I, et al: Papillary serous carcinoma of the peritoneum in women: A clinicopathologic and immunohistochemical study. Cancer. 76:429–436, 1995. 472. Nouwen EJ, Pollet DE, Eerdekens MW, et al: Immunohistochemical localization of placental alkaline phosphatase, carcinoembryonic antigen and cancer antigen 125 in normal and neoplastic human lung. Cancer Res. 46:866–876, 1986. 473. Bateman AC, al-Talib RK, Newman T, et al: Immunohistochemical phenotype of malignant mesothelioma: predictive value of CA-125 and HBME-1 expression. Histopathology. 30:49–56, 1997. 474. Nap M: Immunohistochemistry of CA-125: unusual expression in normal tissues, distribution in the human fetus and questions around its application in diagnostic pathology. Int J Biol Markers. 13:210–215, 1998. 475. O’Brien TJ, Raymond LM, Bannon GA, et al: New monoclonal antibodies identify the glycoprotein carrying the CA-125 epitope. Am J Obst Gynecol. 61:1857–1864, 1991. 476. Okamoto H, Matsuno Y, Noguchi M, et al: Malignant pleural mesothelioma producing chorionic gonadotropin: report of two cases. Am J Surg Pathol. 16:969–974, 1992. 477. McAuley P, Asa SL, Chiv B, et al: Parathyroid hormone-like peptide in normal and neoplastic mesothelial cells. Cancer. 66:1975–1979, 1990. 478. Tateyama H, Eimoto T, Tada T, et al: Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma. Am J Clin Pathol. 111:235–240, 1999. 479. Husain AN, Colby TV, Ordonez NG, et al: Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the International Mesothelioma Interest Group. Arch Pathol Lab Med. 133:1317–1331, 2009. 480. Chirieac L, Pinkus G, Pinkus J: Sarcomatoid malignant mesothelioma: immunohistochemical characteristics of 24 cases. Mod Pathol. 19:305A, Abstract 1422, 2006. 481. Ordonez NG: D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Hum Pathol. 36:372–380, 2005. 482. Hinterberger M, Reineke T, Storz M, et al: D2-40 and calretinin: a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation. Mod Pathol. 20:248–255, 2007. 483. Klebe S, Mahar A, Henderson DW, et al: Malignant mesothelioma with heterologous elements: clinicopathological correlation of 27 cases and literature review. Mod Pathol. 21:1084–1094, 2008. 484. Klebe S, Brownlee NA, Mahar A, et al: Sarcomatoid mesothelioma: a clinical-pathologic correlation of 326 cases. Mod Pathol. 23:470–479, 2010. 485. Krishna J, Haqqani MT: Liposarcomatous differentiation in diffuse pleural mesothelioma. Thorax. 48:409–410, 1993. 486. Dardick I, Al-Jabi M, McCaughey WTE, et al: Ultrastructure of poorly differentiated diffuse epithelial mesotheliomas. Ultrastruct Pathol. 7:151–160, 1984. 487. Hammar SP, Bockus D, Remington F: Metastatic tumors of unknown origin: an ultrastructural analysis of 265 cases. Ultrastruct Pathol. 11:209–250, 1987. 488. Gaber AO, Rice P, Eaton C, et al: Metastatic malignant disease of unknown origin. Am J Surg Pathol. 145:493–497, 1983. 489. Crotty TB, Myers JL, Katzenstein AL, et al: Localized malignant mesothelioma: a clinicopathologic and flow cytometric study. Am J Surg Pathol. 18:357–363, 1994. 490. Allen TC, Cagle PT, Churg AM, et al: Localized malignant mesothelioma. Am J Surg Pathol. 29:866–873, 2005. 491. Talerman A, Chilcote RR, Montero JR, et al: Diffuse malignant peritoneal mesothelioma in a 13-year-old girl. Am J Surg Pathol. 9:73–80, 1985. 492. Nascimento AG, Keeney GL, Fletcher CDM: Deciduoid peritoneal mesothelioma: an unusual phenotype affecting young females. Am J Surg Pathol. 18:439–445, 1994. 493. Orosz Z, Nagy P, Szentirmay Z, et al: Epithelial mesothelioma with deciduoid features. Virchows Arch. 434:263–266, 1999. 494. Ordonez NG: Deciduoid mesothelioma: report of 21 cases with review of the literature. Mod Pathol. 25:1481–1495, 2012.
478.e12
Immunohistology of Lung and Pleural Neoplasms
495. Ordonez NG: Pleomorphic mesothelioma: report of 10 cases. Mod Pathol. 25:1011–1022, 2012. 496. Mayall FG, Gibbs AR: The histology and immunohistochemistry of small cell mesothelioma. Histopathology. 20:47–51, 1992. 497. Falconieri G, Zanconati F, Bussani R, et al: Small cell carcinoma of lung simulating mesothelioma. Pathol Res Pract. 191:1147– 1151, 1995. 498. Krismann M, Müller K-M, Jaworska M, et al: Pathological anatomy and molecular pathology [of mesothelioma]. Lung Cancer. 45S:S29–S33, 2004. 499. Ordonez NG: Mesotheliomas with small cell features: report of eight cases. Modern Pathol. 25:689–698, 2012. 500. Ordonez NG: Mesothelioma with rhabdoid features: an ultrastructural and immunohistochemical study of 10 cases. Mod Pathol. 19:373–383, 2006. 501. Hammar SP, Bockus DE, Remington FL, et al: Mucin-positive epithelial mesotheliomas: a histochemical, immunohistochemical and ultrastructural comparison with mucin-producing pulmonary adenocarcinomas. Ultra Pathol. 20:293–325, 1996. 502. Ordonez NG: Mesotheliomas with crystalloid structures: report of nine cases, including one with oncocytic features. Mod Pathol. 25:272–281, 2012. 503. Briselli M, Mark EJ, Dickersin GR: Solitary fibrous tumors of the pleura: eight new cases and review of 360 cases in the literature. Cancer. 47:2678–2689, 1981. 504. Doucet J, Dardick I, Srigley JR, et al: Localized fibrous tumour of serosal surfaces. Virch Arch Pathol Anat. 409:349–363, 1986. 505. England DM, Hochholzer L, McCarthy MJ: Localized benign and malignant fibrous tumors of the pleura. A clinicopathologic review of 223 cases. Am J Surg Pathol. 13:640–658, 1989. 506. van de Rijn M, Lombard CM, Rouse RV: Expression of CD34 by solitary fibrous tumors of the pleura, mediastinum and lung. Am J Surg Pathol. 18:814–820, 1994. 507. Flint A, Weiss SW: CD-34 and keratin expression distinguishes solitary fibrous tumor (fibrous mesothelioma) of pleura from desmoplastic mesothelioma. Hum Pathol. 26:428–431, 1995. 508. Hasegawa T, Matsuno Y, Shimoda T, et al: Frequent expression of bcl-2 protein in solitary fibrous tumors. Jpn J Clin Oncol. 28:86–91, 1998. 509. Schirosi L, Lanteujoul S, Cavazza A, et al: Pleuro-pulmonary solitary fibrous tumors: a clinicopathologic, immunohistochemical, and molecular study of 88 cases confirming the prognostic value of de Perrot staging system and p53 expression, and evaluating the role of c-kit, BRAF, PDGFRs (α/β), c-met, and EGFR. Am J Surg Pathol. 32:1627–1642, 2008. 510. de Perrot M, Fischer S, Brϋndler MA, et al: Solitary fibrous tumors of the pleura. Ann Thorac Surg. 74:285–293, 2002. 511. Babolini G, Blasi A: The pleural form of primary cancer of the lung. Dis Chest. 29:314–323, 1956. 512. Harwood TR, Gracey DR, Yokoo H: Pseudomesotheliomatous carcinoma of the lung. Am J Clin Pathol. 65:159–167, 1976. 513. Koss M, Travis W, Moran C, et al: Pseudomesotheliomatous adenocarcinoma: a reappraisal. Semin Diag Pathol. 9:117–123, 1992. 514. Hartman C-A, Schutze H: Mesothelioma-like tumors of the pleura: a review of 72 cases. Cancer Res Clin Oncol. 120:331– 347, 1994. 515. Koss MN, Fleming M, Przygodzki RM, et al: Adenocarcinoma simulating mesothelioma: a clinicopathologic and immunohistochemical study of 29 cases. Ann Diagn Pathol. 2:93–102, 1998. 516. Robb JA, Hammar SP, Yokoo H: Pseudomesotheliomatous carcinoma of lung. Lab Invest. 68:134A, 1993. 517. Attanoos RL, Gibbs AR: ‘Pseudomesotheliomatous’ carcinomas of the pleura: a 10-year analysis of cases from the Environmental Lung Disease Research Group, Cardiff. Histopathol. 43:444–452, 2003. 518. Battifora H: Epithelioid hemangioendothelioma imitating mesothelioma. Appl Immunohistochem. 1:220–221, 1993. 519. Lin BT-Y, Colby T, Gown AM, et al: Malignant vascular tumors of the serous membranes mimicking mesothelioma: A report of 14 cases. Am J Surg Pathol. 20:1431–1439, 1996. 520. Gray MH, Rosenberg AE, Dickersin GR, et al: Cytokeratin expression in epithelioid vascular neoplasms. Hum Pathol. 21:212–217, 1990.
521. Pinkard NB, Wilson RW, Lawless N, et al: Calcifying fibrous pseudotumor of pleura: A report of three cases of a newly described entity involving the pleura. Am J Clin Pathol. 105:189– 194, 1996. 522. Wilson RW, Galateau-Salle F, Moran CA: Desmoid tumors of the pleura: a clinicopathologic mimic of localized fibrous tumor. Mod Pathol. 12:9–14, 1999. 523. Andino L, Cagle PT, Murer B, et al: Pleuropulmonary desmoids tumors: immunohistochemical comparison with solitary fibrous tumors and assessment of β-catenin and cyclin D1 expression. Arch Pathol Lab Med. 130:1503–1509, 2006. 524. Moran CA, Travis WD, Rosada-de-Christenson M, et al: Thymomas presenting as pleural tumors: report of eight cases. Am J Surg Pathol. 16:138–144, 1992. 525. Payne CB Jr, Morningstar WA, Chester EH: Thymoma of the pleura masquerading as diffuse mesothelioma. Am Rev Respir Dis. 94:441–446, 1966. 526. Honma K, Shimada K: Metastasizing ectopic thymoma arising in the right thoracic cavity and mimicking diffuse pleural mesothelioma: An autopsy study of a case with review of the literature. Wien Klin Wschr. 98:14–20, 1986. 527. Shih D, Wang J, Tseng H, et al: Primary pleural thymoma. Arch Pathol Lab Med. 121:79–82, 1997. 528. Gaertner E, Zeren H, Fleming MV, et al: Biphasic synovial sarcomas arising in the pleural cavity: a clinicopathologic study of five cases. Am J Surg Pathol. 20:36–45, 1996. 529. Nicholson AG, Goldstraw P, Fischer C: Synovial sarcoma of the pleura and its differentiation from other primary pleural tumors: a clinicopathological and immunohistochemical review of three cases. Histopathol. 33:508–513, 1998. 530. Colwell AS, D’Cunha J, Vargas SO, et al: Synovial sarcoma of the pleura: a clinical and pathologic study of three cases. J Thorac Cardiovasc Surg. 124:828–832, 2002. 531. Ordonez NG, Mahfouz SM, Mackay B: Synovial sarcoma: an immunohistochemical and ultrastructural study. Hum Pathol. 21:733–749, 1990. 532. Yano M, Toyooka S, Tsukuda K, et al: SYT-SSX fusion genes in synovial sarcoma of the thorax. Lung Cancer. 44:391–397, 2004. 533. Litzky LA: Pulmonary sarcomatous tumors. Arch Pathol Lab Med. 132:1104–1117, 2008. 534. Hachitanda Y, Aoyama C, Sato JK, et al: Pleuropulmonary blastoma in childhood. A tumor of divergent differentiation. Am J Surg Pathol. 17:382–391, 1993. 535. Priest JR, McDermott MB, Bhatia S, et al: Pleuropulmonary blastoma: a clinicopathologic study of 50 cases. Cancer. 80:147– 161, 1997. 536. Hill DA, Sadeghi S, Schultz MZ, et al: Pleuropulmonary blastoma in an adult: an initial case report. Cancer. 85:2368–2374, 1999. 537. Celikoglu F, Teirstein AS, Krellenstein DJ, et al: Pleural effusion in non-Hodgkin’s lymphoma. Chest. 101:1357–1360, 1992. 538. Vega F, Padula A, Valbuena JR, et al: Lymphomas involving the pleura: a clinicopathologic study of 34 cases diagnosed by pleural biopsy. Arch Pathol Lab Med. 130:1497–1502, 2006. 539. Fetsch PA, Abati A, Hijazi YM: Utility of the antibodies CA-199, HBME-1 and thrombomodulin in the diagnosis of malignant mesothelioma and adenocarcinoma in cytology. Cancer. 84:101– 108, 1998. 540. Khoor A, Whitsett JA, Stahlman MT, et al: Utility of surfactant protein B precursor and thyroid transcription factor-1 in differentiating adenocarcinoma of the lung from malignant mesothelioma. Hum Pathol. 30:695–700, 1999. 541. Ordonez NG: Role of immunohistochemistry in differentiating epithelial mesothelioma from adenocarcinoma: review and update. Am J Clin Pathol. 112:75–89, 1999. 542. Ordonez NG: Immunohistochemical diagnosis of epithelioid mesotheliomas: a critical review of old markers, new markers. Hum Pathol. 33:953–967, 2002. 543. Ordonez NG: The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. Am J Surg Pathol. 27:1031–1051, 2003. 544. Comin CE, Novelli L, Boddi V, et al: Calretinin, thrombomodulin, CEA and CD15: a useful combination of immunohistochemical markers for differentiating pleural epithelial
References mesothelioma from peripheral pulmonary adenocarcinoma. Hum Pathol. 32:529–536, 2001. 545. Zhang PJ, Genega EM, Tomaszewski JE, et al: The role of calretinin, inhibin, melan-A, BCL-2 and C-kit in differentiating adrenal cortical and medullary tumors: an immunohistochemical study. Mod Pathol. 16:591–597, 2003. 546. Laskin WB, Miettinen M: Epithelioid sarcoma: new insights based on an extended immunohistochemical analysis. Arch Pathol Lab Med. 127:1161–1168, 2003. 547. Pan C, Chen DC, Choo T, et al: Expression of calretinin and other mesothelioma-related markers in thymic carcinoma and thymoma. Hum Pathol. 34:1155–1162, 2003. 548. Fine SW, McClain SA, Li M: Immunohistochemical staining for calretinin is useful for differentiating schannomas from neurofibromas. Am J Clin Pathol. 122:552–559, 2004. 549. Bergmann L, Maurer U, Weidmann E: Wilms tumor gene expression in acute myeloid leukemias. Leuk Lymphoma. 25:435–443, 1997. 550. Sugiyama H: Wilms tumor gene (WT1) as a new marker for the detection of minimal residual disease in leukemia. Leuk Lymphoma. 30:55–61, 1998. 551. Hwang H, Quenneville L, Yaziji H, et al: Wilms tumor gene product: sensitive and contextually specific marker of serous carcinoma of ovarian surface epithelial origin. Appl Immunohistochem Mol Morphol. 12:122–126, 2004. 552. Carpentieri DF, Nichols K, Chou PM, et al: The expression of WT-1 in the differentiation of rhabdomyosarcoma from other pediatric small round blue cell tumors. Mod Pathol. 15:1080– 1086, 2002. 553. Davidson B, Reich R, Lazarovici P, et al: Expression of the nerve growth factor receptor TrkA and p75 in malignant mesothelioma. Lung Cancer. 44:159–165, 2004. 554. van Hengel P, van Geffen F, Kazzaz BA, et al: Atypical carcinoid presenting as mesothelioma. Neth J Med. 58:185–190, 2001. 555. Henderson DW, Atwood HD, Constance TJ, et al: Lymphohistiocytoid mesothelioma: a rare lymphomatoid variant of predominantly sarcomatoid mesothelioma. Ultrastruct Pathol. 12:367–384, 1988. 556. Daya D, McCaughey WTE: Well-differentiated papillary mesothelioma of the peritoneum: a clinicopathologic study of 22 cases. Cancer. 65:292–296, 1990. 557. Yesner R, Hurwitz A: Localized pleural mesothelioma of epithelial type. J Thorac Surg. 26:325–329, 1953. 558. Thickett DR, Armstrong L, Millar AB: Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions. Thorax. 54:707–710, 1999. 559. Golden A, Ash J: Adenomatoid tumors of genital tract. Am J Pathol. 14:63–80, 1990. 560. Isotalo PA, Keeney GL, Sebo TJ, et al: Adenomatoid tumor of the adrenal gland: a clinicopathologic study of five cases and review of the literature. Am J Surg Pathol. 27:969–977, 2003. 561. Overstreet K, Wixum C, Shabaik A, et al: Adenomatoid tumor of the pancreas: a case report with comparison of histology and aspiration cytology. Mod Pathol. 16:613–617, 2003. 562. Argani P, Rosai J: Hyperplastic mesothelial cells in lymph nodes. Report of six cases of a benign process that simulate metastatic involvement by mesothelioma or carcinoma. Am J Surg Pathol. 29:339–346, 1998. 563. Brooks JSJ, LiVolsi VA, Pietra GG: Mesothelial cell inclusions in mediastinal lymph nodes mimicking metastatic carcinoma. Am J Clin Pathol. 93:741–748, 1990. 564. Ordonez NG, Ro JY, Ayal AG: Lesions described as nodular mesothelial hyperplasia are primarily composed of histiocytes. Am J Surg Pathol. 22:285–292, 1998. 565. Sussman J, Rosai J: Lymph node metastasis as the initial manifestation of malignant mesothelioma: Report of six cases. Am J Surg Pathol. 14:819–828, 1990. 566. Kannerstein M, Churg J, McCaughey WTE, et al: Papillary tumors of the peritoneum in women: mesothelioma or papillary carcinoma. Am J Obstet Gynecol. 127:306–314, 1977. 567. Foyle A, Al-Jabi M, McCaughey WTE: Papillary peritoneal tumors in women. Am J Surg Pathol. 5:241–249, 1981. 568. Mills SE, Andersen WA, Fechner RE, et al: Serous surface papillary carcinomas: a clinicopathologic study of 10 cases and comparison with stage III—IV ovarian serous carcinoma. Am J Surg Pathol. 12:827–834, 1988.
478.e13
569. Raju U, Fine G, Greenwald KA, et al: Primary papillary serous neoplasia of the peritoneum: a clinicopathologic and ultrastructural study of eight cases. Hum Pathol. 20:426–436, 1989. 570. Bell DA, Scully RE: Benign and borderline serious lesions of the peritoneum in women. Pathol Annu. 24:(pt 2):1–21, 1989. 571. Rutledge ML, Silva EG, McLemore D, et al: Serous surface carcinoma of the ovary and peritoneum: A flow cytometric study. Pathol Annu. 24:(pt 2):227–235, 1989. 572. Bell DA, Scully RE: Serous boderline tumors of the peritoneum. Am J Surg Pathol. 14:230–239, 1990. 573. Truong LD, Maccato ML, Awalt H, et al: Serous surface carcinoma of the peritoneum: a clinicopathologic study of 22 cases. Hum Pathol. 21:99–110, 1990. 574. Shah IA, Jayram L, Gani OS, et al: Papillary serous carcinoma of the peritoneum in a man. Cancer. 82:860–866, 1998. 575. Biscotti CV, Hart WR: Peritoneal serous micropapillomatosis of low malignant potential (serous borderline tumors of the peritoneum): A clinicopathologic study of 17 cases. Am J Surg Pathol. 16:467–475, 1992. 576. Khoury N, Raju R, Crissman JD, et al: A comparative immunohistochemical study of peritoneal and ovarian tumors, and mesotheliomas. Hum Pathol. 21:811–819, 1990. 577. Taylor DR, Page W, Huges D, et al: Metastatic renal cell carcinoma mimicking pleural mesothelioma. Thorax. 42:901–902, 1987. 578. Ordonez NG, Myhre M, Mackay B: Clear cell mesothelioma. Ultrastruct Pathol. 20:331–336, 1996. 579. Butnor KJ, Nicholson AG, Allred DC, et al: Expression of renal cell carcinoma-associated markers erythropoietin, CD10, and renal cell carcinoma marker in diffuse malignant mesothelioma and metastatic renal cell carcinoma. Arch Pathol Lab Med. 130:823–827, 2006. 580. Kato Y, Tsuta K, Ski K, et al: Immunohistochemical detection of GLUT-1 can discriminate between reactive mesothelium and malignant mesothelioma. Mod Pathol. 20:215–220, 2007. 581. Monaco SE, Shuai Y, Bansal M, et al: The diagnostic utility of p16 FISH and GLUT-1 immunohistochemical analysis in mesothelial proliferations. Am J Clin Pathol. 135:619–627, 2011. 582. Wu M, Sun Y, Li G, et al: Immunohistochemical detection of XIAP in mesothelium and mesothelial lesions. Am J Clin Pathol. 128:783–787, 2007. 583. Shi M, Fraire AE, Chu P, et al: Oncofetal protein IMP3, a new diagnostic biomarker to distinguish malignant mesothelioma from reactive mesothelial proliferation. Am J Surg Pathol. 35:878–882, 2011. 584. Lagana SM, Taub RN, Borczuk AC: Utility of glucose transporter 1 in the distinction of benign and malignant thoracic and abdominal mesothelial lesions. Arch Pathol Lab Med. 136:804– 809, 2012. 585. Attanoos RL, Griffin A, Gibbs AR, et al: The use of immunohistochemistry in distinguishing reactive from neoplastic mesothelium: a novel use for desmin and comparative evaluation with epithelial membrane antigen, p53, platelet-derived growth factor-receptor, P-glycoprotein and Bcl-2. Histopathology. 43:231–238, 2003. 586. Acurio A, Arif Q, Gattuso P, et al: Value of immunohistochemical markers in differentiating benign from malignant mesothelial lesions. Mod Pathol. 21:334A, 2008. 587. Illei PB, Rusch VW, Zakowski MF, et al: Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas. Clin Cancer Res. 9:2108–2113, 2003. 588. Lopez-Rios F, Chuai S, Flores R, et al: Global gene expression profiling of pleural mesotheliomas: overexpression of aurora kinases and P16/CDKN2A deletion as prognostic factors and critical evaluation of microarray-based prognostic prediction. Cancer Res. 66:2970–2979, 2006. 589. Prins JB, Williamson KA, Kamp MM, et al: The gene for the cyclin-dependent-kinase-4 inhibitor, CDKN2A, is preferentially deleted in malignant mesothelioma. Int J Cancer. 75:649–653, 1998. 590. Xio S, Li D, Vijg J, et al: Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene. 11:511–515, 1995. 591. Chiosea S, Krasinskas A, Cagle PT, et al: Diagnostic importance of 9p21 homozygous deletion in malignant mesotheliomas. Mod Pathol. 21:742–747, 2008.
478.e14
Immunohistology of Lung and Pleural Neoplasms
592. Illei PB, Ladanyi M, Rusch VW, et al: The use of CDKN2A deletion as a diagnostic marker for malignant mesothelioma in body cavity effusions. Cancer. 99:51–56, 2003. 593. Dacic S, Kothmaier H, Land S, et al: Prognostic significance of P16/CDKN2A loss in pleural malignant mesotheliomas. Virchows Arch (in press). 594. Allen TC, Popper HH, Kothmaier H, et al: Osteopontin (OPN) and HIF-1 expression in diffuse malignant mesothelioma (DMM) with long-term (>3 year) survival (LS) versus shortterm survival (SS). Mod Pathol. 21:335A, 2008. 595. Chung CT-S, Santos GC, Hwang DM, et al: p16/CDKN2A deletion as a diagnostic marker to distinguish benign from malignant mesothelial proliferations. Mod Pathol. 21:338A, 2008. 596. Xu H, Simon R, Bourne PA, et al: Immunohistochemical analysis of KOC/IMP3 in malignant pleural mesothelioma. Mod Pathol. 21:353A, 2008. 597. Gardner JM, Allen TC, Jagirdar J, et al: Expression of matrix metalloproteinase-7 (MMP-7) in 45 diffuse malignant
mesotheliomas (MM): potential target for therapy. Mod Pathol. 21:342A, 2008. 598. Moore BH, Cagle PT, Allen TC, et al: Topoisomerasae II-alpha, minichromosome maintenance protein 2 (MCM2), and X-linked mammalian inhibitor of apoptosis protein (XIAP) expression in pleural diffuse malignant mesothelioma (PDMM): possible role for chemotherapeutic intervention. Mod Pathol. 21:347A, 2008. 599. Westerhoff M, Faoro L, Loganathan S, et al: Immunohistochemical (IHC) expression of c-Met receptor tyrosine kinase (c-Met) has prognostic significance and its activation is related to phosphorylated protein kinase C β (p-PKC β) in malignant mesothelioma (MM). Mod Pathol. 21:353A, 2008. 600. Kadota K, Suzuki K, Colovos C, et al: A nuclear grading system is a strong predictor of survival in epithelioid diffuse malignant pleural mesothelioma. Mod Pathol. 25:260–271, 2012.
C H A P T E R 1 3
IMMUNOHISTOLOGY OF SKIN TUMORS VICTOR G. PRIETO
Overview 479 Epithelial Tumors of the Skin 479 Cutaneous Lymphohematopoietic Disorders 488 Mesenchymal Tumors of the Skin 494 Special Topics in Cutaneous Immunohistochemistry 506
Overview The skin is a complex microenvironment. The normal structures of the epidermis, dermis, and cutaneous adnexa are morphologically and functionally complicated, and the histologic entities that occur in this tissue compartment are also numerous. Furthermore, cutaneous lesions may also be a part of systemic proliferations, or they may have exact morphologic counterparts in other sites. Principal examples of such disorders are hematolymphoid diseases and mesenchymal tumors. Because the diagnostic and immunohistochemical (IHC) issues pertaining to those conditions are covered in detail elsewhere in this text, comments on them are relatively limited in scope, mainly centering on lesions peculiar to the skin. Finally, as with melanocytic lesions of the skin (see Chapter 7), this chapter primarily focuses on antigenic profiles that can be obtained with routinely processed, formalin-fixed tissues and commercially available reagents.
Epithelial Tumors of the Skin The many forms of differentiated epidermal and adnexal epithelia in the skin result in a potentially confusing nosologic categorization. Despite that diversity, most lesions can be grouped under five basic patterns of differentiation: 1) epidermal, 2) sweat glandular (eccrine and apocrine), 3) sebaceous, 4) follicular, and 5) endocrine. However, not all lesions can be easily classified in these five groups. As an example, a recently described entity, the clear cell carcinoma with comedonecrosis, is
a neoplasm characterized by an aggressive course (local recurrence or distant metastasis) that has features that resemble squamous cell carcinoma (SCC), sebaceous carcinoma, and eccrine carcinoma.1 Reflecting this morphologic diversity, this lesion expresses IHC features of those lesions, including cytokeratins 5 and 6 (CK5/6), the same as in SCC; epithelial membrane antigen (EMA) and CK17 in the clear cells, as in follicular structures or sebaceous carcinoma; and also focal carcinoembryonic antigen (CEA) expression, as in eccrine carcinoma. Cases like this one are best classified as “adnexal,” and then the types of differentiation that they contain may be indicated.2
Epidermal Tumors Tumors with differentiation toward epidermal cells are the most common epithelial neoplasms of the skin. The most important of these are SCC and basal cell carcinoma (BCC). Furthermore, BCC is the most common malignancy in humans. SCC is usually composed of polygonal cells with nuclear atypia and diverse degrees of keratinization, either in the epidermis (in situ) or invasive, and thus it is usually easy to diagnose. However, several microscopic subtypes of SCC may occasionally mimic both glandular and mesenchymal neoplasms in skin: these include adenoid (acantholytic), pleomorphic, small cell, and spindle cell forms.3-5 Because of the latter variants, an understanding of the immunohistologic attributes of squamous carcinoma is important to the differential diagnosis of cutaneous tumors in general. Fortunately, all forms of SCC show similar antigenic profiles. SCC contains an abundance of cytokeratin intermediate filament proteins that range from 40 to 68 kD in molecular weight.6-8 The more well-differentiated cells synthesize high-molecular-weight (HMW) cytokeratin; in contrast, poorly differentiated tumors usually only express low-molecular-weight (LMW) keratin peptides. The concomitant expression of both cytokeratin and vimentin characterizes spindle cell, pleomorphic, and some acantholytic squamous carcinomas and metastatic renal cell carcinoma (RCC; Fig. 13-1).9 Sarcomatoid carcinomas may sometimes express keratin only focally, and the best approach to their recognition is to use a broadly reactive mixture of monoclonal antikeratin as a screening reagent. CK5/6 could 479
480
Immunohistology of Skin Tumors
B
A
C Figure 13-1 A, Sarcomatoid (spindle cell) squamous cell carcinoma of the skin. Diffuse reactivity for vimentin (B) and keratin (C) are shown (antivimentin and antikeratin cocktail; diaminobenzidine and light hematoxylin).
be included as a specific target therein, because it is more selective for squamous differentiation. Another antigen displayed in SCC is EMA. Although variable amounts of this glycoprotein are encountered in most examples of SCC, diffuse expression of EMA is usually seen only in poorly differentiated lesions. Carcinoembryonic antigen (CEA) staining has been described in SCC,10 but in our experience, diffuse reactivity is rare. Also, p63 protein is a nuclear determinant associated with both myoepithelial and epithelial differentiation, and it is seen in squamous, basal cell, and appendageal cutaneous carcinomas (Fig. 13-2). It is a member of the p53 family, which includes the p53, p63, and p73 polypeptides. Squamous carcinomas of the skin lack S-100 protein, chromogranin, synaptophysin, CD99, CD15, and CD57. Further, reactivity with human melanoma black 45 (HMB-45) or anti–melan-A is consistently absent, as are desmin and muscle-specific isoforms of actin in most spindle cell forms of these neoplasms.9 There are no well-characterized proteins that exclusively define malignant epidermal differentiation. Molecules associated with epidermal keratinization, such as filaggrin and involucrin, are preferentially expressed in SCC but can also be seen in keratoacanthomas and a variety of benign keratinocytic proliferations.11-13 Although they may help distinguish SCC from BCC,
those markers cannot separate SCC from adnexal tumors of the skin, particularly those of pilar differentiation.
KEY DIAGNOSTIC POINTS Epithelial Tumors • Adenoid (acantholytic), pleomorphic, small cell, and spindle cell forms are variants of SCC that may mimic other lesions. • SCC is labeled with AE1/AE3, 34βE12, and CK5/6. • Poorly differentiated SCC is labeled with CAM5.2, AE1/AE3, and p63. • In general, SCC is EMA, p63, and CD44 positive and BerEp4 negative. • In general, basal cell carcinoma is EMA negative and BerEP4 positive.
BCC, as with SCC, has several distinctive variants, all of which invoke dissimilar differential diagnoses. Among the better-recognized subtypes of BCC are the morpheaform, adenoid, clear cell, hamartomatous (also referred to as infundibulocystic), and metatypical forms (i.e., with extensive squamous differentiation).14 Of all the subtypes, the ones associated with high rate of
Epithelial Tumors of the Skin
481
Figure 13-2 Positivity for p63 protein in poorly differentiated squamous cell carcinoma of the skin (anti-p63; diaminobenzidine and light hematoxylin).
Figure 13-4 Diffuse immunoreactivity for BerEP4 in basal cell carcinoma (anti-BerEP4; diaminobenzidine and light hematoxylin).
recurrence are those associated with an irregular, infiltrative border (sclerodermoid, morpheaform, infiltrative); small nest size (micronodular); extensive squamous differentiation; and eccrine differentiation. In general, BCC lacks complex patterns of antigenic expression. As for SCC, this tumor displays reactivity for CK polypeptides; however, the molecular weight of these intermediate filaments are typically less than 50 kD.14 In contrast to SCC, EMA is not observed in any variant of pure BCC (Fig. 13-3).15 Occasionally BCC can contain “passenger” melanocytes, and such cells will express melan-A (formerly MART-1) and HMB-45 antigen. Similarly, a few lesions exhibit staining for endocrine-associated peptides including CD56, synaptophysin, and chromogranin A (CG).16,17 The apparent ability of BCC to express such specialized determinants has been used to support the
premise that the cells of that lesion recapitulate the properties of epidermal “stem” cells. Nevertheless, the absence of vimentin, CEA, S-100 protein, CD57, and CD1518 suggests that there may be flaws in this “stem cell hypothesis” as applied to BCC. Indeed, BCC with additional patterns of differentiation such as “eccrine epithelioma,”19 “apocrine epithelioma,”20 and so-called basosebaceous epithelioma14 may show positivity for EMA, CEA, or CD15 in histologically divergent areas. Also interesting is reduced smooth muscle actin (SMA) expression in some BCCs associated with an aggressive behavior.21 Another useful glycoprotein marker present in most BCCs is recognized by the antibody BerEP4, directed at two epitopes, 34 kD and 39 kD, on human epithelial cells (Fig. 13-4).22 In the skin, BerEP4 labels not only BCCs but also the cells of Paget disease, Merkel cell carcinoma, and other selected appendageal neoplasms. It is probably most useful in separating BCC with squamous differentiation from basaloid SCC, which is BerEP4 negative.23 Conversely, squamous tumors bind to the L-Fucose–specific lectin, Ulex europaeus I, which is usually negative in BCC.24 The distinction between basaloid SCC and BCC is diagnostically crucial in certain anatomic locations, such as the anal and perianal skin, because the former has a much worse prognosis.
Sweat Duct Tumors
Figure 13-3 Negative epithelial membrane antigen (EMA) in basal cell carcinoma. Note the positivity in the overlying epidermis (antiEMA; diaminobenzidine and light hematoxylin).
The eccrine and apocrine glandular adnexa comprise the sudoriferous structures of skin. The neoplasms of these structures are histologically diverse but share certain immunohistologic features.25,26 All of these tumors demonstrate CK reactivity and the potential for expression of CEA, tumor-associated glycoprotein 72 (TAG-72, also known as CA72.4; Fig. 13-5), EMA, CD15, and p63.27-30 The last three of these substances are seen in varying proportions in both adenomas and carcinomas. EMA is more often observed in malignant sudoriferous neoplasms than in benign ones; indeed, spiradenoma and cylindroma typically lack this
482
Immunohistology of Skin Tumors
Figure 13-5 Positivity for TAG-72, recognized by antibody B72.3, in sweat gland carcinoma (B72.3; diaminobenzidine and light hematoxylin).
determinant. In contrast, CEA and CD15 are detected in roughly 70% to 80% of all eccrine and apocrine lesions31-33 regardless of their biologic potentials; TAG-72 appears to show a predilection for apocrine lesions and is usually not seen in eccrine tumors.34 This profile proves useful in the separation of sudoriferous neoplasms from other cutaneous glandular and epidermal
A
C
neoplasms. It has also been instrumental in verifying the presence of sweat glandular differentiation in occasional examples of lymphoepithelioma-like carcinoma of the skin, a poorly differentiated tumor that shows histologic similarities to nasopharyngeal lymphoepithelioma.35 Immunophenotypic differences between eccrine and apocrine neoplasms may occasionally be exploited to diagnostic advantage. For instance, eccrine carcinomas express S-100 protein in almost 50% of cases, whereas apocrine carcinomas, including invasive extramammary Paget disease (EMPD), are generally negative for that marker.36 Conversely, the mammary antigen gross cystic disease fluid protein 15 (GCDFP-15) and androgen receptors are more commonly expressed by cutaneous apocrine cells and neoplasms (Fig. 13-6),31,37,38 along with TAG-72. Some studies have described the selective expression of specific CK peptides and other antigens in either eccrine or apocrine lesions. Antibodies to EKH5, EKH6, and IKH-4; chondroitinase ABC–sensitive anionic sites, detected by cationic gold at pH 2.0 after pretreatment with ethylene glycol tetraacetic acid (EGTA); and intercellular canaliculi with high activity of alkaline phosphatase are reportedly specific for eccrine differentiation, whereas antibodies to 70-kD glycoprotein purified from human milk fat globule (HMFG) membranes and HMFG-1 antigen (1.10.F3,
B
Figure 13-6 A, Extramammary Paget disease of the skin. B, Reactivity for gross cystic disease fluid protein-15 (GCDFP-15) is often demonstrated. C, Some cases express ERBB2 (formerly Her2/neu) and thus may be amenable to treatment with Herceptin (anti-ERBB2; diaminobenzidine and light hematoxylin).
Epithelial Tumors of the Skin
against defatted HMFG membranes) would be present only in apocrine cells.39-41 GCDFP-15, CA72.4, androgen receptor, CK7, CD23, and BerEP4 can separate EMPD from melanoma and pagetoid SCC—that is, intraepidermal squamous carcinoma with pagetoid migration that resembles Paget disease and melanoma—because they are present only in the first of those three tumors.5,42 In contrast, SCC expresses HMW keratin, whereas melanoma reacts with HMB-45, melan-A, and antibodies to S-100 protein in this group of lesions (see Chapter 7). In EMPD, there seems to be a differential expression of mucin core proteins (MUCs), because intraepidermal EMPD shows expression of MUC1 and MUC5, whereas the latter is lost in invasive EMPD.43 Cytokeratin subtyping can also help, because primary vulvar EMPD usually expresses CK7 and GCDFP-15, whereas EMPD secondary to anorectal carcinomas usually expresses CK20.44 Furthermore, EMPD secondary to urothelial carcinoma expresses CK7, CK20, and uroplakin III but not GCDFP-15.45-47 Alpha-methylacyl-Co-A-racemase (AMACR) reactivity has been observed in EMPD and also in SCC and melanoma,48 therefore it has less diagnostic value compared with the aforementioned antigens. Also,
A
C
483
highlighting the similarities between sweat gland tumors and mammary carcinomas, EMPD can express ERBB2 (formerly Her2/neu), a fact that may be exploited for therapeutic purposes (see Fig. 13-6).47,49 One difficulty in the diagnosis of sweat gland carcinoma is its distinction from metastatic adenocarcinoma in the skin, although the best way of achieving that goal is still by paying attention to the clinical history. Primary adnexal tumors are typically solitary and slowly growing (present for at least 6 months), whereas metastases are multiple and evolve rapidly. However, data from some studies suggest that p63 reactivity may be helpful,27,28 because expression of p63 by more than 25% of tumor cells has been observed in the majority of sweat gland and apocrine carcinomas, both primary and metastatic,50 but it has not been seen in metastatic adenocarcinomas. A possible pitfall with p63 is that it will be expressed in squamous and urothelial neoplasms.29 Additional markers that may contribute to the separation of primary and secondary carcinomas in the skin are calretinin, CK5/6, and podoplanin.30,51 As with p63, these antigens are mainly seen in primary cutaneous appendage tumors and not in adenocarcinomas metastatic to the skin (Fig. 13-7).
B
Figure 13-7 A, Primary eccrine carcinoma in the skin with papillary features. B, Positivity for p63 is shown in more than 25% of the tumor cells. C, Focal calretinin expression is also apparent (anti-p63 and anticalretinin; diaminobenzidine and light hematoxylin).
484
Immunohistology of Skin Tumors
KEY DIAGNOSTIC POINTS Sweat Gland Tumors • Sweat gland tumors express keratin, CEA, CA72.4, CD15, and p63. • EMA is more common in malignant neoplasms. • Eccrine carcinomas are S-100 positive but apocrine S-100 negative and are positive for GCDFP-15, estrogen receptors, and p63. • Paget disease is positive with CK7, GCDFP-15, BerEP4, and CA72.4; some secondary extramammary Paget disease is CK20 positive. • ERBB2 (formerly Her2/neu) may be expressed in cutaneous adenocarcinomas, including extramammary Paget disease, and thus may be a therapeutic target.
Sebaceous Tumors Although it is a glandular adnexal structure, the sebaceous gland is topographically and ontogenetically related to the hair sheath, hence the similarities between the isthmus of the outer hair sheath and germinative basaloid cells of the sebaceous gland. Also there may be lesions with both basosebaceous and hair sheath elements (as in epithelioma with sebaceous differentiation and sebaceous epithelioma, also called “sebaceoma” by some authors) or the finding of melanocytes in both follicular and sebaceous lesions. Thus in contrast to most eccrine or apocrine tumors, pilar and sebaceous neoplasms commonly express both high- and lowmolecular-weight keratins, including CK17. Many antigens associated with sweat gland tumors—including S-100 protein, CA72.4, GCDFP-15, and CEA—are absent in sebaceous neoplasms. Reactivity for EMA and related substances is often obtained in sebaceous tumors.52 In both benign and malignant sebaceous proliferations, a characteristic microvesicular or “bubbly” cytoplasmic profile of mature sebocytes results in scalloping of a central nucleus
A
(Fig. 13-8). EMA expression in such neoplasms is comparable to that seen in nonneoplastic sebaceous epithelium, in that cytoplasmic lipid vesicles are rimmed by EMA reactivity. Another marker that is fairly specific for sebaceous differentiation is adipophilin,53 a marker seen in the lipid vacuoles characteristic of adipocytes. However, because positivity with antiadipophilin can be seen in other vacuoles in macrophages, to consider that this antibody indicates sebaceous differentiation, it has to label the membrane of the vacuoles (see Fig 13-8). Other diagnostically relevant determinants shared by sebaceous and sweat gland neoplasms are CD15 and BerEP4.22,33 However, an antibody panel directed at EMA, S-100 protein, adipophilin, and CEA should allow for separation between those tumors. In addition, Bayer-Garner and colleagues54 found that all sebaceous tumors were reactive for androgen receptors, whereas Shikata and colleagues55 suggested that sebaceous carcinomas lacked such expression. The pathologic distinction between primary sebaceous carcinoma and metastatic renal cell carcinoma (RCC) is important, because both lesions share a clear cell appearance. Slow evolution of a solitary skin tumor favors a primary lesion. In addition, podoplanin and adipophilin are effective in this context; both are consistently present in sebaceous tumors,30 whereas they are absent in RCC.53,56 CD10 is potentially seen in both lesion categories.57,58 KEY DIAGNOSTIC POINTS Sebaceous Tumors • No expression of S-100 protein, CA72.4, GCDFP-15, or CEA was found, compared with sweat gland tumors, which are positive for these markers. • Sebaceous and sweat gland tumors are CD15 and BerEP4 positive. • EMA and adipophilin are positive. Adipophilin should be seen in the membrane of the cytoplasmic vacuoles to be considered positive.
B
Figure 13-8 Sebaceous carcinoma (A) usually demonstrates diffuse reactivity for epithelial membrane antigen (B), often with a “bubbly” cytoplasmic pattern of staining.
Epithelial Tumors of the Skin
Pilar Tumors The multiplicity of diagnoses assigned to benign pilar tumors reflects the multiple morphologic manifestations of trichogenic differentiation. Unfortunately, most commercially available antibodies fail to select among those various patterns, including lesions with features of the germinal matrix, cortex, inner hair sheath, outer hair sheath, and infundibulum. Indeed, the antigenic profiles of all benign pilar tumors are generally similar: they typically contain BerEP4 antigen, p63, and CK polypeptides more than 50 kD in molecular weight, including CK17,7,59 but except for occasional proliferating pilar tumors, trichogenic neoplasms are EMA negative. CEA, S-100, CD15, CA72.4, the HMB-45 antigen, and GCDFP-15 are also usually not expressed in these tumors. As such, the immunophenotype of pilar tumors is generally similar to that of BCC. Regarding the differentiation between trichoepithelioma (TE) and BCC, several studies have indicated IHC differences between the two. In BCC, Bcl-2 protein is expressed with a diffuse pattern (Fig. 13-9) of the epithelial cells, whereas CD34 is expressed in the peritumoral stromal cells of TE.60,61 Moreover, Lum and Binder62 found that a Ki-67 index of greater than 25% and p21 protein expression were also discriminatory, and both favor a diagnosis of BCC. In contrast, other authors have indicated that IHC is not very helpful in this differential diagnosis.63 Because both TE and BCC are neoplasms with putative differentiation toward follicular structures, it is likely that IHC is not going to be able to absolutely differentiate every case. Furthermore, we recently reported our experience in trying to separate TE from a type of BCC that has advanced follicular differentiation (hamartomatous/ infundibulocystic). As expected, both tumors share many features, but expression of CK20-positive cells (Merkel cells) and CD10-positive stromal cells is more
A
485
frequent in TE, whereas BCC expresses CD10 in the basaloid cells.64 Three lesions that may be very similar histologically are desmoplastic trichoepithelioma (DTE), infiltrative BCC (IBCC), and microcystic adnexal carcinoma (MAC). The epithelial cells in DTE more often show EMA positivity than in IBCC and frequently show neuroendocrine (Merkel) cells with chromogranin A and anti-CK20, as mentioned above (Fig. 13-10).65,66 Peritumoral stromal cells are reactive for stromelysin-3, a proteolytic enzyme, in IBCC but not in DTE,67 and the ducts in MAC will express CEA in their lumina.68,69 The “hard keratins” recognized by the antibodies AE12 and AE13 are selectively expressed in cells that exhibit matrical differentiation. Pilomatricoma is diffusely reactive with AE13, whereas diminutive or abortive hair follicles represent the only reactive population in trichofolliculoma. Tumors with trichilemmal patterns of differentiation, proliferating pilar tumor and tricholemmoma, are negative with AE13 but usually react with AE14 (Fig. 13-11), a monoclonal antibody that recognizes both a cortical sulfur-containing moiety and an LMW cytokeratin.70 The latter keratin is typically expressed by most adnexal epithelia and epidermal basal cells. The small keratotic cysts in desmoplastic trichoepithelioma are AE13 positive, whereas examples of classic trichoepithelioma are reportedly negative. Consistent AE13 labeling is also seen in the small keratin cysts of microcystic adnexal carcinoma, indicating partial pilar differentiation in that tumor (Fig. 13-12).69 The expression of keratin proteins labeled by the anti-hair keratin (HKN) antibodies HKN5, HKN6, and HKN7 and anti–human hair keratin (HHK) probably reflect specialized patterns of pilar differentiation.71 Substances recognized by HKN6/7 are found only in cuticular, cortical, and inner hair sheath cells, whereas HKN5 also labels a cellular component of the outer hair
B
Figure 13-9 A, Positivity for Bcl-2 protein in basal cell carcinoma. B, In contrast with trichoepithelioma, no significant labeling of peritumoral spindle cells is seen (anti-CD34 and anti–Bcl-2; diaminobenzidine and light hematoxylin).
486
Immunohistology of Skin Tumors
A
B
C
D
Figure 13-10 A, Reactivity for chromogranin A is apparent in scattered cell nests in desmoplastic trichoepithelioma. B, Similar reactivity is apparent with CK20. C-D, Note the extensive labeling for CD34 and CD10 in the stromal cells that surround the clusters of trichoepithelioma tumor cells. Those antigens are typically absent in sclerosing basal cell carcinoma, which is the principal differential diagnostic consideration.
Figure 13-11 Immunoreactivity for “pilar type” keratin in lowgrade malignant proliferating pilar tumor, as recognized with antibody AE14 (diaminobenzidine and light hematoxylin).
sheath. Pilomatricomas typically express the HKN6/7 and HHK antigens, and the latter is also present in malignant pilomatricoma.72 In contrast, most other pilar tumors, BCCs, and other epidermal proliferations do not contain these substances. HKN5 is expressed by pilar tumors and BCCs, but it is not expressed in seborrheic keratoses. Along these lines, the detection of HKNs 5, 6, and 7 in some examples of the Borst-Jadassohn (clonal) type of intraepidermal carcinoma suggests that some such lesions may have pilar characteristics.71 With malignant transformation, the immunophenotypes of pilar neoplasms become more complex. Trichilemmal and squamous carcinomas that arise in proliferating pilar tumors (referred to as malignant proliferating pilar tumors)73 often display EMA. It has been reported that trichilemmal carcinomas express CEA,74 although it is unclear whether some of those lesions were actually the more aggressive, newly described entity of adnexal clear cell carcinoma with comedonecrosis.1
Epithelial Tumors of the Skin
A
487
B
Figure 13-12 A, Microcystic adnexal carcinoma of the skin. B, Multifocal positivity with antibody AE13 is shown (diaminobenzidine and light hematoxylin).
KEY DIAGNOSTIC POINTS Pilar Tumors • The pilar immunoprofile is similar to BCC of skin: EMA, CEA, S-100 protein, CD15, and CA72.4 are negative. • Desmoplastic trichoepithelioma versus BCC: EMA, CD15, chromogranin, CK20, and CD10 are largely negative in the cluster of tumor cells of BCC. • BCC stromal cells are stromelysin-3 and CD10 positive. Most lesions lack CD34 expression in the peritumoral dendritic cells.
Endocrine Tumors Primary cutaneous neuroendocrine carcinoma was originally referred to as trabecular carcinoma of the skin, but it is mostly known as Merkel cell carcinoma (MCC). Some authors have postulated that MCC displays neurotactile differentiation that emulates Merkel cells of the normal skin and oral mucosa. Nonneoplastic Merkel cells are reactive for CK20, CG, MOC-31, neurofilament protein (NFP), CD56, met-enkephalin, vasoactive intestinal polypeptide (VIP), and blood group antigen Pr(h), but in general, they appear to lack the ability to synthesize other endocrine determinants consistently. Malignant Merkel cells may express keratins (with pankeratin cocktails), CK20, NFP, CD15, CD56, CD57, EMA, MOC-31 antigen, BerEP4 antigen, chromogranin A, calcitonin, somatostatin, adrenocorticotropic hormone (ACTH), VIP, pancreatic polypeptide, and substance P.75-77 It has also been suggested that MCC is closely related to sweat gland carcinomas, because rare MCC lesions show glandular or squamous differentiation. Because of its capacity for diffuse or medullary patterns of growth and its uniform, occasionally dyshesive small cell constituency, MCC is potentially mistaken for lymphoma and leukemia cutis. Although leukemia/ lymphoma and MCC may express Pax-5 and terminal
deoxynucleotidyl transferase (TdT),78,79 lymphoma is reactive for CD45, whereas MCC is not. Moreover, keratin filaments in Merkel cell carcinomas are often clustered in the perinuclear cytoplasm and yield a characteristic “dot” of chromogenic precipitate (Fig. 13-13). Such an image is simultaneously diagnostic of epithelial and neuroendocrine differentiation in a small cell cutaneous neoplasm. Histologic features cannot reliably distinguish MCC from metastatic small cell neuroendocrine carcinomas. Nonetheless, clinical pathologic correlation and a battery of antibodies to CEA, CK20, NFP, and thyroid transcription factor 1 (TTF-1) are useful in this context. Bronchogenic small cell neuroendocrine carcinoma typically shows reactivity for CEA or TTF-1 (Fig. 13-14) or both, but it lacks CK20 and NFP.80,81 Conversely, CK20 and NFP are often expressed by MCC and are generally absent in extracutaneous neuroendocrine
Figure 13-13 Perinuclear keratin (dotlike) reactivity in Merkel cell carcinoma. That pattern simultaneously identifies the tumor as epithelial and neuroendocrine, and it is only very rarely seen in neuroendocrine tumors outside the skin, mainly in the salivary gland (antikeratin; diaminobenzidine and light hematoxylin).
488
Immunohistology of Skin Tumors
Cutaneous Lymphohematopoietic Disorders Given the histologic diversity of T-cell and B-cell lymphoid and histiocytic proliferations of skin, complete consideration of each of those conditions is well beyond the scope of this chapter; indeed, detailed information on the immunohistology of hematopoietic lesions in general is provided in other chapters in this book. Definitive determinations of cell lineage in several of these disorders are often difficult or impossible to make, if only their morphologic and clinical attributes are considered. This discussion emphasizes selected aspects of immunophenotyping that have special relevance to hematologic lesions of the skin. Figure 13-14 Diffuse nuclear positivity for thyroid transcription factor 1 in metastatic small cell neuroendocrine carcinoma arising in the lung and involving the skin. That marker is exceptionally seen in Merkel cell carcinoma (diaminobenzidine and light hematoxylin).
carcinomas. A more recently described antigen, the Merkel cell polyomavirus (MCPyV) has not been reported to be expressed in other neuroendocrine carcinomas.82 Another differential diagnosis is with Ewing sarcoma/ primitive neuroectodermal tumor (ES/PNET) primary or metastatic to the skin.83,84 Although these tumor types share possible reactivity for CD56, CD57, FLI-1, synaptophysin, and CD99 (Fig. 13-15, A), expression of pankeratin and CK20 is unusual in PNETs. KEY DIAGNOSTIC POINTS Endocrine Tumors • Merkel cell carcinoma is positive for CK20 (dot pattern), neurofilament protein, CD15, CD56, CD57, chromogranin, and various neuroendocrine hormones. Rare cases express TTF-1. • Pulmonary small cell carcinoma metastatic in skin is CEA and TTF-1 positive and CK20 negative. • In cutaneous Ewing sarcoma/PNET, CK is focal if positive, vimentin is positive, and CK20 and EMA are negative.
MCC may also be mistaken for BCC.85 The potential for endocrine differentiation in the latter tumor has already been discussed above, but it is never as diffuse or global as that seen in MCC. Moreover, CK20 and EMA are not seen in BCC, in contrast with MCC, and the perinuclear keratin reactivity pattern of MCC is absent in BCC. Zembowicz and colleagues86 have described an endocrine mucin-producing sweat gland carcinoma. That lesion histologically resembles mucinous carcinoma, rather than MCC, and it is immunoreactive for estrogen and progesterone receptor proteins in addition to endocrine markers. Expected immunoreactivity patterns in various cutaneous epithelial tumors are shown in Figure 13-15, B.
Lymphoma and Leukemia in the Skin Virtually all examples of lymphoma and leukemia cutis are reactive for CD45 in paraffin sections,87 which allows for the exclusion of histologically similar nonhematologic cellular proliferations. In addition, several lineage-selective reagents may be used that include CD3, 4, 5, 7, 8, and 43 and CD45RO as effective T-cell markers; CD20, 4KB5, CD45R, CD79a, Pax-5, CD179, and cyclin D1 as B-cell markers; CD68, MAC387, factor XIIIa (FXIIIa), and cathepsin B as histiocyte/monocyte markers; and CD30 and ALK-1 as selective markers in primary and secondary anaplastic large cell lymphomas (ALCLs; Fig. 13-16).88-92 Hodgkin lymphoma of the skin is extraordinarily rare, therefore it is not considered here, except to say that ALCL appears to be separable from that entity because of its common reactivity for CD99 and not for CD15.93 We should keep in mind that some T-cell lymphomas that express CD30 may also express CD15.94 A recent study has suggested the use of a panel of Pax-5, Oct2, and BOB.1, because anti–Pax-5 is strongly positive in classic Hodgkin disease, anti-Oct2 may be focally positive, and anti-BOB.1 is negative; ALCL is negative for all three B-cell markers.95 Among the other markers listed earlier, most are closely restricted to their respective lymphoid lineages. However, CD43 may be seen in selected B-cell lesions, T-cell infiltrates, and myeloid proliferations.96 Because cutaneous myelomonocytic infiltrates are typically CD43 positive but lack CD45,97 “CD43-only” lesions should be considered likely myeloid leukemia cutis (extramedullary myeloid tumor, granulocytic sarcoma), a diagnosis that can be further supported with other granulocyte- and monocyte-related markers such as myeloperoxidase, CD117, and cathepsin-B (Fig. 13-17).92,98 In young children, lymphoblastic leukemia/ lymphoma may also present with a CD43-only phenotype in the skin. The cells will also express CD10 (Fig. 13-18), CD99, Pax-5, TdT, and CD179.99-101 Expression of these markers supports a diagnosis of a lymphoblastic infiltrate. One important limitation of the immunophenotypic analysis of paraffin sections is that the markers listed earlier do not distinctly differentiate between benign and malignant lymphohistiocytes. This problem is made
Epithelial Tumors of the Skin
489
A 100 SCC BCC SGC
90
SBC PC NEC
80
70
60
50
40
30
20
10
0 CK5/6
B
AE13
AE14
PER CK
BerEP4
EMA
CEA
S100
ARP
p63
TAG-72
CGA
SYN
CD56
Figure 13-15 A, Immunoreactivity for CD99 in primitive neuroectodermal tumor of the subcutis can also be seen in Merkel cell carcinoma. B, Expected immunoreactivity patterns in various carcinomas in the skin. SCC, Squamous cell carcinoma; BCC, basal cell carcinoma; SGC, sweat gland carcinoma; SBC, sebaceous carcinoma; PC, pilar carcinoma; NEC, neuroendocrine carcinoma; PER CK, perinuclear globular reactivity for keratin; EMA, epithelial membrane antigen; CEA, carcinoembryonic antigen; S100, S-100 protein; ARP, androgen receptor protein; TAG-72, tumor-associated glycoprotein-72; CGA, chromogranin A; SYN, synaptophysin.
490
Immunohistology of Skin Tumors
A B
C
even more vexing by the recent recognition that cutaneous lymphoid hyperplasia and lymphoma cutis probably represent a continuum rather than mutually exclusive entities.102,103 Hence it may not always be possible to distinguish between those disorders, even with the use of other techniques, such as analysis of gene rearrangement of T-cell receptors. In any event, the presence of functionally and immunophenotypically mature lymphoid follicles and a
A
Figure 13-16 A, Anaplastic large cell lymphoma (ALCL) of the skin. B, Expression of CD30 is shown. C, Cases of systemic ALCL that involve the skin also usually express the ALK-1 protein (diaminobenzidine and light hematoxylin).
mixed inflammatory infiltrate indicate benign cutaneous B-cell infiltrates and only rarely B-cell lymphomas. Many cutaneous B-cell malignancies also show monotypic cell-surface or cytoplasmic immunoglobulin kappa or lambda light chains.91 Commercially available anti-κ and anti-λ IHC or in situ reagents may work poorly in fixed tissue but are usually sensitive enough to detect monoclonality in some B-cell and plasma cell proliferations.104 Heavy- or light-chain immunoglobulin gene
B
Figure 13-17 A, Myeloid leukemia cutis. B, Multifocal reactivity for myeloperoxidase is shown (diaminobenzidine and light hematoxylin).
Cutaneous Lymphohematopoietic Disorders
491
B
A
Figure 13-18 A, Lymphoblastic lymphoma of the skin. B, Diffuse labeling for CD10 is shown (diaminobenzidine and light hematoxylin).
rearrangements can also be detected in many cutaneous B-cell lymphomas.105 IHC may help to diagnose T-cell neoplasms, because T-cell lymphomas may show a predominance of CD4 or CD8 cells, such as in mycosis fungoides (MF) or in subcutaneous T-cell lymphoma (Fig. 13-19).106,107 Loss
of pan–T-cell markers CD3, CD5, CD43, and CD7 especially may be observed.108 Unfortunately, some examples of cutaneous lymphoma do not exhibit aberrant phenotypes, whereas benign infiltrates may occasionally show abnormal antigenic profiles. Reactive conditions such as nickel allergy may present with a
A
B
C
Figure 13-19 A, Mycosis fungoides. B, Partial or complete deletion of CD7 is often shown in the neoplastic lymphoid cells. C, Diffuse CD4 reactivity is retained (anti-CD7 and anti-CD8; diaminobenzidine and light hematoxylin).
492
Immunohistology of Skin Tumors
CD4-positive, CD7-negative infiltrate, thus mimicking MF. T-cell gene rearrangement may also be helpful in distinguishing benign from malignant T-cell processes.109 Certain aberrant B-cell phenotypes may also have diagnostic value. The most well-known example is the coexpression of CD5 or CD43 with CD20 in B-cell infiltrates.110
Special Pseudoneoplastic Lymphoid Lesions of the Skin EPIDERMOTROPIC INFILTRATES RESEMBLING MYCOSIS FUNGOIDES
Actinic reticuloids and selected drug-induced dermatitides mimic MF histologically, mainly those that show lichenoid and spongiotic patterns, such as with dilantin, carbamazepine, and hydrochlorothiazide therapy.111-113 This is so because they feature exocytosis and colonization of the epidermis by lymphocytes, and such cells commonly demonstrate at least a modest degree of nuclear atypia. Nearly universally, these elements are also T cells, as are the proliferating cells of MF. As mentioned before, predominance of CD4 or CD8 may be helpful in distinguishing benign and malignant T-cell processes. Most reactive conditions will have an approximately equal CD4/CD8 ratio, particularly in the intraepidermal lymphocytes. Actinic reticuloid shows a predominantly CD8-positive infiltrate in those intraepidermal lymphocytes. CD7 is the pan–T-cell antigen cluster most often lost in MF, but it can also be absent in reactive conditions (see above). Drug-induced pseudo-MF may have an immunophenotype similar to chronic spongiotic dermatitis, or it may simulate the profile of MF perfectly, including possible rearrangements of the T-cell receptor.111,114 In the final analysis, withdrawal of all medications and continued clinical surveillance over time may be the only means to distinguish drug-induced dermatitis from MF. DEEP LYMPHOID INFILTRATES SIMULATING SMALL CELL OR MIXED B-CELL LYMPHOMAS
Generally speaking, “bottom-heavy” deep lymphoid infiltrates of the dermis and subcutis suggest the diagnosis of a B-cell lymphoma.115 In general, benign dermal processes are composed principally of T lymphocytes, or they show a roughly equal admixture of B and T cells. Such lesions include Jessner infiltrates, deep cutaneous lupus erythematosus, and cutaneous lymphoid hyperplasia (CLH). Frozen section studies or in situ hybridization (ISH) analyses are helpful if they show restriction of lambda or kappa light-chain immunoglobulin expression by the constituent B cells. By convention, this profile must demonstrate a ratio of at least 10 : 1 when one light chain is compared with the other. B-cell lymphoma is the likely diagnosis when CD20 and CD43 are coexpressed by the same population of lymphocytes, when more than 75% of the infiltrate is marked as B cells, and when more than 30% of the cells are positive for proliferating cell nuclear antigen or
Figure 13-20 Labeling for Bcl-2 protein is seen only inconsistently in primary follicular lymphomas of the skin and is principally represented in low-grade tumors (diaminobenzidine and light hematoxylin).
Ki-67.110 Bcl-2 protein is an inhibitor of apoptosis, and it is overexpressed in B cells that demonstrate a t(14;18) chromosomal translocation.116 Such translocation is characteristically seen in systemic follicular lymphomas (Fig. 13-20). However, because of their T-cell content, many examples of CLH and MF contain numerous Bcl-2–positive cells and also some cutaneous B-cell lymphomas. Therefore the pathologist must restrict attention only to the follicular areas of CLH and cutaneous follicular lymphoma; in both cases, the B cells in the follicular regions are Bcl-2 negative.117-119 CUTANEOUS LARGE CELL LYMPHOID PROLIFERATIONS THAT SIMULATE LARGE B-CELL LYMPHOMA
Occasional examples of follicular CLH may contain such a strikingly large number of reactive immunoblasts that they mimic the microscopic appearance of a nodular B-cell lymphoma of the large cell type.115 Similarly, Kikuchi disease (KD)—a rare benign lymphoproliferative disorder seen much more often in Asia than in the United States—also comprises large atypical lymphoid elements that may cause confusion with a malignant process.120 A broad panel of antibodies may be especially helpful in cases of florid follicular CLH. A large number of B cells are present among the large-cell population, but features of nodal hyperplasia are also present: mantles, interfollicular zones rich in T cells, and accentuation of Ki-67 reactivity in the central aspects of the follicular aggregates. Anti-CD30 may detect scattered, large cells, as in most benign processes.121 KD features large, atypical mononuclear cells in the dermis admixed with neutrophils and zones of necrosis; as such, it is a potential imitator of a subtype of ALCL.120,122 KD is apparently a true histiocytic
Cutaneous Lymphohematopoietic Disorders
proliferation and is correspondingly labeled by the monoclonal antibody MAC387 and by those in the CD68 group (e.g., KP-1) but not by CD20 or pan–T-cell reagents.123
Langerhans Cell Histiocytosis/ Granulomatosis (“Histiocytosis X”) As with other prototypical hematopoietic infiltrates of the skin, Langerhans cell histiocytosis/granulomatosis (LCH) is often readily diagnosed when all of its expected clinicopathologic features are present.124 LCH is typically a disorder of children and often presents as a multifocal macular or papular eruption represented by vaguely defined or nodular histologic infiltrates of histiocytoid cells with characteristically folded or grooved nuclei. Patients with LCH often have prior or concurrent disease in extracutaneous sites, but LCH may occasionally present as a solitary skin lesion. Adults may develop limited cutaneous LCH as well.125 In each of these circumstances, the clinical and morphologic differential diagnosis may include lymphoma cutis, melanoma, and poorly differentiated epithelial and mesenchymal lesions. This review will not enter into the controversy about the relationship between LCH and other Langerhans cell–rich processes such as Letterer-Siwe and HandChristian-Schuller disease. The most accepted of these lesions is known as eosinophilic granuloma, a term used for isolated lesions rich in Langerhans cells that can occur in almost any location. Regarding the neoplastic nature of LCH, lesions may be monoclonal and may even contain a BRAF V600D mutation.126 Regardless of its mode of presentation in the skin, LCH has a consistent immunophenotype. As with nonneoplastic intraepidermal Langerhans cells, the constituent cells of LCH are usually reactive for S-100 protein, fascin, CD1a, langerin, and CD31 (Fig. 13-21).127,128 KEY DIAGNOSTIC POINTS Pseudoneoplastic Lymphoid Proliferations • CD4-positive intraepidermal lymphocytes that are negative for CD3, CD5, CD7, CD43, or CD45R9O are more likely to be seen in mycosis fungoides than in benign conditions. • B-cell lymphoma is more likely with coexpression of CD20 and CD43, when greater than 75% of lymphocytes are B cells, and if Ki-67 is greater than 30% of cells. • Cells in Kikuchi disease are true histiocytes: they are negative for B- and T-cell markers and positive for CD68 and MAC387. • Langerhans histiocytosis cells express S-100 protein, fascin, CD1a, and CD31.
S-100 protein reactivity is otherwise limited to a few examples of non-LCH histiocytic infiltrates, including reticulum cell proliferations and extranodal cutaneous infiltrates of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). Additional evidence of Langerhans cell differentiation may be gained from
493
the ultrastructural demonstration of Birbeck granules in the lesional cells, but those inclusions are not seen in all examples of LCH.129 Two other cutaneous histiocytoses that typically affect children, congenital self-healing reticulohistiocytosis (CSHR) and benign cephalic histocytosis (BCH) resemble LCH.130,131 The former is characterized by a proliferation of Langerhans cells and shares most immunophenotypic and ultrastructural attributes with LCH. In fact, CSHR is generally regarded as a localized form of LCH. In contrast, the infiltrates of BCH are generally circumscribed, limited to the superficial dermis, and lack the nuclear features of Langerhans cells as well as CD1a reactivity or synthesis of Birbeck granules.132
CD-30 Lymphoproliferative Disorders Among all lymphohematopoietic lesions of the skin, one group is characterized by a relatively good prognosis and variable numbers of CD30 cells. The two main components are lymphomatoid papulosis and ALCL, sometimes referred to as regressing atypical histiocytosis (RAH). In 1982, Flynn and colleagues133 described a distinctive lesion of the skin composed of large, anaplastic, pleomorphic lymphoid cells that resembled Hodgkin cells and involved the dermis and subcutis. Despite the worrisome histologic appearance of this infiltrate, affected patients commonly followed a favorable clinical course; the cutaneous lesions regressed spontaneously, and periods of apparent remission were relatively long. However, some of these patients later developed frankly aggressive cutaneous and extracutaneous lesions.134,135 Immunohistochemically, the cells of LYP/ALCL are usually reactive for CD3, CD5, CD30, CD43, CD45, CD45RO, and CD99 (Fig. 13-22).93,134-136 This phenotype supports the interpretation of most of these lesions (~80%) as T-cell neoplasms. The remaining cases demonstrate null-cell differentiation. Primary ALCLs in the skin are typically negative for the ALK-1 protein, whereas systemic ALCLs that secondarily involved the skin had the (2;5)(p23;q35) translocation and were ALK-1 reactive. Lymphomatoid papulosis (LYP) typically follows an indolent course but may arise in virtually any cutaneous or mucocutaneous surface, although axial sites are most common. The typical lesions are small papules that may undergo central hemorrhage and necrosis before healing spontaneously. Some may persist indefinitely with common local or distant recurrences.137 Histologically, LYP is a superficial and deep perivascular and interstitial infiltrate, often with a wedgeshaped configuration that may have a lichenoid component in the superficial dermis. The overlying epidermis is often spongiotic and parakeratotic, but it may be ulcerated. The infiltrate is mixed with lymphocytes, macrophages, eosinophils, neutrophils, and plasma cells. However, at least some tumor cells in LYP have highly atypical cytologic features similar to those seen in ALCL; in some cases, the large cells may be multinucleated and may contain atypical mitotic figures. Lesions with mixed infiltrate that contain scattered, large,
494
Immunohistology of Skin Tumors
A
B
C
Figure 13-21 A, Langerhans cell histiocytosis (LCH) of the skin (“histiocytosis X”). B, CD1a expression is shown. C, Reactive Langerhans cells in non-LCH show a more dendritic morphology (diaminobenzidine and light hematoxylin).
atypical lymphocytes are classified as type A LYP. Lesions predominantly composed of hyperchromatic, convoluted lymphocytes similar to those seen in MF are classified as type B LYP. Less commonly, the large cells in LYP may form tumoral nodules or sheets, which are known as type C LYP. The main difference between type C LYP and ALCL is the multiplicity of lesions and rapidly regressing course of type C LYP. In the vast majority of LYP lesions, the large cells express CD4. However, some LYP lesions show a predominance of CD8 in those large, atypical cells, which is type D LYP.138-140 Because of the degree of cytologic atypia and CD8 expression, such lesions may be confused with cytotoxic aggressive cutaneous lymphoma, and some authors have suggested the designation type E LYP for those lesions with tropism to dermal vessels that mimics angiocentric lymphoma.141 Detection of clonal T-cell receptor gene rearrangements in LYP further solidifies the conclusion that it is inherently a malignancy,142 because such rearrangements are uncommon in benign reactive T-cell infiltrates. Eventually, approximately 10% to 20% of patients with LYP will eventually develop a systemic lymphoma,143,144 and it has been reported that those cases
with rearrangement of the T-cell receptor may be more likely to develop frank lymphoma during follow-up.145
Mesenchymal Tumors of the Skin Fibroblastic or myofibroblastic, fibrohistiocytic, muscular, neural, epithelial, and vascular lesions may be seen as primary tumors in the dermis and subcutis. IHC is often helpful in establishing the correct diagnosis in such spindle, polygonal, epithelioid, and small cell lesions of the skin.
Fibroblastic/Myofibroblastic Neoplasms Some fibroblastic or myofibroblastic differentiation may occur in the skin: this may be seen as infantile digital fibroma/digital fibromatosis (IDF),146 congenital superficial hemangiopericytoma, and “standard” and sclerosing epithelioid fibrosarcoma.147 The first of those tumors typically occurs on the hands of children; in many instances, it is multifocal, appearing as dome-shaped/ polypoid superficial nodules. IDF is composed of cytologically bland spindle cells that involve the superficial
Mesenchymal Tumors of the Skin
A
Figure 13-22 A and B, Lymphomatoid papulosis of the skin. C, Numerous CD30-positive cells are shown (diaminobenzidine and light hematoxylin).
dermis and dermal adnexa, with scattered mitotic figures and paranuclear cytoplasmic inclusions. Ultrastructural analysis has shown that IDF is mostly composed of myofibroblasts;148 accordingly, it expresses vimentin, muscle-specific isoforms of actin (which is also expressed in the intracytoplasmic inclusions), and calponin. They also express desmin and, rarely, caldesmon, nuclear β-catenin, or CD34.146 IDF commonly recurs, although some cases regress spontaneously. Congenital hemangiopericytoma is a neoplasm in young children, occasionally in the skin, that receives its name from the “staghorn” vascular pattern, similar to that seen in hemangiopericytomas/solitary fibrous tumors in adults. Congenital hemangiopericytoma shares clinical and histologic features with myofibromatoses of childhood and solitary acquired myofibroma of adults.149,150 Unlike true hemangiopericytomas, which lack musclespecific actin (MSA), congenital hemangiopericytomas are usually diffusely reactive for MSA (Fig. 13-23).150 Fibrosarcoma (FS) of the skin has been reported in association with scars from burns, surgery, smallpox vaccinations, and other injection sites.151 This very uncommon neoplasm is composed exclusively of fibroblastic elements and expresses only vimentin, to the exclusion of other specialized mesenchymal determinants (Fig. 13-24). A more common source of FS in the skin is the dedifferentiation (clonal transformation) of dermatofibrosarcoma protuberans (DFSP), which will be discussed later. A more recently described neoplasm
495
B
C is epithelioid FS. This peculiar tumor was described by Meis-Kindblom and colleagues147 as a mesenchymal neoplasm with epithelioid features that resembles infiltrating carcinoma. A slight predominance is seen for males, at all ages, and neoplasms occur most commonly in the lower extremities and limb girdles followed by the trunk; sizes range from 2 cm to more than 20 cm. The original series reported an aggressive behavior: persistent disease or local recurrences in 53% of patients and metastases in 43%. Histologically these tumors are characterized by uniform, small, round to ovoid epithelioid cells with sparse, clear cytoplasm; tumors are arranged in nests and cords, and mitotic figures are rare. Intervening stroma is dense and resembles osteoid or cartilage, with occasional myxoid areas, calcification, and bone.147,152 Tumor cells strongly express vimentin and may express EMA, p53, Bcl-2, S-100 protein, and less commonly keratins (with AE1/AE3 and CAM5.2).147
Fibrohistiocytic Neoplasms Some disagreement surrounds the meaning (and validity) of the term fibrohistiocytic. Although some authors consider it a “wastebasket” term, most pathologists and dermatopathologists use it to describe lesions that have epithelioid and spindle cell morphology and that express histiocytic markers such as FXIIIa, MAC387, CD68, and CD163. In the skin, we may include in this group lesions such as atypical fibroxanthoma (AFX),
496
Immunohistology of Skin Tumors
A
B
Figure 13-23 A, Hemangiopericytoma of the dermis and subcutis is currently regarded as a form of myofibromatosis. B, Reactivity for actins, shown here, is therefore expected (anti–Bcl-2; diaminobenzidine and light hematoxylin).
malignant fibrous histiocytoma (MFH), dermatofibroma (DF), and DFSP. In these lesions, some of the tumors cells may express specialized hematopoietic determinants that include CD14, CD16, CD18, CD36, CD43, CD68, and human lymphocyte antigen (HLA)-DR, all markers associated with bone marrow–derived monocytes and histiocytes.153,154 Cellular proteases such as α-l-antichymotrypsin and cathepsin-B are regularly present in AFX, MFH, and DFSP155 but are also found in many other spindle cell tumors of the skin.156 An antibody to a cytoplasmic determinant (the L1 antigen) characteristic of functionally mature monocytes and macrophages, MAC387 is also frequently is observed in multinucleated and, less commonly, in fusiform elements in MFH, AFX, and DFSP. However, this marker may be observed in virtually every other type of malignant cutaneous neoplasm.157 FXIIIa, a coagulation factor expressed by fibroblasts and dermal dendrocytes, is seen in DF (see below), AFX, and MFH but may be encountered in other sarcomas, granular cell tumors, and neurofibromas.158-160
A
As might be expected, MFH, AFX, DF, and DFSP demonstrate diffuse reactivity for vimentin. MSAs and caldesmon have also been detected in some of these neoplasms, particularly in AFX and DF.161-164 CD34 positivity separates DFSP and its variant—giant cell fibroblastoma, which is virtually always diffusely reactive for that marker (Fig. 13-25)165,166—from most other spindle cell neoplasms of the dermis and subcutis, with the exception of selected peripheral nerve sheath tumors (PNSTs), acral fibromyxoma, and spindle cell lipoma.163,166-168 Other tumors seen also in the skin typically show CD34 expression, including giant cell angiofibroma, cutaneous solitary fibrous tumor, sclerotic fibroma, and fibrous papule of the nose.169-173 CD34 analysis is usually not needed to establish the diagnosis of DFSP, but it may be helpful in small biopsies. In the differential diagnosis between DFSP and DF, it is worth mentioning that DF sometimes shows peripheral-lesional “leading-edge” positivity for CD34 (Fig. 13-26).163 Another group of lesions appear to share clinical, morphologic, and immunophenotypic features
B
Figure 13-24 A, Fibrosarcoma of the dermis and subcutis. B, Diffuse vimentin positivity is shown (diaminobenzidine and light hematoxylin).
Mesenchymal Tumors of the Skin
A
497
B
Figure 13-25 A, Dermatofibrosarcoma protuberans. B, Global reactivity for CD34 is typically shown (diaminobenzidine and light hematoxylin).
with both DF and DFSP,174 and these lesions were termed indeterminate cutaneous fibrohistiocytic lesions (Fig. 13-27); they are more commonly located on the trunk and may recur (as DFSP does), show epidermal hyperplasia (as DF does) and storiform pattern (more commonly seen in DFSP), and show expression of both CD34 and FXIIIa. Because of their possible recurrent behavior, it is recommended to completely excise such lesions. Fibrosarcoma ex DFSP is a special variant of DFSP. Two immunoprofiles may be observed in this “composite” tumor: it may be diffusely reactive for CD34 throughout, or the fibrosarcomatous component may lack that marker, producing biphasic immunoreactivity.175,176 Thus in such lesions, it may be important to examine additional sections or, when dealing with needle biopsies, to recommend other biopsies to detect the standard DFSP areas. Another subtype of DFSP in which CD34 labeling is diagnostically beneficial is its “atrophic” form. In that lesion, rather than the characteristic bulky and protuberant proliferation of spindle cells of classic DFSP, an “alternating stair-step” pattern is apparent in spindle cells of the subcutis.177 This atrophic DFSP is also diffusely positive for CD34. It has been proposed that MFH represents a histologic pattern that is a common final pathway of differentiation for several modes of mesenchymal neoplasia. That concept was first espoused by Brooks.178 Furthermore, some authors, particularly those from Fletcher’s group, have discouraged the term and have suggested using the name undifferentiated pleomorphic sarcoma for
such lesions. However, extensive literature about this type of sarcoma describes their clinical behavior and management, and clinicians are familiar with the terminology; therefore we still use this term in our practice. An unqualified diagnosis of MFH can be made immunohistologically only if the pathologist is dealing with a pleomorphic malignant tumor that lacks epithelial, myogenous, neural, and endothelial markers. Such tumors usually show expression of CD68, MAC387, and other so-called histiocytic markers, but because those markers can also be seen in other neoplasms, no “proactive” determinants define MFH at present. Regarding AFX, most authors regard it as a special superficial variant of MFH, and thus both lesions have a similar immunophenotype. CD99 has been touted by some authors as a helpful marker of AFX,179 although a number of other soft tissue tumors are also positive, including DFSP.180 CD10 is also commonly expressed in AFX,181 but it is also expressed in spindle cell squamous carcinomas and melanomas.182,183 For a diagnosis of AFX, we propose using an IHC panel that includes a pankeratin cocktail, p63, S-100 protein, SMA, CD10, and CD68. We establish a diagnosis of AFX in a cutaneous neoplasm composed of spindled and epithelioid pleomorphic cells, with numerous mitotic figures (some of them of atypical shape) and lacking expression of keratin, p63, S-100 protein, and SMA while expressing CD10 and CD68. A possible pitfall is the expression, albeit very uncommon, of HMB-45 antigen or melan-A in AFX, thus mimicking melanoma.184 Regarding the different prognoses of AFX and MFH, in addition to the smaller size and more superficial location of AFX, it has
498
Immunohistology of Skin Tumors
A
B
C
Figure 13-26 Dermatofibromas (A) express factor XIIIa (FXIIIa; B). When using anti-CD34 (C), it is restricted to the border of the tumor, likely showing dermal dendritic cells in contrast to the expected diffuse pattern in dermatofibrosarcoma protuberans (diaminobenzidine and light hematoxylin).
been suggested that the relatively benign behavior of AFX may be due in part to absence of HRAS, KRAS, and NRAS gene mutations.185 Dermatofibromas and their variants—nodular histiocytoma, xanthogranuloma, transitional histiocytoma, nodular subepidermal fibroma, aneurysmal cutaneous fibrous histiocytoma, palisading fibrous histiocytoma, and epithelioid reticulohistiocytoma—all share reactivity for vimentin, FXIIIa, CD10, CD68, CD163, and stromelysin-3.163 An important morphologic variant of dermatofibroma is the epithelioid fibrous histiocytoma, characterized by a dermal proliferation of epithelioid cells arranged in an expansile pattern of growth that spares the epidermis. Because of the presence of mitotic figures, when these lesions are interpreted as melanocytic, they can be diagnosed as melanomas.186 These lesions express the same markers as dermatofibromas (FXIIIa, CD163, CD68, etc.). As mentioned in Chapter 7, it is important to recognize that S-100 protein is
expressed by dendritic cells in the dermis, therefore it is possible to misinterpret them as part of the neoplastic proliferation and thus consider the lesion to be melanocytic rather than fibrohistiocytic (Fig. 13-28).
KEY DIAGNOSTIC POINTS Fibrohistiocytic Tumors • Dermatofibrosarcoma protuberans is diffusely CD34 positive and FXIIIa negative; dermatofibroma is CD34 negative but positive for FXIIIa; the leading edge of dermatofibromas may be CD34 positive. • Malignant fibrous histiocytoma and atypical fibroxanthoma have no specific immunoprofile. They may show expression of CD10, CD99, and CD68, but their diagnosis is mainly based upon the lack of expression of epithelial, smooth muscle, and melanocytic markers.
Mesenchymal Tumors of the Skin
A
C
Tumors with “Pure” or Partial Smooth Muscle Differentiation As is true of extracutaneous leiomyomas and leiomyosarcomas, those lesions in the skin express vimentin, desmin, caldesmon, and muscle-related actins (Fig. 13-29).187,188 However, it is important to consider that expression of actin is not pathognomonic of smooth muscle differentiation. Thus myofibroblastic proliferations also show actin expression, including cellular scars, posttraumatic spindle cell nodules, and nodular fasciitis. Therefore it is essential to correlate the histologic and IHC features in the context of a panel of immunomarkers. A few peculiarities exist regarding cutaneous smooth muscle tumors. Roughly 30% of dermal leiomyomas and leiomyosarcomas are S-100 protein reactive, and a similar number of subcutaneous lesions express CD57.189 It has been speculated that those findings reflect pilar smooth muscular and vascular smooth muscular differentiation, respectively. It is important to consider this possible cross-reactivity to avoid rendering a diagnosis of a neural lesion in such smooth muscle
499
B
Figure 13-27 Indeterminate fibrohistiocytic lesion (fibrohistiocytic lesion of unknown malignant potential). A, Lesions have epidermal hyperplasia, storiform pattern, and invasion of the subcutaneous tissue. CD34 (B) and factor XIIIa (FXIIIa; C) are expressed by a dual population of cells (diaminobenzidine and light hematoxylin).
tumors. Another possible source of misdiagnosis is the occasional detection of keratin in cutaneous smooth muscle tumors.190,191 Glomus tumors, glomangiomas, myopericytomas, and glomangiosarcomas are also related to tumors of smooth muscle, in that they show features of specialized perivascular smooth muscle (pericytic) differentiation.192-194 However, they differ from other myogenous tumors in that desmin positivity is usually absent,168,194 and they are distinguished from pure vascular lesions by the absence of endothelial cell markers such as D2-40 and CD31. So-called PEComas, tumors of perivascular epithelioid cells (PECs), are neoplasms originally described in the lung, kidney, and soft tissue that sometimes involve the skin.195 They are composed of epithelioid and fusiform cells with variably granular or clear cytoplasm (Fig. 13-30). Tumor cells express MSA, desmin and melanocytic markers (HMB-45 antigen, tyrosinase, and melan-A).196 With others, we prefer the term myomelanocytoma, because to date expression of melanocytic markers has not been described in normal cells located in a perivascular location.
500
Immunohistology of Skin Tumors
A
C
Nerve Sheath Tumors The most common cutaneous nerve sheath tumors are neurofibromas, and when multiple, they may occur in the setting of von Recklinghausen neurofibromatosis. Schwannomas (neurilemmomas), granular cell tumors, perineuriomas, and neurothekeomas are other neural tumors that occur in the skin.197 The malignant counterpart, cutaneous malignant peripheral nerve sheath
A
B
Figure 13-28 Atypical fibroxanthoma: ulcerated cellular proliferation with cytologic atypia (A) with benign, interspersed dendritic cells that express S-100 protein (B) and show strong, diffuse expression of CD10 (C; diaminobenzidine and light hematoxylin).
tumor (MPNST), only rarely occurs in the skin.198,199 Most MPNSTs arise primarily in the dermis or subcutaneous tissues, but deeply located lesions may secondarily involve the skin. The immunophenotypic attributes that typify most peripheral nerve sheath tumors (PNSTs) include staining for vimentin along with S-100 protein, CD56, or CD57 (Fig. 13-31).200 Detection of the latter three markers, alone or in combination, provides for the reliable
B
Figure 13-29 A, Leiomyosarcoma of the subcutis. B, Diffuse labeling for desmin is shown (diaminobenzidine and light hematoxylin).
Mesenchymal Tumors of the Skin
501
protein. However, menigiomas lack claudin-1, a tight junction–associated protein.213,214 Another differential diagnosis would be with spindle cell epithelioid sarcoma, a rare variant of epithelioid sarcoma composed of relatively bland spindle cells. In contrast to perineurioma, epithelioid sarcoma is strongly positive for keratin. Malignant perineuriomas are a very rare variant of MPNST; these lesions show an infiltrative pattern of growth, malignant cytology, and mitotic figures. They also express EMA and may express focal CD34.215,216 Neurothekeomas (NTKs; Fig. 13-34) are a group of neoplasms composed of spindle and epithelioid cells arranged in more or less myxoid stromas. They have been subdivided into two main groups: conventional NTK (nerve sheath myxoma) and cellular NTK.197,217 The former is likely to be a real PNST, with bland spindle cells embedded in a markedly myxoid stroma and expressing S-100 protein.218 Cellular NTK may be a so-called fibrohistiocytic lesion, because in addition to SMA, it expresses actin, NKI/C3, and CD68.219 In some instances prominent myxoid change is noted in the
Figure 13-30 Myomelanocytoma of the skin.
identification of PNSTs in tumors that lack myogenous and epithelial markers. IHC against neuronal differentiation (neurofilaments, peripherin) may be also helpful in distinguishing neuroma, neurofibroma, and schwannoma. When comparing the number of axons present in the lesion, neuroma has an axon/cell ratio of 1 : 1, neurofibroma’s is 4 : 1, and schwannomas only show rare axons at the periphery of the lesions. Other markers also expressed in nerve sheath tumors include glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and nerve growth factor receptor. Most granular cell tumors (GCTs) of the skin show neural differentiation, but granular morphology can also be seen in leiomyoma, leiomyosarcoma, BCC, dermatofibroma, and angiosarcoma.201-203 Accordingly, most granular cell tumors (80%) express S-100 protein,204 CD56, or CD57. GCTs also share expression of FXIIIa with neurofibroma but not schwannoma. Furthermore, rare GCT lesions show spindle cell morphology with keloidal collage and thus mimic dermatofibroma.205 Other markers occasionally seen in GCTs are protein gene product 9.5 (PGP9.5), calretinin, and inhibin (Fig. 13-32).206 Malignant GCTs also tend to exhibit nerve sheath differentiation. Perineuriomas are uncommon in the skin.207-209 They may demonstrate bland spindle cells that form vaguely concentric whorls in a partially myxoid stroma, whereas others may resemble dermatofibroma, solitary fibrous tumor, or storiform collagenoma.210 They may also show a reticular or plexiform architecture.211,212 Perineural cells express EMA and lack S-100 protein and keratin; analogously, perineuriomas show a similar profile (Fig. 13-33), along with claudin-1 expression. The main differential diagnosis is with cutaneous meningiomas, which also express EMA but not keratin or S-100
A
B Figure 13-31 A, Malignant peripheral nerve sheath tumor of the dermis in a patient with neurofibromatosis. B, The tumor shows patchy labeling for S-100 protein (diaminobenzidine and light hematoxylin).
502
Immunohistology of Skin Tumors
A
C stroma of cellular NTK, the myxoid variant of cellular NTK; this variant has basically the same IHC pattern as the standard cellular NTK. Two interesting markers expressed in this variant are microphthalmia transcription factor (MiTF) and S-100A6, one of the S-100 group of proteins.220,221 The main differential diagnosis of cellular NTK is with melanocytic lesions, in particular melanoma. Cytomorphologic features are similar: nests or small groups of epithelioid cells are seen with focal nucleoli and scattered mitotic figures. Furthermore, both melanoma and cellular NTK express MiTF and S-100A6. However, standard S-100 protein and other,
KEY DIAGNOSTIC POINTS Peripheral Nerve Sheath Tumors • S-100 distribution is dependent on pattern. • Tumors are typically CD56 and CD57 positive. • Granular cell tumors and neurofibromas are positive with FXIIIa, calretinin, and inhibin. • Perineuriomas express EMA and claudin-1 but are negative for other markers. • Cellular neurothekeomas express NKI/C3, S-100A6, and MiTF.
B
Figure 13-32 A and B, Granular cell tumor of the skin. C, Immunoreactivity for S-100 protein is shown (anti-calretinin; diaminobenzidine and light hematoxylin).
more specific melanocytic markers (HMB-45 antigen, melan-A) are not detected in cellular NTK.
Vascular Neoplasms Several markers are associated with endothelial differentiation, but so far none is pathognomonic for endothelial cells. Because of the large number of vascular tumors, this chapter will consider them as a group, with special comments on selected lesions. The antigens generally associated with endothelial differentiation include factor VIII–related antigen (von Willebrand factor [vWF]); CD31, CD34, CD141 (thrombomodulin), FLI-1, vascular endothelial growth factor receptor 3 (VEGFR3), podoplanin, and fucoserich cell membrane binding sites for Ulex europaeus I lectin (Fig. 13-35).222,223 Some of these markers may be more specific for lymphatic or endothelial differentiation. However, because this differentiation has no relevant practical utility in diagnostic dermatopathology, it will not be addressed further. Some endothelial tumors may histologically simulate carcinomas or nonvascular sarcomas with myogenic differentiation. Moreover, epithelioid vascular neoplasms may express keratin, thus it is essential to include other antigens of epithelial differentiation in the IHC panel of epithelioid vascular lesions.
Mesenchymal Tumors of the Skin
A
Figure 13-33 A and B, Cutaneous perineuroma. C, Diffuse positivity for epithelial membrane antigen is shown (diaminobenzidine and light hematoxylin).
A
503
B
C
B
Figure 13-34 A, Cellular neurothekeoma shows a histiocytic phenotype in this case. B, Diffuse labeling of the tumor cells for CD68 is apparent, as is an absence of other specialized determinants (diaminobenzidine and light hematoxylin).
504
Immunohistology of Skin Tumors
A
for that marker, albeit some cases may have weak labeling of only scattered cells in the lesion. In contrast, other lesions that mimic KS do not express HHV-8.224-226 A group of vascular lesions has been designated hemangioendotheliomas and are generally considered to be “borderline.” These lesions have immunophenotypes similar to other vascular neoplasms (CD31, CD34, podoplanin). However, Billings and colleagues have described a peculiar tumor in this group that histologically simulates epithelioid sarcoma with large, epithelioid, and spindle cells in a fibrous stroma. This lesion has been named epithelioid sarcoma–like hemangioendothelioma (ESLH; Fig. 13-37).227 ESLH shares expression of keratin with true epithelioid sarcoma but paradoxically lacks CD34, which is seen in only 50% of epithelioid sarcoma and in most other hemangioendotheliomas. As with other vascular tumors, ESLH expresses CD31 and FLI-1.227
B Figure 13-35 A, Cutaneous angiosarcoma. B, Immunoreactivity for CD31 is shown (diaminobenzidine and light hematoxylin).
KEY DIAGNOSTIC POINTS Vascular Neoplasms • The best panel includes CD31, CD141, CD34, and FLI-1 (Ulex europaeus may be also used). • Epithelioid angiosarcomas often show expression of keratin with both AE1/AE3 and CAM5.2, which emphasizes the need to use an immunohistochemical panel to avoid possible diagnostic pitfalls. • Antibody HHV-8-LNA (human herpesvirus latent nuclear antigen) is highly sensitive and specific for Kaposi sarcoma. • Epithelioid sarcoma–like hemangioendothelioma is positive for CD31, FLI-1, and occasionally keratin but is negative for CD34, whereas epithelioid sarcoma is consistently positive for keratin and CD34.
IHC has a limited role in the diagnosis of Kaposi sarcoma (KS). In its “patch” stage, KS is easily recognizable on morphologic grounds alone, whereas in its late, spindle cell or “nodular” stage, the tumor cells in KS tend to lose endothelial-related markers. However, human herpesvirus 8 (HHV-8) latent nuclear antigen 1 is highly selective for most KS variants (Fig. 13-36). The majority of KS cases (80% in our experience) are labeled
A
B Figure 13-36 A, Kaposi sarcoma of the skin. B, Nuclear positivity for human herpesvirus 8 latent nuclear antigen 1 is shown (diaminobenzidine and light hematoxylin).
Mesenchymal Tumors of the Skin
505
Regarding angiosarcomas, IHC may be helpful not only in detecting expression of endothelial markers (CD31 or CD34) by the tumor cells but also in highlighting the characteristic irregular pattern of growth of the tumor cells that infiltrate collagen at the periphery of the lesion.
Epithelioid Sarcoma
Figure 13-37 Epithelioid sarcoma–like hemangioendothelioma of the subcutis. This tumor expresses CD31 but lacks CD34. As with other epithelioid vascular lesions, it is reproducibly positive for keratin.
A
C
Epithelioid sarcoma is a polygonal-cell neoplasm of the dermis and subcutis that may assume varied microscopic appearances,228 therefore it may be confused with other lesions. When it manifests a predominantly solid pattern of growth, epithelioid sarcoma may be mistaken for metastatic carcinoma or malignant melanoma; alternatively, those cases with a granulomatous pattern of growth, with palisading of neoplastic cells around areas of necrosis, are very similar to cutaneous necrobiotic granulomas, particularly rheumatoid nodules and granuloma annulare.229 Some rare cases may have clefting of the stroma that resembles angiosarcoma.227 Among epithelioid mesenchymal tumors of the superficial soft tissues, only epithelioid sarcoma consistently expresses keratin (Fig. 13-38);230 synovial sarcomas also express keratin, but they are more typically
B
Figure 13-38 A-B, Epithelioid sarcoma of the subcutis. C, Diffuse expression of keratin is shown (diaminobenzidine and light hematoxylin).
506
Immunohistology of Skin Tumors
situated in the deep soft tissues. The potential immunophenotypic overlap between epithelioid sarcoma and metastatic carcinoma is accentuated by the presence of CK7 and EMA in 70% to 80% of epithelioid sarcoma cases and even CA-125 in some examples.231,232 Approximately 50% of epithelioid sarcoma cases express CD34 positivity,233 and that marker is rare in carcinoma. In contrast, CK5/6, p63 protein, and E-cadherin are expressed in SCC but not in epithelioid sarcoma.234-236 In contrast with melanoma or clear cell sarcoma (melanoma of soft parts), fewer than 15% of epithelioid sarcoma cases express S-100 protein and are consistently negative for HMB-45 antigen, tyrosinase, and melan-A.228 As mentioned earlier, the distinction between epithelioid sarcoma and ESLH principally relies on a demonstration of specific endothelial markers in ESLH, with CD31 and FLI-1 being the most discriminatory.
Small Round Cell Mesenchymal Tumors Small cell neoplasms of the skin and subcutis may include Merkel cell carcinoma; small cell squamous carcinoma, eccrine carcinoma, and melanoma; extraskeletal ES/PNET; lymphoma; or rhabdomyosarcoma (RMS; Fig. 13-39). Mesenchymal tumors in that group constitute a small minority. In the absence of keratin, RMS consistently expresses desmin, myogenin, and MSA, whereas ES/PNET uniformly exhibits CD99 reactivity
+
Polyphenotypic PNET
DES/MSA/MYG
+
–
and may express CD56, CD57, and synaptophysin and may focally express keratin.237,238 Neither of those tumor groups is reactive for EMA, CD45, S-100 protein, CEA, or HMB-45.
Special Topics in Cutaneous Immunohistochemistry Estrogen and Progesterone Receptor Proteins Selected hormone receptor proteins were originally targeted as diagnostic discriminants between eccrine carcinomas and histologically similar metastases of mammary carcinoma. However, estrogen receptor protein (ERP) and progesterone receptor protein (PRP) are common in benign and malignant eccrine/apocrine cutaneous neoplasms (Fig. 13-40).239-241 On the other hand, apocrine adenocarcinoma and EMPD typically lack ERP or PRP and instead express androgen receptors.38
Oncogenes and Other Possibly Prognostic Markers Two oncogenes that have been studied in detail in the skin are epidermal growth factor receptor (EGFR) and the Erbb2 (neu) oncogene. The latter is a 185-kD Small cell neuroendocrine carcinoma –
CD56/SYN/CGA
Small cell adenoCA
+
CD15/MOC31/TAG-72 Small cell squamous CA
–
+
CK
–
+
Malignant lymphoma or leukemia
+
CD45
–
EMA
+
Malignant melanoma
S100/HMB45/ MART1/MITF
–
Rhabdomyosarcoma
–
VIM
+
–
+ DES/MSA/MYG
CD56/SYN/CGA/CD99
–
–
ES/PNET +
Technically inadequate specimen
Undiff. sarcoma –
CD56/SYN/ CGA/CD99
+
ES/PNET
Figure 13-39 Algorithm showing the sequence of immunohistologic interpretation associated with diagnosis of small cell undifferentiated tumors of the skin and subcutis. adenoCA, Adenocarcinoma; CA, carcinoma; CGA, chromogranin A; CK, cytokeratin; DES, desmin; EMA, epithelial membrane antigen; ES, Ewing sarcoma; MiTF, microphthalmia transcription factor; MSA, muscle-specific actin; MYG, myogenin; PNET, primitive neuroectodermal tumor; SYN, synaptophysin; TAG-72, tumor-associated glycoprotein 72; Undiff., undifferentiated; VIM, vimentin.
Special Topics in Cutaneous Immunohistochemistry
transmembrane glycoprotein with tyrosine kinase activity that is functionally and structurally related to the 175-kD EGFR gene product. In normally differentiated and developing cells, native forms of both EGFR and Erbb2 are expressed, but each may be altered or overexpressed in certain epithelial neoplasms. Not surprisingly, EGFR expression is characteristic of SCC and some BCC (Fig. 13-41),242 but it may also be encountered in benign keratinocytic proliferations, including acrochordons and seborrheic keratosis, and in adnexal carcinomas of the skin.241 The receptor is increased in density in adnexal carcinomas in pregnant women, in patients who are taking exogenous estrogenic or progestational hormones, and in those who have dysplastic nevus syndrome.243 In addition, Erbb2 is overexpressed in some aggressive breast carcinomas and some malignant cutaneous neoplasms, and it may be associated with poor prognosis.244 Similarly, EMPD may express Erbb2.49,245 CD44 is a cell-surface protein involved in cell-to-cell and cell-to-matrix adhesion and in lymphocyte-homing
Figure 13-40 Positivity for estrogen receptor protein in primary eccrine carcinoma of the skin (diaminobenzidine and light hematoxylin).
507
Figure 13-41 Diffuse reactivity for epidermal growth factor receptor in poorly differentiated squamous cell carcinoma of the skin (diaminobenzidine and light hematoxylin).
activity. It is expressed in epidermal keratinocytes, hair follicles, and sebaceous and eccrine cells. CD44 expression has also been reported in a variety of tumors, and expression of CD44 has been correlated with more aggressive behavior. For example, BCCs do not exhibit immunolabeling for CD44, in contrast to SCCs and metastatic adenocarcinomas, in which almost 100% of cells are labeled.246 CD44 expression by Merkel cell carcinoma also may correlate with metastatic risk.247 In summary, IHC plays a very important role as an adjunct in the evaluation of neoplastic cutaneous lesions. To avoid possible pitfalls in the diagnosis, the IHC results should be always correlated with the clinical and histologic findings. In the diagnostic use of IHC, some antibodies can be used as surrogates of diagnosis and to detect therapy targets. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Chaudhry IH, Zembowicz A: Adnexal clear cell carcinoma with comedonecrosis: clinicopathologic analysis of 12 cases. Arch Pathol Lab Med. 131(11):1655–1664, 2007. 2. Prieto VG, Ivan D: The use of immunohistochemistry in the differential diagnosis of primary cutaneous adnexal neoplasms and metastatic adenocarcinomas to the skin. Diagn Histopathol. 16(9):409–416, 2010. 3. Lohmann CM, Solomon AR: Clinicopathologic variants of cutaneous squamous cell carcinoma. Adv Anat Pathol. 8(1):27–36, 2001. 4. Petter G, Haustein UF: Histologic subtyping and malignancy assessment of cutaneous squamous cell carcinoma. Dermatol Surg. 26(6):521–530, 2000. 5. Carvalho J, Fullen D, Lowe L, et al: The expression of CD23 in cutaneous non-lymphoid neoplasms. J Cutan Pathol. 34(9):693– 698, 2007. 6. Dotto JE, Glusac EJ: p63 is a useful marker for cutaneous spindle cell squamous cell carcinoma. J Cutan Pathol. 33(6):413– 417, 2006. 7. Reis-Filho JS, Simpson PT, Martins A, et al: Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray. Virchows Arch. 443(2):122–132, 2003. 8. Sigel JE, Skacel M, Bergfeld WF, et al: The utility of cytokeratin 5/6 in the recognition of cutaneous spindle cell squamous cell carcinoma. J Cutan Pathol. 28(10):520–524, 2001. 9. Wick MR, Fitzgibbon J, Swanson PE: Cutaneous sarcomas and sarcomatoid neoplasms of the skin. Semin Diagn Pathol. 10(2):148–158, 1993. 10. Egawa K, Honda Y, Ono T, et al: Immunohistochemical demonstration of carcinoembryonic antigen and related antigens in various cutaneous keratinous neoplasms and verruca vulgaris. Br J Dermatol. 139(2):178–185, 1998. 11. Hashimoto T, Inamoto N, Nakamura K, et al: Involucrin expression in skin appendage tumours. Br J Dermatol. 117(3):325–332, 1987. 12. Kanitakis J: Expression of involucrin in skin appendage tumours. Br J Dermatol. 118(4):585–586, 1988. 13. Nozoe T, Oyama T, Takenoyama M, et al: Significance of immunohistochemical expression of p27 and involucrin as the marker of cellular differentiation of squamous cell carcinoma of the esophagus. Oncology. 71(5–6):402–410, 2006. 14. Strutton GM: Pathological variants of basal cell carcinoma. Australas J Dermatol. 38(Suppl 1):S31–S35, 1997. 15. Wick MR, Swanson PE: Primary adenoid cystic carcinoma of the skin. A clinical, histological, and immunocytochemical comparison with adenoid cystic carcinoma of salivary glands and adenoid basal cell carcinoma. Am J Dermatopathol. 8(1):2–13, 1986. 16. George E, Swanson PE, Wick MR: Neuroendocrine differentiation in basal cell carcinoma. An immunohistochemical study. Am J Dermatopathol. 11(2):131–135, 1989. 17. Foschini MP, Eusebi V: Divergent differentiation in endocrine and nonendocrine tumors of the skin. Semin Diagn Pathol. 17(2):162–168, 2000. 18. Grando SA, Schofield OM, Skubitz AP, et al: Nodular basal cell carcinoma in vivo vs in vitro. Establishment of pure cell cultures, cytomorphologic characteristics, ultrastructure, immunophenotype, biosynthetic activities, and generation of antisera. Arch Dermatol. 132(10):1185–1193, 1996. 19. Sanchez NP, Winkelmann RK: Basal cell tumor with eccrine differentiation (eccrine epithelioma). J Am Acad Dermatol. 6(4 Pt 1):514–518, 1982. 20. Sakamoto F, Ito M, Sato S, et al: Basal cell tumor with apocrine differentiation: apocrine epithelioma. J Am Acad Dermatol. 13(2 Pt 2):355–363, 1985. 21. Uzquiano MC, Prieto VG, Nash JW, et al: Metastatic basal cell carcinoma exhibits reduced actin expression. Mod Pathol. 21(5):540–543, 2008. 22. Jimenez FJ, Burchette JL Jr, Grichnik JM, et al: Ber-EP4 immunoreactivity in normal skin and cutaneous neoplasms. Mod Pathol. 8(8):854–858, 1995. 23. Swanson PE, Fitzpatrick MM, Ritter JH, et al: Immunohistologic differential diagnosis of basal cell carcinoma, squamous cell
507.e1
carcinoma, and trichoepithelioma in small cutaneous biopsy specimens. J Cutan Pathol. 25(3):153–159, 1998. 24. Ko T, Muramatsu T, Shirai T: Distribution of lectin UEA-I in trichilemmal carcinoma, squamous cell carcinoma, and other epithelial tumors of the skin. J Dermatol. 23(6):389–393, 1996. 25. Alsaad KO, Obaidat NA, Ghazarian D: Skin adnexal neoplasms– part 1: an approach to tumours of the pilosebaceous unit. J Clin Pathol. 60(2):129–144, 2007. 26. Obaidat NA, Alsaad KO, Ghazarian D: Skin adnexal neoplasms– part 2: an approach to tumours of cutaneous sweat glands. J Clin Pathol. 60(2):145–159, 2007. 27. Qureshi HS, Ormsby AH, Lee MW, et al: The diagnostic utility of p63, CK5/6, CK 7, and CK 20 in distinguishing primary cutaneous adnexal neoplasms from metastatic carcinomas. J Cutan Pathol. 31(2):145–152, 2004. 28. Ivan D, Hafeez Diwan A, Prieto VG: Expression of p63 in primary cutaneous adnexal neoplasms and adenocarcinoma metastatic to the skin. Mod Pathol. 18(1):137–142, 2005. 29. Kanitakis J, Chouvet B: Expression of p63 in cutaneous metastases. Am J Clin Pathol. 128(5):753–758, 2007. 30. Liang H, Wu H, Giorgadze TA, et al: Podoplanin is a highly sensitive and specific marker to distinguish primary skin adnexal carcinomas from adenocarcinomas metastatic to skin. Am J Surg Pathol. 31(2):304–310, 2007. 31. Swanson PE, Cherwitz DL, Neumann MP, et al: Eccrine sweat gland carcinoma: an histologic and immunohistochemical study of 32 cases. J Cutan Pathol. 14(2):65–86, 1987. 32. Metze D, Grunert F, Neumaier M, et al: Neoplasms with sweat gland differentiation express various glycoproteins of the carcinoembryonic antigen (CEA) family. J Cutan Pathol. 23(1):1– 11, 1996. 33. Ansai S, Koseki S, Hozumi Y, et al: An immunohistochemical study of lysozyme, CD-15 (Leu M1), and gross cystic disease fluid protein-15 in various skin tumors. Assessment of the specificity and sensitivity of markers of apocrine differentiation. Am J Dermatopathol. 17(3):249–255, 1995. 34. Santos-Juanes J, Bernaldo de Quiros JF, Galache Osuna C, et al: Apocrine carcinoma, adenopathies, and raised TAG-72 serum tumor marker. Dermatol Surg. 30(4 Pt 1):566–569, 2004. 35. Wick MR, Swanson PE, LeBoit PE, et al: Lymphoepitheliomalike carcinoma of the skin with adnexal differentiation. J Cutan Pathol. 18(2):93–102, 1991. 36. Ramachandra S, Gillett CE, Millis RR: A comparative immunohistochemical study of mammary and extramammary Paget’s disease and superficial spreading melanoma, with particular emphasis on melanocytic markers. Virchows Arch. 429(6):371– 376, 1996. 37. Mazoujian G, Margolis R: Immunohistochemistry of gross cystic disease fluid protein (GCDFP-15) in 65 benign sweat gland tumors of the skin. Am J Dermatopathol. 10(1):28–35, 1988. 38. Diaz de Leon E, Carcangiu ML, Prieto VG, et al: Extramammary Paget disease is characterized by the consistent lack of estrogen and progesterone receptors but frequently expresses androgen receptor. Am J Clin Pathol. 113(4):572–575, 2000. 39. Saga K: Histochemical and immunohistochemical markers for human eccrine and apocrine sweat glands: an aid for histopathologic differentiation of sweat gland tumors. J Investig Dermatol Symp Proc. 6(1):49–53, 2001. 40. Eto H, Hashimoto K, Kobayashi H, et al: Monoclonal antikeratin antibody: production, characterization, and immunohistochemical application. J Invest Dermatol. 84(5):404–409, 1985. 41. Ishihara M, Mehregan DR, Hashimoto K, et al: Staining of eccrine and apocrine neoplasms and metastatic adenocarcinoma with IKH-4, a monoclonal antibody specific for the eccrine gland. J Cutan Pathol. 25(2):100–105, 1998. 42. Battles OE, Page DL, Johnson JE: Cytokeratins, CEA, and mucin histochemistry in the diagnosis and characterization of extramammary Paget’s disease. Am J Clin Pathol. 108(1):6–12, 1997. 43. Yoshii N, Kitajima S, Yonezawa S, et al: Expression of mucin core proteins in extramammary Paget’s disease. Pathol Int. 52(56):390–399, 2002. 44. Lau J, Kohler S: Keratin profile of intraepidermal cells in Paget’s disease, extramammary Paget’s disease, and pagetoid squamous cell carcinoma in situ. J Cutan Pathol. 30(7):449–454, 2003.
507.e2
Immunohistology of Skin Tumors
45. Nowak MA, Guerriere-Kovach P, Pathan A, et al: Perianal Paget’s disease: distinguishing primary and secondary lesions using immunohistochemical studies including gross cystic disease fluid protein-15 and cytokeratin 20 expression. Arch Pathol Lab Med. 122(12):1077–1081, 1998. 46. Ohnishi T, Watanabe S: The use of cytokeratins 7 and 20 in the diagnosis of primary and secondary extramammary Paget’s disease. Br J Dermatol. 142(2):243–247, 2000. 47. Perrotto J, Abbott JJ, Ceilley RI, et al: The role of immunohistochemistry in discriminating primary from secondary extramammary Paget disease. Am J Dermatopathol. 32(2):137–143, 2010. 48. Mayes DC, Patterson JW, Ramnani DM, et al: alpha-methylacyl coenzyme A racemase is immunoreactive in extramammary Paget disease. Am J Clin Pathol. 127(4):567–571, 2007. 49. Plaza JA, Torres-Cabala C, Ivan D, et al: HER-2/neu expression in extramammary Paget disease: a clinicopathologic and immunohistochemistry study of 47 cases with and without underlying malignancy. J Cutan Pathol. 36(7):729–733, 2009. 50. Ivan D, Nash JW, Prieto VG, et al: Use of p63 expression in distinguishing primary and metastatic cutaneous adnexal neoplasms from metastatic adenocarcinoma to skin. J Cutan Pathol. 34(6):474–480, 2007. 51. Plumb SJ, Argenyi ZB, Stone MS, et al: Cytokeratin 5/6 immunostaining in cutaneous adnexal neoplasms and metastatic adenocarcinoma. Am J Dermatopathol. 26(6):447–451, 2004. 52. Latham JA, Redfern CP, Thody AJ, et al: Immunohistochemical markers of human sebaceous gland differentiation. J Histochem Cytochem. 37(5):729–734, 1989. 53. Ostler DA, Prieto VG, Reed JA, et al: Adipophilin expression in sebaceous tumors and other cutaneous lesions with clear cell histology: an immunohistochemical study of 117 cases. Mod Pathol. 23(4):567–573, 2010. 54. Bayer-Garner IB, Givens V, Smoller B: Immunohistochemical staining for androgen receptors: a sensitive marker of sebaceous differentiation. Am J Dermatopathol. 21(5):426–431, 1999. 55. Shikata N, Kurokawa I, Andachi H, et al: Expression of androgen receptors in skin appendage tumors: an immunohistochemical study. J Cutan Pathol. 22(2):149–153, 1995. 56. Browning L, Bailey D, Parker A: D2-40 is a sensitive and specific marker in differentiating primary adrenal cortical tumours from both metastatic clear cell renal cell carcinoma and phaeochromocytoma. J Clin Pathol. 61(3):293–296, 2008. 57. Perna AG, Smith MJ, Krishnan B, et al: CD10 is expressed in cutaneous clear cell lesions of different histogenesis. J Cutan Pathol. 32(5):348–351, 2005. 58. Bahrami S, Malone JC, Lear S, et al: CD10 expression in cutaneous adnexal neoplasms and a potential role for differentiating cutaneous metastatic renal cell carcinoma. Arch Pathol Lab Med. 130(9):1315–1319, 2006. 59. Manivel C, Wick MR, Mukai K: Pilomatrix carcinoma: an immunohistochemical comparison with benign pilomatrixoma and other benign cutaneous lesions of pilar origin. J Cutan Pathol. 13(1):22–29, 1986. 60. Basarab T, Orchard G, Russell-Jones R: The use of immunostaining for bcl-2 and CD34 and the lectin peanut agglutinin in differentiating between basal cell carcinomas and trichoepitheliomas. Am J Dermatopathol. 20(5):448–452, 1998. 61. Kirchmann TT, Prieto VG, Smoller BR: CD34 staining pattern distinguishes basal cell carcinoma from trichoepithelioma. Arch Dermatol. 130(5):589–592, 1994. 62. Lum CA, Binder SW: Proliferative characterization of basal-cell carcinoma and trichoepithelioma in small biopsy specimens. J Cutan Pathol. 31(8):550–554, 2004. 63. Poniecka AW, Alexis JB: An immunohistochemical study of basal cell carcinoma and trichoepithelioma. Am J Dermatopathol. 21(4):332–336, 1999. 64. Bhambri A, Prieto VG: Immunohistochemistry Helps Distinguish Hamartomatous Basal Cell Carcinoma from Trichoepithelioma. Am J Dermatopathol. 32(4):404, 2010. 65. Hartschuh W, Schulz T: Merkel cells are integral constituents of desmoplastic trichoepithelioma: an immunohistochemical and electron microscopic study. J Cutan Pathol. 22(5):413–421, 1995. 66. Abesamis-Cubillan E, El-Shabrawi-Caelen L, LeBoit PE: Merkel cells and sclerosing epithelial neoplasms. Am J Dermatopathol. 22(4):311–315, 2000.
67. Thewes M, Worret WI, Engst R, et al: Stromelysin-3—A potent marker for histopathologic differentiation between desmoplastic trichoepithelioma and morphealike basal cell carcinoma. Am J Dermatopathol. 20(2):140–142, 1998. 68. Hoang MP, Dresser KA, Kapur P, et al: Microcystic adnexal carcinoma: an immunohistochemical reappraisal. Mod Pathol. 21(2):178–185, 2008. 69. Wick MR, Cooper PH, Swanson PE, et al: Microcystic adnexal carcinoma. An immunohistochemical comparison with other cutaneous appendage tumors. Arch Dermatol. 126(2):189–194, 1990. 70. Lynch MH, O’Guin WM, Hardy C, et al: Acidic and basic hair/ nail (“hard”) keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to “soft” keratins. J Cell Biol. 103(6 Pt 2):2593–2606, 1986. 71. Ito M, Tazawa T, Shimizu N, et al: Cell differentiation in human anagen hair and hair follicles studied with anti-hair keratin monoclonal antibodies. J Invest Dermatol. 86(5):563–569, 1986. 72. Tateyama H, Eimoto T, Tada T, et al: Malignant pilomatricoma. An immunohistochemical study with antihair keratin antibody. Cancer. 69(1):127–132, 1992. 73. Amaral AL, Nascimento AG, Goellner JR: Proliferating pilar (trichilemmal) cyst. Report of two cases, one with carcinomatous transformation and one with distant metastases. Arch Pathol Lab Med. 108(10):808–810, 1984. 74. Swanson PE, Marrogi AJ, Williams DJ, et al: Tricholemmal carcinoma: clinicopathologic study of 10 cases. J Cutan Pathol. 19(2):100–109, 1992. 75. Jensen K, Kohler S, Rouse RV: Cytokeratin staining in Merkel cell carcinoma: an immunohistochemical study of cytokeratins 5/6, 7, 17, and 20. Appl Immunohistochem Mol Morphol. 8(4):310–315, 2000. 76. Kurokawa M, Nabeshima K, Akiyama Y, et al: CD56: a useful marker for diagnosing Merkel cell carcinoma. J Dermatol Sci. 31(3):219–224, 2003. 77. Mott RT, Smoller BR, Morgan MB: Merkel cell carcinoma: a clinicopathologic study with prognostic implications. J Cutan Pathol. 31(3):217–223, 2004. 78. Buresh CJ, Oliai BR, Miller RT: Reactivity with TdT in Merkel cell carcinoma: a potential diagnostic pitfall. Am J Clin Pathol. 129(6):894–898, 2008. 79. Dong HY, Liu W, Cohen P, et al: B-cell specific activation protein encoded by the PAX-5 gene is commonly expressed in Merkel cell carcinoma and small cell carcinomas. Am J Surg Pathol. 29(5):687–692, 2005. 80. Lau SK, Luthringer DJ, Eisen RN: Thyroid transcription factor-1: a review. Appl Immunohistochem Mol Morphol. 10(2):97–102, 2002. 81. Leech SN, Kolar AJO, Barrett PD, et al: Merkel cell carcinoma can be distinguished from metastatic small cell carcinoma using antibodies to cytokeratin 20 and thyroid transcription factor 1. J Clin Pathol. 54(9):727–729, 2001. 82. Lewis JS Jr, Duncavage E, Klonowski PW: Oral cavity neuroendocrine carcinoma: a comparison study with cutaneous Merkel cell carcinoma and other mucosal head and neck neuroendocrine carcinomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 110(2):209–217, 2010. 83. Hasegawa SL, Davison JM, Rutten A, et al: Primary cutaneous Ewing’s sarcoma: immunophenotypic and molecular cytogenetic evaluation of five cases. Am J Surg Pathol. 22(3):310–318, 1998. 84. Taylor GB, Chan YF: Subcutaneous primitive neuroectodermal tumour in the abdominal wall of a child: Long-term survival after local excision. Pathology. 32(4):294–298, 2000. 85. Ball NJ, Tanhuanco-Kho G: Merkel cell carcinoma frequently shows histologic features of basal cell carcinoma: a study of 30 cases. J Cutan Pathol. 34(8):612–619, 2007. 86. Zembowicz A, Garcia CF, Tannous ZS, et al: Endocrine mucinproducing sweat gland carcinoma: twelve new cases suggest that it is a precursor of some invasive mucinous carcinomas. Am J Surg Pathol. 29(10):1330–1339, 2005. 87. Kurtin PJ, Pinkus GS: Leukocyte Common Antigen—a Diagnostic Discriminant between Hematopoietic and Nonhematopoietic Neoplasms in Paraffin Sections Using MonoclonalAntibodies—Correlation with Immunological Studies and
References Ultrastructural-Localization. Hum Pathol. 16(4):353–365, 1985. 88. Andrade RE, Wick MR, Frizzera G, et al: Immunophenotyping of hematopoietic malignancies in paraffin sections. Hum Pathol. 19(4):394–402, 1988. 89. Falini B, Mason DY: Proteins encoded by genes involved in chromosomal alterations in lymphoma and leukemia: clinical value of their detection by immunocytochemistry. Blood. 99(2):409–426, 2002. 90. Fartasch M, Goerdt S, Hornstein OP: [Possibilities and limits of paraffin-embedded cell markers in diagnosis of primary cutaneous histiocytosis]. Hautarzt. 46(3):144–153, 1995. 91. Cerroni L, Smolle J, Soyer HP, et al: Immunophenotyping of cutaneous lymphoid infiltrates in frozen and paraffin-embedded tissue sections: a comparative study. J Am Acad Dermatol. 22(3):405–413, 1990. 92. Audouin J, Comperat E, Le Tourneau A, et al: Myeloid sarcoma: clinical and morphologic criteria useful for diagnosis. Int J Surg Pathol. 11(4):271–282, 2003. 93. Sung CO, Ko YH, Park S, et al: Immunoreactivity of CD99 in non-Hodgkin’s lymphoma: unexpected frequent expression in ALK-positive anaplastic large cell lymphoma. J Korean Med Sci. 20(6):952–956, 2005. 94. Gorczyca W, Tsang P, Liu Z, et al: CD30-positive T-cell lymphomas co-expressing CD15: an immunohistochemical analysis. Int J Oncol. 22(2):319–324, 2003. 95. Cho RJ, McCalmont TH, Ai WZ, et al: Use of an expanded immunohistochemical panel to distinguish cutaneous Hodgkin lymphoma from histopathologic imitators. J Cutan Pathol. 39(6):651–658, 2012. 96. Segal GH, Stoler MH, Tubbs RR: The “CD43 only” phenotype. An aberrant, nonspecific immunophenotype requiring comprehensive analysis for lineage resolution. Am J Clin Pathol. 97(6):861–865, 1992. 97. Ritter JH, Goldstein NS, Argenyi Z, et al: Granulocytic sarcoma: an immunohistologic comparison with peripheral T-cell lymphoma in paraffin sections. J Cutan Pathol. 21(3):207–216, 1994. 98. Quintanilla-Martinez L, Zukerberg LR, Ferry JA, et al: Extramedullary tumors of lymphoid or myeloid blasts. The role of immunohistology in diagnosis and classification. Am J Clin Pathol. 104(4):431–443, 1995. 99. Schmitt IM, Manente L, Di Matteo A, et al: Lymphoblastic lymphoma of the pre-B phenotype with cutaneous presentation. Dermatology. 195(3):289–292, 1997. 100. Torlakovic E, Torlakovic G, Nguyen PL, et al: The value of antipax-5 immunostaining in routinely fixed and paraffin-embedded sections: a novel pan pre-B and B-cell marker. Am J Surg Pathol. 26(10):1343–1350, 2002. 101. Kiyokawa N, Sekino T, Matsui T, et al: Diagnostic importance of CD179a/b as markers of precursor B-cell lymphoblastic lymphoma. Mod Pathol. 17(4):423–429, 2004. 102. Nihal M, Mikkola D, Horvath N, et al: Cutaneous lymphoid hyperplasia: a lymphoproliferative continuum with lymphomatous potential. Hum Pathol. 34(6):617–622, 2003. 103. Gilliam AC, Wood GS: Cutaneous lymphoid hyperplasias. Semin Cutan Med Surg. 19(2):133–141, 2000. 104. Magro C, Crowson AN, Porcu P, et al: Automated kappa and lambda light chain mRNA expression for the assessment of B-cell clonality in cutaneous B-cell infiltrates: its utility and diagnostic application. J Cutan Pathol. 30(8):504–511, 2003. 105. Leinweber B, Colli C, Chott A, et al: Differential diagnosis of cutaneous infiltrates of B lymphocytes with follicular growth pattern. Am J Dermatopathol. 26(1):4–13, 2004. 106. Nuckols JD, Shea CR, Horenstein MG, et al: Quantitation of intraepidermal T-cell subsets in formalin-fixed, paraffinembedded tissue helps in the diagnosis of mycosis fungoides. J Cutan Pathol. 26(4):169–175, 1999. 107. Takeshita M, Imayama S, Oshiro Y, et al: Clinicopathologic analysis of 22 cases of subcutaneous panniculitis-like CD56- or CD56+ lymphoma and review of 44 other reported cases. Am J Clin Pathol. 121(3):408–416, 2004. 108. Murphy M, Fullen D, Carlson JA: Low CD7 expression in benign and malignant cutaneous lymphocytic infiltrates: experience with an antibody reactive with paraffin-embedded tissue. Am J Dermatopathol. 24(1):6–16, 2002.
507.e3
109. Lukowsky A, Muche JM, Mobs M, et al: Evaluation of T-cell clonality in archival skin biopsy samples of cutaneous T-cell lymphomas using the biomed-2 PCR protocol. Diagn Mol Pathol. 19(2):70–77, 2010. 110. Ritter JH, Adesokan PN, Fitzgibbon JF, et al: Paraffin Section Immunohistochemistry as an Adjunct to Morphologic Analysis in the Diagnosis of Cutaneous Lymphoid Infiltrates. J Cutan Pathol. 21(6):481–493, 1994. 111. Magro CM, Crowson AN, Kovatich AJ, et al: Drug-induced reversible lymphoid dyscrasia: a clonal lymphomatoid dermatitis of memory and activated T cells. Hum Pathol. 34(2):119–129, 2003. 112. Gramvussakis S, George SA: Chronic actinic dermatitis (photosensitivity dermatitis/actinic reticuloid syndrome): beneficial effect from hydroxyurea. Br J Dermatol. 143(6):1340, 2000. 113. Clark S, Duvic M, Prieto VG: Mycosis Fungoides-Like Reaction in a Patient Treated w/Gleevec. J Cutan Pathol. 28(10):563, 2001. 114. Bignon YJ, Souteyrand P: Genotyping of cutaneous T-cell lymphomas and pseudolymphomas. Curr Probl Dermatol. 19:114– 123, 1990. 115. Ceballos KM, Gascoyne RD, Martinka M, et al: Heavy multinodular cutaneous lymphoid infiltrates: clinicopathologic features and B-cell clonality. J Cutan Pathol. 29(3):159–167, 2002. 116. Utz GL, Swerdlow SH: Distinction of follicular hyperplasia from follicular lymphoma in B5-fixed tissues: comparison of MT2 and bcl-2 antibodies. Hum Pathol. 24(11):1155–1158, 1993. 117. Mirza I, Macpherson N, Paproski S, et al: Primary cutaneous follicular lymphoma: an assessment of clinical, histopathologic, immunophenotypic, and molecular features. Am J Clin Oncol. 20(3):647–655, 2002. 118. Kim BK, Surti U, Pandya AG, et al: Primary and secondary cutaneous diffuse large B-cell lymphomas: a multiparameter analysis of 25 cases including fluorescence in situ hybridization for t(14;18) translocation. Am J Surg Pathol. 27(3):356–364, 2003. 119. Hoefnagel JJ, Vermeer MH, Jansen PM, et al: Bcl-2, Bcl-6 and CD10 expression in cutaneous B-cell lymphoma: further support for a follicle centre cell origin and differential diagnostic significance. Br J Dermatol. 149(6):1183–1191, 2003. 120. Lee CS, Lim HW: Cutaneous diseases in Asians. Dermatol Clin. 21(4):669–677, 2003. 121. Cepeda LT, Pieretti M, Chapman SF, et al: CD30-positive atypical lymphoid cells in common non-neoplastic cutaneous infiltrates rich in neutrophils and eosinophils. Am J Surg Pathol. 27(7):912–918, 2003. 122. Yen HR, Lin PY, Chuang WY, et al: Skin manifestations of Kikuchi-Fujimoto disease: case report and review. Eur J Pediatr. 163(4–5):210–213, 2004. 123. Kuo TT: Kikuchi’s disease (histiocytic necrotizing lymphadenitis). A clinicopathologic study of 79 cases with an analysis of histologic subtypes, immunohistology, and DNA ploidy. Am J Surg Pathol. 19(7):798–809, 1995. 124. Lieberman PH, Jones CR, Steinman RM, et al: Langerhans cell (eosinophilic) granulomatosis. A clinicopathologic study encompassing 50 years. Am J Surg Pathol. 20(5):519–552, 1996. 125. Singh A, Prieto VG, Czelusta A, et al: Adult langerhans cell histiocytosis limited to the skin. Dermatology. 207(2):157–161, 2003. 126. Kansal R, Quintanilla-Martinez L, Datta V, et al: Identification of the V600D mutation in Exon 15 of the BRAF oncogene in congenital, benign langerhans cell histiocytosis. Genes Chromosomes Cancer. 52(1):99–106, 2013. 127. Slone SP, Fleming DR, Buchino JJ: Sinus histiocytosis with massive lymphadenopathy and Langerhans cell histiocytosis express the cellular adhesion molecule CD31. Arch Pathol Lab Med. 127(3):341–344, 2003. 128. Lau SK, Chu PG, Weiss LM: Immunohistochemical expression of Langerin in Langerhans cell histiocytosis and non-Langerhans cell histiocytic disorders. Am J Surg Pathol. 32(4):615–619, 2008. 129. Favara BE, Feller AC, Pauli M, et al: Contemporary classification of histiocytic disorders. The WHO Committee On Histiocytic/ Reticulum Cell Proliferations. Reclassification Working Group
507.e4
Immunohistology of Skin Tumors
of the Histiocyte Society. Med Pediatr Oncol. 29(3):157–166, 1997. 130. Kapur P, Erickson C, Rakheja D, et al: Congenital self-healing reticulohistiocytosis (Hashimoto-Pritzker disease): ten-year experience at Dallas Children’s Medical Center. J Am Acad Dermatol. 56(2):290–294, 2007. 131. Jih DM, Salcedo SL, Jaworsky C: Benign cephalic histiocytosis: a case report and review. J Am Acad Dermatol. 47(6):908–913, 2002. 132. Gianotti R, Alessi E, Caputo R: Benign cephalic histiocytosis: a distinct entity or a part of a wide spectrum of histiocytic proliferative disorders of children? A histopathological study. Am J Dermatopathol. 15(4):315–319, 1993. 133. Flynn KJ, Dehner LP, Gajl-Peczalska KJ, et al: Regressing atypical histiocytosis: a cutaneous proliferation of atypical neoplastic histiocytes with unexpectedly indolent biologic behavior. Cancer. 49(5):959–970, 1982. 134. Drews R, Samel A, Kadin ME: Lymphomatoid papulosis and anaplastic large cell lymphomas of the skin. Semin Cutan Med Surg. 19(2):109–117, 2000. 135. Kadin ME, Carpenter C: Systemic and primary cutaneous anaplastic large cell lymphomas. Semin Hematol. 40(3):244–256, 2003. 136. Turner ML, Gilmour HM, McLaren KM, et al: Regressing atypical histiocytosis: report of two cases with progression to high grade T-cell non-Hodgkin’s lymphoma. Hematol Pathol. 7(1):33– 47, 1993. 137. Brown JR, Skarin AT: Clinical mimics of lymphoma. Oncologist. 9(4):406–416, 2004. 138. Cardoso J, Duhra P, Thway Y, et al: Lymphomatoid papulosis type D: a newly described variant easily confused with cutaneous aggressive CD8-positive cytotoxic T-cell lymphoma. Am J Dermatopathol. 34(7):762–765, 2012. 139. Magro CM, Crowson AN, Morrison C, et al: CD8+ lymphomatoid papulosis and its differential diagnosis. Am J Clin Pathol. 125(4):490–501, 2006. 140. Saggini A, Gulia A, Argenyi Z, et al: A variant of lymphomatoid papulosis simulating primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma. Description of 9 cases. Am J Surg Pathol. 34(8):1168–1175, 2010. 141. Kempf W, Kazakov DV, Scharer L, et al: Angioinvasive lymphomatoid papulosis: a new variant simulating aggressive lymphomas. Am J Surg Pathol. 37(1):1–13, 2013. 142. Steinhoff M, Hummel M, Anagnostopoulos I, et al: Single-cell analysis of CD30+ cells in lymphomatoid papulosis demonstrates a common clonal T-cell origin. Blood. 100(2):578–584, 2002. 143. Kadin ME, Levi E, Kempf W: Progression of lymphomatoid papulosis to systemic lymphoma is associated with escape from growth inhibition by transforming growth factor-beta and CD30 ligand. Ann NY Acad Sci. 941:59–68, 2001. 144. Liu HL, Hoppe RT, Kohler S, et al: CD30+ cutaneous lymphoproliferative disorders: the Stanford experience in lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. J Am Acad Dermatol. 49(6):1049–1058, 2003. 145. de Souza A, el-Azhary RA, Camilleri MJ, et al: In search of prognostic indicators for lymphomatoid papulosis: a retrospective study of 123 patients. J Am Acad Dermatol. 66(6):928–937, 2012. 146. Laskin WB, Miettinen M, Fetsch JF: Infantile digital fibroma/ fibromatosis: a clinicopathologic and immunohistochemical study of 69 tumors from 57 patients with long-term follow-up. Am J Surg Pathol. 33(1):1–13, 2009. 147. Meis-Kindblom JM, Kindblom LG, Enzinger FM: Sclerosing epithelioid fibrosarcoma. A variant of fibrosarcoma simulating carcinoma. Am J Surg Pathol. 19(9):979–993, 1995. 148. Bhawan J, Bacchetta C, Joris I, et al: A myofibroblastic tumor. Infantile digital fibroma (recurrent digital fibrous tumor of childhood). Am J Pathol. 94(1):19–36, 1979. 149. Beham A, Badve S, Suster S, et al: Solitary myofibroma in adults: clinicopathological analysis of a series. Histopathology. 22(4):335– 341, 1993. 150. Mentzel T, Calonje E, Nascimento AG, et al: Infantile hemangiopericytoma versus infantile myofibromatosis. Study of a series
suggesting a continuous spectrum of infantile myofibroblastic lesions. Am J Surg Pathol. 18(9):922–930, 1994. 151. Diaz-Cascajo C, Borghi S, Weyers W, et al: Fibroblastic/ myofibroblastic sarcoma of the skin: a report of five cases. J Cutan Pathol. 30(2):128–134, 2003. 152. Antonescu CR, Rosenblum MK, Pereira P, et al: Sclerosing epithelioid fibrosarcoma: a study of 16 cases and confirmation of a clinicopathologically distinct tumor. Am J Surg Pathol. 25(6):699–709, 2001. 153. Strauchen JA, Dimitriu-Bona A: Malignant fibrous histiocytoma. Expression of monocyte/macrophage differentiation antigens detected with monoclonal antibodies. Am J Pathol. 124(2):303– 309, 1986. 154. Mechtersheimer G: Towards the phenotyping of soft tissue tumours by cell surface molecules. Virchows Arch A Pathol Anat Histopathol. 419(1):7–28, 1991. 155. Crocker J, Burnett D, Jones EL: Immunohistochemical demonstration of cathepsin B in the macrophages of benign and malignant lymphoid tissues. J Pathol. 142(1):87–94, 1984. 156. Mechtersheimer G, Staudter M, Majdic O, et al: Expression of HLA-A,B,C, beta 2-microglobulin (beta 2m), HLA-DR, -DP, -DQ and of HLA-D-associated invariant chain (Ii) in soft-tissue tumors. Int J Cancer. 46(5):813–823, 1990. 157. Loftus B, Loh LC, Curran B, et al: Mac387—Its Nonspecificity as a Tumor-Marker or Marker of Histiocytes. Histopathology. 19(3):251–255, 1991. 158. Cerio R, Spaull J, Oliver GF, et al: A study of factor XIIIa and MAC 387 immunolabeling in normal and pathological skin. Am J Dermatopathol. 12(3):221–233, 1990. 159. Gray MH, Smoller BR, McNutt NS, et al: Neurofibromas and neurotized melanocytic nevi are immunohistochemically distinct neoplasms. Am J Dermatopathol. 12(3):234–241, 1990. 160. Silverman JS, Tamsen A: High grade malignant fibrous histiocytomas have bimodal cycling populations of factor XIIIa+ dendrophages and dedifferentiated mesenchymal cells possibly derived from CD34+ fibroblasts. Cell Vis. 5(1):73–76, 1998. 161. Longacre TA, Smoller BR, Rouse RV: Atypical fibroxanthoma. Multiple immunohistologic profiles. Am J Surg Pathol. 17(12):1199–1209, 1993. 162. Hasegawa T, Hasegawa F, Hirose T, et al: Expression of smooth muscle markers in so called malignant fibrous histiocytomas. J Clin Pathol. 56(9):666–671, 2003. 163. Prieto VG, Reed JA, Shea CR: Immunohistochemistry of dermatofibromas and benign fibrous histiocytomas. J Cutan Pathol. 22(4):336–341, 1995. 164. OConnell JX, Trotter MJ: Fibrosarcomatous dermatofibrosarcoma protuberans with myofibroblastic differentiation: A histologically distinctive variant (vol 9, pg 273, 1996). Mod Pathol. 9(7):803, 1996. 165. Goldblum JR: Giant cell fibroblastoma: a report of three cases with histologic and immunohistochemical evidence of a relationship to dermatofibrosarcoma protuberans. Arch Pathol Lab Med. 120(11):1052–1055, 1996. 166. Wick MR, Ritter JH, Lind AC, et al: The pathological distinction between “deep penetrating” dermatofibroma and dermatofibrosarcoma protuberans. Semin Cutan Med Surg. 18(1):91–98, 1999. 167. Abenoza P, Lillemoe T: CD34 and factor XIIIa in the differential diagnosis of dermatofibroma and dermatofibrosarcoma protuberans. Am J Dermatopathol. 15(5):429–434, 1993. 168. Mentzel T, Hugel H, Kutzner H: CD34-positive glomus tumor: clinicopathologic and immunohistochemical analysis of six cases with myxoid stromal changes. J Cutan Pathol. 29(7):421–425, 2002. 169. Hanft VN, Shea CR, McNutt NS, et al: Expression of CD34 in sclerotic (“plywood”) fibromas. Am J Dermatopathol. 22(1):17– 21, 2000. 170. Cowper SE, Kilpatrick T, Proper S, et al: Solitary fibrous tumor of the skin. Am J Dermatopathol. 21(3):213–219, 1999. 171. Hardisson D, Cuevas-Santos J, Contreras F: Solitary fibrous tumor of the skin. J Am Acad Dermatol. 46(2 Suppl Case Reports):S37–S40, 2002. 172. Shea CR, Salob S, Reed JA, et al: CD34-reactive fibrous papule of the nose. J Am Acad Dermatol. 35(2 Pt 2):342–345, 1996.
References 173. Requena L, Gutierrez J, Sanchez Yus E: Multiple sclerotic fibromas of the skin. A cutaneous marker of Cowden’s disease. J Cutan Pathol. 19(4):346–351, 1992. 174. Horenstein MG, Prieto VG, Nuckols JD, et al: Indeterminate fibrohistiocytic lesions of the skin: is there a spectrum between dermatofibroma and dermatofibrosarcoma protuberans? Am J Surg Pathol. 24(7):996–1003, 2000. 175. Diaz-Cascajo C, Weyers W, Borrego L, et al: Dermatofibrosarcoma protuberans with fibrosarcomatous areas: a clinicopathologic and immunohistochemic study in four cases. Am J Dermatopathol. 19(6):562–567, 1997. 176. Mentzel T, Beham A, Katenkamp D, et al: Fibrosarcomatous (“high-grade”) dermatofibrosarcoma protuberans: clinicopathologic and immunohistochemical study of a series of 41 cases with emphasis on prognostic significance. Am J Surg Pathol. 22(5):576–587, 1998. 177. Davis DA, Sanchez RL: Atrophic and plaquelike dermatofibrosarcoma protuberans. Am J Dermatopathol. 20(5):498–501, 1998. 178. Brooks JJ: The significance of double phenotypic patterns and markers in human sarcomas. A new model of mesenchymal differentiation. Am J Pathol. 125(1):113–123, 1986. 179. Monteagudo C, Calduch L, Navarro S, et al: CD99 immunoreactivity in atypical fibroxanthoma: a common feature of diagnostic value. Am J Clin Pathol. 117(1):126–131, 2002. 180. Diwan AH, Skelton HG 3rd, Horenstein MG, et al: Dermatofibrosarcoma protuberans and giant cell fibroblastoma exhibit CD99 positivity. J Cutan Pathol. 35(7):647–650, 2008. 181. de Feraudy S, Mar N, McCalmont TH: Evaluation of CD10 and procollagen 1 expression in atypical fibroxanthoma and dermatofibroma. Am J Surg Pathol. 32(8):1111–1122, 2008. 182. Hall JM, Saenger JS, Fadare O: Diagnostic Utility of P63 and CD10 in Distinguishing Cutaneous Spindle Cell/Sarcomatoid Squamous Cell Carcinomas and Atypical Fibroxanthomas. Int J Clin Exp Pathol. 1(6):524–530, 2008. 183. Hultgren TL, DiMaio DJ: Immunohistochemical staining of CD10 in atypical fibroxanthomas. J Cutan Pathol. 34(5):415– 419, 2007. 184. Smith-Zagone MJ, Prieto VG, Hayes RA, et al: HMB-45 (gp103) and MART-1 expression within giant cells in an atypical fibroxanthoma: a case report. J Cutan Pathol. 31(3):284–286, 2004. 185. Vollmer RT, Humphrey PA, Swanson PE, et al: Invasion of the bladder by transitional cell carcinoma: its relation to histologic grade and expression of p53, MIB-1, c-erb B-2, epidermal growth factor receptor, and bcl-2. Cancer. 82(4):715–723, 1998. 186. Singh Gomez C, Calonje E, Fletcher CD: Epithelioid benign fibrous histiocytoma of skin: clinico-pathological analysis of 20 cases of a poorly known variant. Histopathology. 24(2):123–129, 1994. 187. Watanabe K, Kusakabe T, Hoshi N, et al: h-Caldesmon in leiomyosarcoma and tumors with smooth muscle cell-like differentiation: its specific expression in the smooth muscle cell tumor. Hum Pathol. 30(4):392–396, 1999. 188. Jensen ML, Jensen OM, Michalski W, et al: Intradermal and subcutaneous leiomyosarcoma: a clinicopathological and immunohistochemical study of 41 cases. J Cutan Pathol. 23(5):458– 463, 1996. 189. Swanson PE, Stanley MW, Scheithauer BW, et al: Primary cutaneous leiomyosarcoma. A histological and immunohistochemical study of 9 cases, with ultrastructural correlation. J Cutan Pathol. 15(3):129–141, 1988. 190. Iwata J, Fletcher CD: Immunohistochemical detection of cytokeratin and epithelial membrane antigen in leiomyosarcoma: a systematic study of 100 cases. Pathol Int. 50(1):7–14, 2000. 191. Buonaccorsi JN, Plaza JA: Role of CD10, wide-spectrum keratin, p63, and podoplanin in the distinction of epithelioid and spindle cell tumors of the skin: an immunohistochemical study of 81 cases. Am J Dermatopathol. 34(4):404–411, 2012. 192. Park JH, Oh SH, Yang MH, et al: Glomangiosarcoma of the hand: a case report and review of the literature. J Dermatol. 30(11):827–833, 2003. 193. Mentzel T, Dei Tos AP, Sapi Z, et al: Myopericytoma of skin and soft tissues—Clinicopathologic and immunohistochemical study of 54 cases. Am J Surg Pathol. 30(1):104–113, 2006.
507.e5
194. Kaye VM, Dehner LP: Cutaneous glomus tumor. A comparative immunohistochemical study with pseudoangiomatous intradermal melanocytic nevi. Am J Dermatopathol. 13(1):2–6, 1991. 195. Mentzel T, Reisshauer S, Rutten A, et al: Cutaneous clear cell myomelanocytic tumour: a new member of the growing family of perivascular epithelioid cell tumours (PEComas). Clinicopathological and immunohistochemical analysis of seven cases. Histopathology. 46(5):498–504, 2005. 196. Liegl B, Hornick JL, Fletcher CD: Primary cutaneous PEComa: distinctive clear cell lesions of skin. Am J Surg Pathol. 32(4):608– 614, 2008. 197. Requena L, Sangueza OP: Benign neoplasms with neural differentiation: a review. Am J Dermatopathol. 17(1):75–96, 1995. 198. George E, Swanson PE, Wick MR: Malignant peripheral nerve sheath tumors of the skin. Am J Dermatopathol. 11(3):213–221, 1989. 199. Leroy K, Dumas V, Martin-Garcia N, et al: Malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1: a clinicopathologic and molecular study of 17 patients. Arch Dermatol. 137(7):908–913, 2001. 200. Swanson PE, Scheithauer BW, Wick MR: Peripheral nerve sheath neoplasms. Clinicopathologic and immunochemical observations. Pathol Annu. 30(Pt 2):1–82, 1995. 201. LeBoit PE, Barr RJ, Burall S, et al: Primitive polypoid granularcell tumor and other cutaneous granular-cell neoplasms of apparent nonneural origin. Am J Surg Pathol. 15(1):48–58, 1991. 202. Hitchcock MG, Hurt MA, Santa Cruz DJ: Cutaneous granular cell angiosarcoma. J Cutan Pathol. 21(3):256–262, 1994. 203. Zelger BG, Steiner H, Kutzner H, et al: Granular cell dermatofibroma. Histopathology. 31(3):258–262, 1997. 204. Le BH, Boyer PJ, Lewis JE, et al: Granular cell tumor: immunohistochemical assessment of inhibin-alpha, protein gene product 9.5, S100 protein, CD68, and Ki-67 proliferative index with clinical correlation. Arch Pathol Lab Med. 128(7):771–775, 2004. 205. Cheng SD, Usmani AS, DeYoung BR, et al: Dermatofibroma-like granular cell tumor. J Cutan Pathol. 28(1):49–52, 2001. 206. Fine SW, Li M: Expression of calretinin and the alpha-subunit of inhibin in granular cell tumors. Am J Clin Pathol. 119(2):259– 264, 2003. 207. Skelton HG, Williams J, Smith KJ: The clinical and histologic spectrum of cutaneous fibrous perineuriomas. Am J Dermatopathol. 23(3):190–196, 2001. 208. Mentzel T: [Cutaneous perineurioma. Clinical and histological findings and differential diagnosis]. Pathologe. 24(3):207–213, 2003. 209. Fetsch JF, Miettinen M: Sclerosing perineurioma: a clinicopathologic study of 19 cases of a distinctive soft tissue lesion with a predilection for the fingers and palms of young adults. Am J Surg Pathol. 21(12):1433–1442, 1997. 210. Hornick JL, Fletcher CDM: Soft tissue perineurioma— Clinicopathologic analysis of 81 cases including those with atypical histologic features. Am J Surg Pathol. 29(7):845–858, 2005. 211. Mentzel T, Kutzner H: Reticular and plexiform perineurioma: clinicopathological and immunohistochemical analysis of two cases and review of perineurial neoplasms of skin and soft tissues. Virchows Arch. 447(4):677–682, 2005. 212. Zelger B, Weinlich G: Perineuroma. A frequently unrecognized entity with emphasis on a plexiform variant. Adv Clin Path. 4(1):25–33, 2000. 213. Folpe AL, Billings SD, McKenney JK, et al: Expression of claudin-1, a recently described tight junction-associated protein, distinguishes soft tissue perineurioma from potential mimics. Am J Surg Pathol. 26(12):1620–1626, 2002. 214. Rankine AJ, Filion PR, Platten MA, et al: Perineurioma: a clinicopathological study of eight cases. Pathology. 36(4):309–315, 2004. 215. Mitchell A, Scheithauer BW, Doyon J, et al: Malignant perineurioma (malignant peripheral nerve sheath tumor with perineural differentiation). Clin Neuropathol. 31(6):424–429, 2012. 216. Zamecnik M, Michal M: Malignant peripheral nerve sheath tumor with perineurial cell differentiation (malignant perineurioma). Pathol Int. 49(1):69–73, 1999.
507.e6
Immunohistology of Skin Tumors
217. Laskin WB, Fetsch JF, Miettinen M: The “neurothekeoma”: immunohistochemical analysis distinguishes the true nerve sheath myxoma from its mimics. Hum Pathol. 31(10):1230– 1241, 2000. 218. Fetsch JF, Laskin WB, Miettinen M: Nerve sheath myxoma: a clinicopathologic and immunohistochemical analysis of 57 morphologically distinctive, S-100 protein- and GFAP-positive, myxoid peripheral nerve sheath tumors with a predilection for the extremities and a high local recurrence rate. Am J Surg Pathol. 29(12):1615–1624, 2005. 219. Hornick JL, Fletcher CD: Cellular neurothekeoma: detailed characterization in a series of 133 cases. Am J Surg Pathol. 31(3):329–340, 2007. 220. Page RN, King R, Mihm MC Jr, et al: Microphthalmia transcription factor and NKI/C3 expression in cellular neurothekeoma. Mod Pathol. 17(2):230–234, 2004. 221. Plaza JA, Torres-Cabala C, Evans H, et al: Immunohistochemical expression of S100A6 in cellular neurothekeoma: clinicopathologic and immunohistochemical analysis of 31 cases. Am J Dermatopathol. 31(5):419–422, 2009. 222. Chang MW: Updated classification of hemangiomas and other vascular anomalies. Lymphat Res Biol. 1(4):259–265, 2003. 223. O’Hara CD, Nascimento AG: Endothelial lesions of soft tissues: a review of reactive and neoplastic entities with emphasis on low-grade malignant (“borderline”) vascular tumors. Adv Anat Pathol. 10(2):69–87, 2003. 224. Cheuk W, Wong KO, Wong CS, et al: Immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish Kaposi sarcoma from its mimickers. Am J Clin Pathol. 121(3):335–342, 2004. 225. Robin YM, Guillou L, Michels JJ, et al: Human herpesvirus 8 immunostaining: a sensitive and specific method for diagnosing Kaposi sarcoma in paraffin-embedded sections. Am J Clin Pathol. 121(3):330–334, 2004. 226. O’Hara CD, Nascimento AG: Endothelial lesions of soft tissues: a review of reactive and neoplastic entities with emphasis on low-grade malignant (“borderline”) vascular tumors. Adv Anat Pathol. 10(2):69–87, 2003. 227. Billings SD, Folpe AL, Weiss SW: Epithelioid sarcomalike hemangioendothelioma. Am J Surg Pathol. 27(1):48–57, 2003. 228. Manivel JC, Wick MR, Dehner LP, et al: Epithelioid sarcoma. An immunohistochemical study. Am J Clin Pathol. 87(3):319– 326, 1987. 229. Wick MR, Manivel JC: Epithelioid sarcoma and isolated necrobiotic granuloma: a comparative immunocytochemical study. J Cutan Pathol. 13(4):253–260, 1986. 230. Wakely P, Jr: Epithelioid/granular soft tissue lesions: correlation of cytopathology and histopathology. Ann Diagn Pathol. 4(5):316–328, 2000. 231. Humble SD, Prieto VG, Horenstein MG: Cytokeratin 7 and 20 expression in epithelioid sarcoma. J Cutan Pathol. 30(4):242– 246, 2003. 232. Kato H, Hatori M, Kokubun S, et al: CA125 expression in epithelioid sarcoma. Jpn J Clin Oncol. 34(3):149–154, 2004.
233. Tardio JC: CD34-reactive tumors of the skin. An updated review of an ever-growing list of lesions. J Cutan Pathol. 36(1):89–102, 2009. 234. Lin L, Skacel M, Sigel JE, et al: Epithelioid sarcoma: an immunohistochemical analysis evaluating the utility of cytokeratin 5/6 in distinguishing superficial epithelioid sarcoma from spindled squamous cell carcinoma. J Cutan Pathol. 30(2):114–117, 2003. 235. Laskin WB, Miettinen M: Epithelioid sarcoma: new insights based on an extended immunohistochemical analysis. Arch Pathol Lab Med. 127(9):1161–1168, 2003. 236. Laskin WB, Miettinen M: Epithelial-type and neural-type cadherin expression in malignant noncarcinomatous neoplasms with epithelioid features that involve the soft tissues. Arch Pathol Lab Med. 126(4):425–431, 2002. 237. Nicholson SA, McDermott MB, Swanson PE, et al: CD99 and cytokeratin-20 in small-cell and basaloid tumors of the skin. Appl Immunohistochem Mol Morphol. 8(1):37–41, 2000. 238. Devoe K, Weidner N: Immunohistochemistry of small roundcell tumors. Semin Diagn Pathol. 17(3):216–224, 2000. 239. Wick MR, Ockner DM, Mills SE, et al: Homologous carcinomas of the breasts, skin, and salivary glands. A histologic and immunohistochemical comparison of ductal mammary carcinoma, ductal sweat gland carcinoma, and salivary duct carcinoma. Am J Clin Pathol. 109(1):75–84, 1998. 240. Wallace ML, Longacre TA, Smoller BR: Estrogen and progesterone receptors and anti-gross cystic disease fluid protein 15 (BRST-2) fail to distinguish metastatic breast carcinoma from eccrine neoplasms. Mod Pathol. 8(9):897–901, 1995. 241. Busam KJ, Tan LK, Granter SR, et al: Epidermal growth factor, estrogen, and progesterone receptor expression in primary sweat gland carcinomas and primary and metastatic mammary carcinomas. Mod Pathol. 12(8):786–793, 1999. 242. Springer EA, Robinson JK: Patterns of epidermal growth factor receptors in basal and squamous cell carcinoma. J Dermatol Surg Oncol. 17(1):20–24, 1991. 243. Ellis DL, Nanney LB, King LE Jr: Increased epidermal growth factor receptors in seborrheic keratoses and acrochordons of patients with the dysplastic nevus syndrome. J Am Acad Dermatol. 23(6 Pt 1):1070–1077, 1990. 244. Hasebe T, Mukai K, Yamaguchi N, et al: Prognostic value of immunohistochemical staining for proliferating cell nuclear antigen, p53, and c-erbB-2 in sebaceous gland carcinoma and sweat gland carcinoma: comparison with histopathological parameter. Mod Pathol. 7(1):37–43, 1994. 245. Wolber RA, Dupuis BA, Wick MR: Expression of c-erbB-2 oncoprotein in mammary and extramammary Paget’s disease. Am J Clin Pathol. 96(2):243–247, 1991. 246. Prieto VG, Reed JA, McNutt NS, et al: Differential expression of CD44 in malignant cutaneous epithelial neoplasms. Am J Dermatopathol. 17(5):447–451, 1995. 247. Penneys NS, Shapiro S: Cd44 Expression in Merkel CellCarcinoma May Correlate with Risk of Metastasis. J Cutan Pathol. 21(1):22–26, 1994.
C H A P T E R 1 4
IMMUNOHISTOLOGY OF THE GASTROINTESTINAL TRACT ALYSSA M. KRASINSKAS, JEFFREY D. GOLDSMITH
Overview 508 Biology of Antigens: General and Tissue Specific 508 Diagnostic Immunohistochemistry 510 Genomic Applications 537 Theranostic Applications 537 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications 539 Summary 539
Overview This chapter divides the discussion of immunohistochemistry (IHC) of the luminal gastrointestinal (GI) tract into three sections: 1) epithelial pathology, 2) neuroendocrine lesions, and 3) spindle cell lesions. We have attempted to compile the innumerable IHC studies that have been applied to these organs into a cogent, useful, and relevant text.
Carcinomas that stain with predominantly HMWCK antibodies include squamous cell carcinomas (SCCs) of the esophagus and anus.
β-Catenin β-Catenin is an 88-kD member of the catenin family of proteins, which are important constituents of the cytoskeleton; β-catenin is important in gene expression and is a component of the Wnt signaling cascade. In certain conditions, when the normal degradation of β-catenin is disrupted, this protein accumulates in the cytoplasm and abnormally translocates to the nucleus, where it can disrupt normal gene expression.2,3
Cytokeratin 7 Cytokeratin 7 (CK7) is an intermediate filament protein expressed predominantly by ductal epithelial cells of the pancreatobiliary tract, renal collecting ducts, and proximal GI tract. Expression of CK7 is limited to subtypes of adenocarcinomas and SCCs that arise within nonkeratinized mucosa.
Cytokeratin 20
Biology of Antigens: General and Tissue Specific This section includes a brief discussion of antigens/ epitopes often used in GI pathology. Table 14-1 shows representative assay conditions for these antibodies. Note that the conditions listed in this table are meant to be guides. All new antibodies must be thoroughly validated by the laboratory performing the test.
General Cytokeratins For the purposes of diagnostic IHC, carcinomas that stain with low- and high-molecular-weight cytokeratins (HMWCKs) include adenocarcinomas of the esophagus and stomach. Those that stain predominantly with lowmolecular-weight CK (LMWCK) antibodies include colorectal adenocarcinomas, neuroendocrine tumors, and high-grade neuroendocrine carcinomas (NECs).1 508
In the small intestine, CK20 stains only the highly differentiated small bowel villous enterocytes (CK18 stains the more immature, basilar, proliferative zone cells). In the colon, CK20 stains only the surface epithelial cell layer, and CK20 staining is more extensive and stronger in small bowel neoplasms than in colonic carcinomas.4
CDX-2 CDX-2 is a homeobox gene that is an integral component of intestinal cell proliferation and differentiation.5 It appears to function as a tumor suppressor gene in colorectal and some pancreatobiliary and gastric adenocarcinomas.6
Chromogranin A This protein is an acidic soluble glycoprotein found within neurosecretory granules.7,8 Chromogranin A
Biology of Antigens: General and Tissue Specific
509
TABLE 14-1 Assay Conditions for Representative Antibodies Antibody
Clone
Dilution
Retrieval
β-catenin
17C2
1 : 200
pH 6.1
KIT (CD117)
104D2
1 : 200
pH 9.0
CDX-2
CDX2-88
1 : 50
pH 6.1
Chromogranin A
DAK-A3
1 : 800
pH 9.0
Cytokeratin 7
OV-TL 12/30
1 : 400
Proteinase K
Cytokeratin 20
Ks20.8
1 : 25
Proteinase K
COX-2
SP-21
1 : 100
pH 8.5
MLH1
CM220C
1 : 10
Proprietary solution
MOC-31
MOC-31
1 : 25
Proteinase K
MSH2
FE11
1 : 25
Proprietary solution
MSH6
70834
1 : 100
pH 9.0
MUC1
Ma95
1 : 100
pH 8.5
MUC2
Ccp58
1 : 25
pH 8.5
MUC5AC
CLH2
1 : 100
pH 8.5
MUC6
CLH5
1 : 25
pH 8.5
p53
DO-7
1 : 25
pH 9.0
p63
4A4
1 : 50
pH 9.0
PMS2
A16-4
1 : 10
pH 9.0
Synaptophysin
Sv38
1 : 100
pH 6.1
Villin
CWWB1
1 : 100
Citrate, pH 6.0
COX-2, Cyclooxygenase 2.
undergoes posttranslational modification, which varies between GI sites and their associated tumors.9 Chromogranin is more specific but less sensitive than synaptophysin.8 Most nonneoplastic neuroendocrine (NE) lesions and low-grade NE neoplasms diffusely and strongly stain with chromogranin, and this is proportional to the number of intracytoplasmic neurosecretory granules. However, some carcinoids stain weakly with chromogranin, which may reflect differences in the type of amines contained within the tumor cell cytoplasm.
Cyclooxygenase 2 Cyclooxygenase 2 (COX-2) is the rate-limiting enzyme in the production of various prostaglandins from arachidonic acid.10 It is expressed in a variety of neoplasms, including colorectal,11,12 gastric,13 and pancreatic carcinomas.14-16
KIT (CD117) The antibody against this protein stains the transmembrane and cytoplasmic KIT protein, which is a type 3 tyrosine kinase receptor. This antibody is useful for identifying GI stromal tumors (GISTs).
DNA Mismatch Repair Proteins MLH1, MSH2, MSH6, and PMS2 These antibodies are directed at protein components of the mismatch repair (MMR) complex. These proteins function as heterodimers; MLH1 associates with PMS2, and MSH2 associates with MSH6. As such, these pairs of proteins can show pairwise loss of expression. Decreased or absent staining is indicative of quantitative protein deficiencies or mutated protein.17
MOC-31 MOC-31 is one of myriad monoclonal antibodies against the epithelial adhesion molecule, Ep-CAM.18,19 MOC-31 is expressed in a host of benign epithelia and is expressed in many carcinomas, including those derived from the colon, stomach, breast, pancreas, ovary, and bile ducts.20,21
Mucin Core Polypeptides Mucin core polypeptides (MUCs) are the backbone molecule of GI tract mucin and are responsible for the mucus-gel layer, which covers the mucosa.22 MUC1 is normally expressed by enterocytes and intestinal goblet cells, MUC2 is normally secreted by intestinal goblet
510
Immunohistology of the Gastrointestinal Tract
cells, MUC5AC is expressed by gastric foveolar mucus cells and neoplastic goblet cells, and MUC6 is secreted by gastric antral and fundic gland cells.
Neuroendocrine Secretory Protein-55 Neuroendocrine secretory protein-55 (NESP-55) is a recently described member of the granin family of proteins that are localized to dense core secretory granules present in various endocrine cells.23,24 It is highly expressed in the adrenal medulla, pituitary gland, and brain.25
p53 Protein Normal p53 protein has an extremely short half-life and is found in small quantities inside cells. As such, p53 can be detected in small quantities in normal cells by using IHC. The p53 protein from abnormal p53 genes has a longer half-life than normal p53 protein, it accumulates inside cells, and it can be detected with anti-p53 antibodies, in which case there is strong diffuse p53 staining, which is an indication of mutation. In theory, p53 overexpression, as a surrogate marker for p53 gene mutations, is an IHC test for neoplasia (dysplasia). However, this is not the case in practice, because correlation between staining and gene abnormalities is not precise.26
p63 Protein This molecule is a member of the p53 family of proteins; it exists as multiple protein variants, which are a result of alternative transcript splicing events. The relative concentrations of these protein variants affect the expression and functionality of wild-type p63 and p53 proteins.27 The p63 protein is expressed in a nuclear pattern in various myoepithelia and is present in the basal layer of squamous epithelium.28,29
Synaptophysin This protein is a membrane glycoprotein found in calcium channels of cells. Its expression is independent of chromogranin A.8
Villin Villin is a brush border, microfilament-associated, actinbinding protein related to rootlet formation. Staining in colorectal adenocarcinomas is diffusely cytoplasmic with brush border accentuation.30-33
columnar metaplasia of the gastroesophageal junction. This finding is distinct from intestinal metaplasia of the gastric cardia,34 and IHC has been used to distinguish these two mucosae with variable success.35-43 Published in 2003, a consensus conference on the morphology of BE stated that IHC stains—including cytokeratins 7 and 20, MUCs, and CDX-2—were not essential for the diagnosis.44 Thus the use of IHC to diagnose BE or distinguish it from gastric cardia mucosa with intestinal metaplasia is currently investigational and should not be used in routine clinical practice. Dysplasia in Barrett Esophagus
Numerous molecular alterations associated with the development of neoplasia within BE have been described.45-51 Many of these genetic alterations were used as the foundation for IHC to assist in the morphologic diagnosis and grading of dysplasia;45,48,49 Of these altered proteins, p53 is the only antibody with any established utility in the diagnosis of dysplasia; in fact, p53 immunoreactivity in BE is weak and focal over a morphologic spectrum of cases that includes reactive dysplasia, indeterminate for dysplasia, and low-grade dysplasia. Cells of high-grade epithelial dysplasia usually show strong and diffuse nuclear staining (Fig. 14-1).52 As a result, some authors have suggested that p53 nuclear reactivity can be used as an adjunct to the histologic evaluation of dysplasia in BE.53 The clinical utility of p53 is limited, however, because of substantial overlap of the patterns of p53 reactivity in epithelia negative for dysplasia, those that show reactive changes, and epithelia with low-grade dysplasia.26,54,55 It appears that p53 is most useful in biopsies distorted by crush artifact, tangential sectioning, ulceration, or fragmentation, and it helps to distinguish between BE with reactive cytologic features and high-grade epithelial dysplasia. However, the pathologist should not use p53 staining as a sole criterion to establish the diagnosis of dysplasia in BE. KEY DIAGNOSTIC POINTS Barrett Esophagus • Immunohistochemistry is not reliable for distinguishing Barrett esophagus from intestinal metaplasia of the gastric cardia. • Expression of p53 can help distinguish high-grade dysplasia from nondysplastic columnar mucosa if the result is extensively and strongly positive; however, p53 expression must be combined with histologic features to make a diagnosis of dysplasia.
Diagnostic Immunohistochemistry Epithelial Lesions of the Gastrointestinal Tract ESOPHAGUS Barrett Esophagus and the Gastric Cardia
Barrett esophagus (BE) is defined as intestinal metaplasia in association with endoscopically recognized
Esophageal Adenocarcinoma
Esophageal adenocarcinomas typically express cytokeratins AE1/AE3, CAM5.2, CK19, and CK7; a minority of cases express CK20. CDX-2 expression is variable, and although many tumors may show focal positivity, a significant minority are completely negative, and only a few are uniformly positive.56 Villin expression is more uniform and is present in approximately 75% of tumors.56
Diagnostic Immunohistochemistry
511
Esophageal Squamous Cell Carcinoma
A
B Figure 14-1 A, High-grade dysplasia in Barrett esophagus. B, Strong and diffuse nuclear staining with p53 is typically shown. However, overlap can be substantial in the patterns of p53 reactivity in epithelial cells that are reactive and in those that show lowgrade dysplasia.
Most studies have found that esophageal adenocarcinomas are immunophenotypically identical to adenocarcinomas of the gastric cardia (see below).57-60 Some50,61,62 but not all36 studies suggest that esophageal and gastric cardia adenocarcinomas have different CK7/CK20 staining patterns, based on statistical comparison of a large number of cases. However, the overlap in staining patterns between the two groups in these studies is substantial, resulting in limited use of CK7/CK20 staining to differentiate esophageal adenocarcinomas from proximal gastric adenocarcinomas.
SCCs generally stain strongly with medium-molecularweight and HMW cytokeratins; expression of lowmolecular-weight (LMW) keratins is typically weak (Fig. 14-2). Accordingly, most SCCs stain diffusely and strongly with CK antibodies CAM5.2, AE1/AE3, 34βE12, CK5/6, CK14, and CK19.63-71 Cytokeratin 34βE12 usually produces stronger and more diffuse staining than CK5/6 (Fig. 14-3). The intensity of CK19 expression increases with higher tumor grade. Approximately 70% of low-grade SCCs stain with CK19 in less than half of the neoplastic cells, whereas almost all high-grade neoplasms are diffusely and strongly reactive.66 Squamous carcinoma in situ is also positive for CK19, whereas benign squamous mucosa is negative (Fig. 14-4). Other diagnostically useful antibodies that are typically strongly positive are p63, which stains in a nuclear pattern,72 thrombomodulin, epithelial membrane antigen (EMA), and selected monoclonal carcinoembryonic antigens (CEAs).73,74 Nonreactive antibodies include CK7, CK20, 35βH11, BerEP4, thyroid transcription factor 1 (TTF1), and Wilms tumor 1 (WT1).70,71,75,76 Although CK7 is included in this group, approximately 15% to 30% of SCCs have occasional and scattered clusters of cells that are CK7 positive.66,67 However, if a positive result is defined as expression in more than 50% of tumor cells, esophageal SCCs are considered CK7/CK20 negative. Primary pulmonary SCC can occasionally be distinguished from esophageal SCC with TTF-1. Although both neoplasms are usually nonreactive, occasional pulmonary SCCs may show extensive and strong nuclear TTF-1 staining, whereas esophageal SCCs are consistently negative for TTF-1.77 It is occasionally important to distinguish between poorly differentiated SCC and poorly differentiated adenocarcinoma, and p63 and cytokeratins 7, 20, and 5/6 are useful in this context.67 SCCs, including poorly differentiated nonkeratinizing neoplasms, are CK7 and CK20 negative and positive with CK5/6 and p63, whereas adenocarcinomas of the esophagus, stomach, and lung are typically CK7 and CK20 positive but negative with CK5/6 and p63. Distinction between SCC and thymic carcinoma is also occasionally required. CD5 can be diffusely and strongly positive in primary thymic carcinoma and may be nonreactive in esophageal SCCs. Importantly, 100 80 60
11
0
βH 35
K2 C
K7 C
7 K1 C
2
K5 /7 C K1 9
C
E1
E3
34 β
1/ A
5. 2
0
AE
• Esophageal adenocarcinoma is immunophenotypically similar to proximal gastric adenocarcinoma. Currently, no reliable immunohistochemical panel is available to distinguish these two entities.
20
AM
Esophageal Adenocarcinoma
40
C
KEY DIAGNOSTIC POINTS
Figure 14-2 Cytokeratin reactivity in esophageal squamous cell carcinoma.
512
Immunohistology of the Gastrointestinal Tract
Squamous Cell Carcinoma Variants
A
B Figure 14-3 A, Esophageal squamous cell carcinoma stains diffusely and strongly with cytokeratin (CK) 34βE12. Inset: Central keratin whorl is clearly outlined against the strongly staining tumor cells that surround it. B, In contrast, CK5/6 staining is predominantly confined to the central keratinized cell whirls. Inset: Rare isolated tumor cells also stain strongly.
selective CD5 reactivity of thymic carcinomas is highly dependent on the pH of the antigen retrieval solution and the antibody clone. Some CD5 antibodies diffusely and strongly stain both thymic carcinomas and esophageal squamous carcinomas.78,79 Mesothelioma can occasionally be morphologically and clinically similar to poorly differentiated, nonkeratinizing, primary esophageal SCC. Both neoplasms are immunoreactive with calretinin and CK5/6, leaving WT1 as a positive diagnostic marker for mesothelioma.80
Basaloid Squamous Cell Carcinoma. Basaloid squamous cell carcinoma (BSCC) is a morphologic and genetic variant of poorly differentiated SCC.81 Mixed basaloid/classic SCC or neoplasms of mixed BSCC/ adenocarcinoma may be seen. Bcl-2 has been reported to stain BSCC, but it is nonreactive in poorly differentiated, conventional SCC.82 CK5/6, CK monoclonalOSCAR, CK13, CK14, CK19, AE1/3 and p63 typically are diffusely and strongly reactive in BSCC, whereas cytokeratins CAM5.2 and 35βH11 are often negative or weakly immunoreactive.83-89 Typically, the central cells within each nest are strongly positive for CK5/6 and p63. Moreover, the pseudopalisading, single cell layer at the periphery of carcinoma nests typically shows myoepithelial differentiation, including reactivity with CK19, S-100 protein, and smooth muscle actin (SMA).85 Similar to some high-grade breast carcinomas, IHC features of myoepithelial cell differentiation can be diffuse.90 Adenoid Cystic Carcinoma. Most reported esophageal adenoid cystic carcinomas are BSCCs; true adenoid cystic carcinomas of the esophagus are extremely rare.85 Esophageal salivary gland–type adenoid cystic carcinomas stain diffusely and strongly with CAM5.2 and AE1/ AE3. In addition, 34βE12 and CEA stain the ductaltype cells, whereas S-100, actin, and vimentin stain the basaloid-type cells.85-87 BSCCs with a solid growth pattern, those with a cribriform growth pattern that mimics adenoid cystic carcinoma, salivary gland-type adenoid cystic carcinomas, and high-grade NECs are often morphologically similar and may be difficult to separate, especially in small biopsy fragments. IHC is useful in this context (Fig. 14-5). CK5/6, CK7, 34βE12, CK19, p63, CEA, chromogranin, and synaptophysin are useful for distinguishing among these three entities.63,86 CK7 is often the single positive marker in high-grade NEC. Care should be given to avoid misinterpreting the nonspecific
KEY DIAGNOSTIC POINTS Esophageal SCC • SCCs stain strongly and diffusely with CAM5.2, AE1/3, CK5/6, and p63. • CK7, CK20, and CEA are either negative or focally positive in poorly differentiated SCCs, whereas these antibodies are strongly positive in poorly differentiated adenocarcinomas. • CD5 testing, if performed correctly, can be used to distinguish thymic carcinoma from esophageal SCC.
Figure 14-4 Cytokeratin 19 strongly stains the squamous cell carcinoma in situ on the right, whereas the benign epithelium on the left is nonreactive.
Diagnostic Immunohistochemistry
513
Helicobacter pylori infection. In patients with celiac disease, the density of surface intraepithelial lymphocytes (IELs) is usually lower than that seen in the duodenum. Gastric IELs are T lymphocytes that stain with CD45RO, CD3, CD7, CD8, and T cell–restricted intracellular antigen 1 (TIA-1).97,98
100 80 60 40 20 0 BSCC CK5/6, p63 CK7
ACC
HG NEC
CK19, 34βE12 Synaptophysin, chromogranin
Figure 14-5 Differential staining patterns of basaloid-patterned esophageal carcinomas. ACC, Adenoid cystic carcinoma; BSCC, basaloid squamous cell carcinoma; CK, cytokeratin; HG NEC, highgrade neuroendocrine carcinoma.
synaptophysin staining of necrotic debris found in BSCCs as true cytoplasmic granular immunoreactivity. KEY DIAGNOSTIC POINTS SCC Variants • Central areas or nests of BSCC are CK5/6 and p63 positive, whereas the peripheral rim of palisading cells may be CK19 positive. • Most adenoid cystic–like carcinomas are BSCCs. True adenoid cystic carcinomas of the esophagus are extremely rare. • High-grade (small cell) neuroendocrine carcinoma is diffusely synaptophysin and CK7 positive.
Esophageal Carcinomas with Spindle Cell or Mesenchymal Differentiation
Esophageal carcinomas can rarely show spindle cell or mesenchymal differentiation; however, the spindle cell component is often associated with recognizable epithelial differentiation. Not surprisingly, this mesenchymal differentiation shows decreased cytokeratin expression. The cytokeratin clones OSCAR and CK5/6 produce the strongest and most diffuse immunoreactivity. CK OSCAR is the preferred antibody for distinguishing neoplastic spindle cells from reactive myofibroblasts; CAM5.2, 35βH11, and AE1/AE3 are usually nonreactive.91-94 Among these three antibodies, AE1/AE3 produces the strongest and most diffuse staining.95 Neoplastic spindle cells can stain with actin antibodies; however, desmin is usually negative, providing the IHC distinction from leiomyosarcoma. True spindle cell rhabdomyosarcomatous differentiation can also occur, in which the cells stain with pan–muscle actin, desmin, and other markers of rhabdomyoblastic differentiation.91,96 STOMACH Nonneoplastic Conditions
Lymphocytic Gastritis. Lymphocytic gastritis is usually a manifestation of celiac disease and, occasionally, of
Helicobacter pylori. Proton-pump inhibitor and H. pylori eradication medications decrease the density of H. pylori organisms and alter their shape from spiral to coccoid.99-101 Coccoid-shaped H. pylori can be difficult to distinguish from small mucin globules or extracellular debris on modified Giemsa or other histochemical stains. IHC is a more reliable and sensitive method for detecting H. pylori, especially when the organisms are few in number, or when they are coccoid in shape (Fig. 14-6).100-105 Additionally, most H. pylori antibodies cross-react with H. heilmannii, which can occasionally be useful when the morphology of these organisms does not unequivocally allow for their identification on routine stains.106 Atrophic Gastritis, Autoimmune Type. IHC can be a useful adjunct in the diagnosis of autoimmune gastritis (AIG), most cases of which show hyperplasia of the enterochromaffin-like (ECL) cell compartment secondary to hypergastrinemia. This phenomenon can be highlighted using synaptophysin and/or chromogranin IHC. Normal mucosa shows occasional synaptophysin- and/or chromogranin-positive cells within the epithelial compartment. In AIG, intraepithelial linear arrays and/or extraepithelial nodules of synaptophysin- or chromograninpositive ECL cells may be seen (Fig. 14-7).107-109 In addition to the detection of ECL hyperplasia, IHC for gastrin can be helpful in separating atrophic “antralized” oxyntic mucosa, with complete loss of both parietal and chief cells, from true antral mucosa. In fully developed AIG, these two types of mucosa can be difficult to separate on routine histology. Gastrin-staining cells are present in the antrum and are absent in
Figure 14-6 Gastric antral mucosa with numerous Helicobacter pylori organisms. Many of the microorganisms are insinuated between the cell membranes of adjacent columnar mucus cells. The inset images across the top of the figure demonstrate the broad range of bacterial shapes that include coccoid, spiral, bacilliform, and barbell varieties.
514
Immunohistology of the Gastrointestinal Tract
A
B
C
D
Figure 14-7 Autoimmune gastritis (AIG). A, This biopsy from the gastric body lacks oxyntic glands; instead, the mucosa has the appearance of antral mucosa. However, this is not true antral mucosa; no G cells are present on the gastrin immunostain (not shown). B, Synaptophysin highlights enterochromaffin-like (ECL) cell hyperplasia within this atrophic body mucosa. Both forms of ECL cell hyperplasia, linear (more than five contiguous positive cells) and nodular (extraepithelial, small, round clusters), are present in this image. When interpreting immunostains in cases of possible AIG, it is important to ignore areas of intestinal metaplasia, because metaplastic G cells or EC cells can often be present within the intestinal-type epithelium, and they can stain with gastrin (C, arrows) and synaptophysin (D, arrows; true ECL cell hyperplasia is also present in the lower right of the figure).
atrophic oxyntic mucosa.109-111 Of note, when assessing gastric biopsies for the presence of body-predominant atrophic gastritis, three stains—synaptophysin, chromogranin, and gastrin—must be assessed in areas free of intestinal metaplasia, because the intestinal metaplasia contains its own NE cells that may be gastrin positive (see Fig. 14-7). Fundic Gland Polyps. Fundic gland polyps occur as sporadic lesions and may be associated with long-term
proton-pump inhibitor therapy. Additionally, fundic gland polyps may arise in association with familial adenomatous polyposis (FAP) and Zollinger-Ellison syndromes. Morphologic distinctions between the sporadic and syndromic polyps can be subtle. CK7 has been reported to stain sporadic polyps and Zollinger-Ellison syndrome–associated polyps, whereas β-catenin expression has been described in sporadic polyps but not in FAP-associated polyps.112-115 IHC is not routinely used to evaluate fundic gland polyps.
Diagnostic Immunohistochemistry
KEY DIAGNOSTIC POINTS Stomach: Nonneoplastic Conditions • T-cell markers can be used to highlight IELs in suspected cases of lymphocytic gastritis associated with celiac disease or Helicobacter pylori infection. • IHC staining for H. pylori is useful when treatmentassociated changes reduce the number of organisms or alter their normal appearance. • In cases of suspected AIG, gastrin IHC can be used to distinguish antral from atrophic body or fundic (oxyntic) mucosa; synaptophysin and chromogranin IHC is useful in the detection of enterochromaffin-like cell hyperplasia, a characteristic feature of AIG.
Gastric Adenocarcinoma
Although it is important for pathologists to characterize gastric adenocarcinomas into one of the two main morphologic subtypes, intestinal or diffuse/poorly cohesive/ signet-ring cell type,116 the IHC staining pattern of these two subtypes is similar. As a result, both types are discussed together in this sectio. Gastric adenocarcinomas stain with various cytokeratins (Fig. 14-8). The reactivity of several other antibodies is listed in Table 14-2, some of which will be discussed in more detail. Cytokeratins. Gastric adenocarcinomas are diffusely and strongly positive for AE1/AE3 and 35βH11.70 Cytokeratin CAM5.2 produces diffuse strong staining in approximately two thirds of these neoplasms and produces weak to moderate, patchy staining in the other third.117 Cytokeratins 18 and 19 are diffusely and strongly positive.50,118-121 Cytokeratin 7. CK7 expression is an important marker of committed gastric epithelial cells and gastric adenocarcinoma. Approximately 50% of gastric adenocarcinomas are strongly positive in a diffuse or patchy distribution; 30% have rare clusters of strongly reactive cells, and 20% are weakly positive or are negative (Fig. 14-9).118-120,122-125 Cytokeratin 20. Approximately 40% of gastric adenocarcinomas are strongly positive for CK20 in a patchy or diffuse distribution, 20% are weakly positive in a patchy distribution, and 40% are negative (Fig. 14-10).118,119,124-127
515
Cytokeratins 7 and 20 Coordinate Staining. Gastric adenocarcinoma is extremely heterogeneous in its CK7/20 coordinate staining patterns. The search for a predominant pattern has been complicated by the use of different cutoff points at which staining is considered positive. The percentage of positively staining cells in the literature ranges from 1% to 25%. Given these findings, no predominant pattern of coordinate CK7/20 staining is apparent; a significant minority of gastric adenocarcinomas stain with each of the four possible CK7/20 patterns. Approximately 35% of gastric adenocarcinomas are CK7 and CK20 positive, 25% are CK7 negative and CK20 positive, 25% are CK7 positive and CK20 negative, and 15% are CK7 and CK20 negative.1,118,119,124,128-132 These percentages vary by as much as 30%, depending on the cutoff point used in the study. CDX-2 and Villin. Several studies examined CDX-2 expression in gastric adenocarcinomas. CDX-2 appears to be variably expressed (percent positivity ranges from 20% to 90%), but even when present, its expression tends to be heterogeneous in diffuse-type cancers as compared with strong and diffuse staining in gland-forming adenocarcinomas.56,133-135 Villin also has variable expression and may be slightly more reliable than CDX-2.136 Apomucins. Overall, the various mucin stains may not be as helpful as the pathologist would like. The gastric mucin MUC5AC is positive but only in 38% to 70% of cases.134-138 MUC6, another gastric mucin, is only positive in 30% to 40% of cases,120,134-138 and MUC2 is positive in up to 50% of cases; MUC4 has inconsistent results in the literature, staining from 57% to 100% of cases, and MUC1 stains only a minority of cases.137,139 Estrogen and Progesterone Receptors. The issue of estrogen receptor (ER) positivity in gastric adenocarcinomas has been debated for many years. Faint ER staining of gastric adenocarcinomas was initially interpreted to be a false-positive reaction. Nuclear staining was later deemed a true positive, with the lack of staining considered to be a false-negative result. The IHC detection of low-level ER expression is dependent on the
KEY DIAGNOSTIC POINTS Gastric Adenocarcinoma
100 80 60 40 20
C K 35 18 βH C 11 AM AE 5.2 1/ AE 3 C K1 9 C K7 C K7 CK C +/C 20 K7 K 2 C –/C 0+ K7 K + 20 C /CK + K7 –/ 20– C K2 0– C K1 C 7 K5 34 /6 βE 12
0
Figure 14-8 Cytokeratin (CK) expression in gastric adenocarcinoma.
• Many cytokeratins stain gastric adenocarcinoma diffusely and strongly. • Gastric adenocarcinoma is immunophenotypically similar to esophageal adenocarcinoma, and no reliable immunohistochemical panel is currently available to distinguish these two entities. • The CK7/20 coordinate staining pattern is not useful in distinguishing gastric adenocarcinoma from other adenocarcinomas. • Weak estrogen receptor or progesterone receptor staining in a metastatic adenocarcinoma does not rule out a primary gastric carcinoma.
516
Immunohistology of the Gastrointestinal Tract
TABLE 14-2 Expression of Various Antibodies (Excluding Cytokeratins) in Gastric Adenocarcinomas Antibody
Reactivity
Comments
References
AMACR more commonly expressed in intestinal-type and high-grade dysplasia
458, 459
AMACR (p504s)
+
B72.3
+
117, 169
BerEP4
+
117
CA-125
S
CA 19-9
+
233
Calretinin
−
460
CD99
S
Staining confined to intestinal-type neoplasms
461
CDX-2
S
Heterogeneous and variable staining seen in 20% to 90% of cases
56, 133-135
CEA
+
Monoclonal antibody more discriminatory
60, 117
DAS-1
S
138
EMA
+
221
GCDFP-15
−
Rare (<1%) signet-ring cells can be positive
126, 171
HepPar1
S
High-grade and signet-ring cells can be focally positive
155, 156
Inhibin-α
−
462
MUC1
R
135, 137
MUC2
S
MUC4
S
MUC5AC
+
MUC6
S
p63
R
Patchy staining seen in high-grade and squamoid carcinomas
65
S-100
S
About 20% of neoplasms can have focal positivity
463
Single cells or small foci evident
Up to 50% of cases can be positive
118, 176
134-138 137, 139
38% to 70% of cases can be positive
134-138 120, 134-138
TTF-1
−
77, 252
Villin
S
56, 136
Vimentin
R
Positive staining seen in spindle cell (sarcomatoid) carcinomas
152, 154, 310
Reactivity: −, negative; R, rarely positive; S, sometimes positive; +, positive. AMACR, α-methylacyl-Co-A racemase; CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen; GCDFP-15, gross cystic disease fluid protein 15; TTF-1, thyroid transcription factor 1.
antibody clone and IHC procedure, but in most studies, gastric adenocarcinomas are negative for ER.135,138,140-144 Well-differentiated adenocarcinomas are reactive more often than are poorly differentiated and undifferentiated neoplasms.145 Gastric adenocarcinomas are also generally negative for progesterone receptor (PR), but a few studies have reported infrequent staining.126,138,141,146 Although typically negative, focal or diffuse weak staining with ER or PR does not exclude a gastric primary. Gastric Adenocarcinoma Variants
Lymphoepithelial-Like Carcinoma. Gastric lymphoepithelial-like carcinomas are undifferentiated (medullary type) carcinomas with a lymphocyte-rich stroma. These tumors are often associated with either EpsteinBarr virus (EBV, Fig. 14-11; this will be discussed later) or a high level of microsatellite instability (MSIH). The MSI designation characterizes a group of carcinomas that develop as a result of deficiencies
of the DNA MMR complex. Microsatellite-unstable adenocarcinomas can be syndromic, such as with hereditary nonpolyposis colorectal cancer (HNPCC), or they can be sporadic. Antibodies against MLH1, MSH2, MSH6, and PMS2, proteins of the DNA MMR complex, can detect MSI by their lack of staining in tumor cell nuclei (for a more complete discussion see “Colorectal Adenocarcinoma with Microsatellite Instability” below). Syndromic patients can show a loss of any of these antibodies, most commonly MSH2. Almost all MSI-H gastric adenocarcinomas are sporadic and show loss of MLH1 protein.147,148 Authors who have classified gastric adenocarcinomas according to cell type have found that tumors with a foveolar phenotype are often microsatellite unstable, whereas carcinomas with an intestinal phenotype are usually microsatellite stable. Intestinal-type carcinomas harbor deletions of tumor suppressor genes that can be demonstrated by staining with p53.149,150
Diagnostic Immunohistochemistry
A
Figure 14-9 A, Gastric adenocarcinoma, intestinal (glandular) type. Cytokeratin 7 (CK7) is diffusely and strongly reactive. B, Almost all of the gastric signet-ring cell adenocarcinoma cells strongly stain with CK7. C, The glandular region of this gastric adenocarcinoma (right) is strongly CK7 positive, whereas the adjacent signet-ring cells are negative to weakly reactive. Patchy or variegated CK7 staining is characteristic of gastric adenocarcinoma.
Spindle Cell Differentiation (Sarcomatoid Carcinoma). Similar to the colon and esophagus, spindle cell differentiation has been described in gastric carcinomas. These neoplasms usually stain with vimentin and EMA and stain variably with cytokeratin.151-154 See “Esophagus,” in the earlier section “Epithelial Lesions of the Gastrointestinal Tract,” for additional discussion. Yolk Sac, Hepatoid, and Choriocarcinomatous Differentiation. So-called yolk sac, clear cell, and hepatoid differentiation are frequent in gastric carcinomas. Although areas of yolk sac and/or hepatoid differentiation
A
517
B
C in an otherwise typical adenocarcinoma are common, pure tumors are rare. The morphology of these areas histologically resembles yolk sac or hepatocellular carcinoma. Single cells or small clusters of cells strongly stain with α-fetoprotein (AFP) in areas with yolk sac differentiation. Additionally, tumors with hepatoid differentiation may stain with HepPar1 in its typical, granular staining pattern.155-158 Focal immunoreactivity with HepPar1 and/or AFP can also be seen in otherwise typical intestinal-type and signet-ring cell adenocarcinomas.117,159,160 Thus AFP or HepPar1 staining alone is not sufficient for the diagnosis of yolk sac or hepatoid
B
Figure 14-10 A, Gastric signet-ring cells are relatively inconspicuous within the lamina propria. B, Cytokeratin 20 reveals the numerous neoplastic signet-ring cells, which were not easily seen on hematoxylin and eosin stain.
518
Immunohistology of the Gastrointestinal Tract
A
B
C
D
Figure 14-11 Lymphoepithelioma-like gastric carcinoma. A, The large neoplastic cells have prominent nucleoli and blend in with the background inflammatory cells. B, An AE1/AE3 cytokeratin stain highlights the carcinoma cells. C, In this neoplasm, MLH1 is lost in the tumor cell nuclei as compared with the intact (positive) staining of lymphocyte and stromal cell nuclei. D, In this different example, the tumor cell nuclei are positive for Epstein-Barr virus mRNA (in situ hybridization), and the inflammatory and stromal cells are negative.
differentiation. Additionally, yolk sac or hepatoid differentiation has no prognostic significance. Choriocarcinoma-like differentiation may also be present in otherwise usual-type adenocarcinomas. These foci usually stain with β–human chorionic gonadotropin (β-hCG) and placental alkaline phosphatase (PLAP). β-hCG staining of usual-type intestinal or signet-ring adenocarcinomas is common: approximately 33% stain strongly with polyclonal β-hCG, and 60% are immunoreactive with the monoclonal antibody.161-163 Gastric Adenocarcinoma with Neuroendocrine Differentiation. Typical gastric adenocarcinomas of either the intestinal or signet-ring cell type may stain with chromogranin and synaptophysin without histologic evidence of neuroendocrine differentiation.8,164,165 Staining with either antibody can be extensive in this setting and can be increased by using more sensitive methodologies.166 Staining with chromogranin or synaptophysin is so common that it could be considered within the normal immunophenotype of gastric adenocarcinoma. Gastric tumors without morphologic features of neuroendocrine differentiation that stain with synaptophysin or chromogranin should not be considered NECs but
rather adenocarcinomas that express neuroendocrine markers. Key Diagnostic Panels
Metastatic Breast Carcinoma Versus Primary Gastric Signet-Ring Cell Carcinoma. Antibodies: ER, MUC1, GCDFP-15, mammaglobin, monoclonal CEA, CDX-2/ villin, CK20, and HepPar1 (Fig. 14-12). Gastric signetring cell carcinoma and metastatic lobular breast carcinoma can be morphologically similar, and IHC can be useful in this differential diagnosis. Gross cystic disease fluid protein 15 (GCDFP-15) and mammaglobin are positive in approximately 50% and 75% of breast carcinomas, respectively,167 whereas these markers are negative in gastric carcinoma.126,168-172 Although several authors have reported ER positivity in gastric carcinoma, almost all recent studies have reported uniformly negative staining (see earlier discussion under “Gastric Adenocarcinoma”). The vast majority of lobular/signet-ring cell carcinomas of breast show positive ER staining in most of the cells.135,138,168 Expression of markers of intestinal differentiation, such as villin and CDX-2, are supportive of a gastric primary, whereas breast ductal carcinomas are villin and CDX-2
Diagnostic Immunohistochemistry
519
adenocarcinomas and are negative in gastric adenocarcinomas.177,178,181-183 Approximately 60% of gastric adenocarcinomas are CDX-2 positive, whereas lung adenocarcinomas, except the colloid variant, are CDX-2 negative.56,174,184,185
100 80 60 40 20
EA m
C
X2 D
Pa ep
Gastric Adenocarcinoma Variants
Gastric carcinoma
• Lymphoepithelial-like gastric carcinomas have a unique morphologic pattern, and most show either loss of MLH1 or expression of EBV by in situ hybridization. • Other morphologic variants of gastric carcinoma have been described that include spindle cell, yolk sac, hepatoid, and choriocarcinoma variants. These variants have a corresponding immunohistochemical staining pattern that helps to confirm the morphologic impression. • Some antibodies, such as AFP, HepPar1, and β-hCG, that stain morphologic variants such as AFP, HepPar1, and β-hCG can also stain the cells of morphologically typical adenocarcinomas. Immunostaining alone should not be used as evidence of variant differentiation without morphologic correlation. • Chromogranin and synaptophysin can also be positive in typical adenocarcinomas. Thus staining with neuroendocrine markers is not sufficient evidence for the diagnosis of neuroendocrine carcinoma.
Figure 14-12 Metastatic breast carcinoma versus gastric signetring cell carcinoma. ER, Estrogen receptor; CK, cytokeratin; GCDFP, gross cystic disease fluid protein; Mammo, mammoglobin; mCEA, monoclonal carcinoembryonic antigen.
negative.30,32,56,133,144,173,174 Gastric signet-ring cell carcinomas are often CK20 positive, compared with 2% of gastric carcinomas.137,138,144 Monoclonal CEA (mCEA) is diffusely positive in gastric adenocarcinoma and is negative in breast carcinoma. Other CEA antibodies share epitopes and can be positive in both neoplasms. Pancreatobiliary Versus Gastric Adenocarcinoma. Antibodies: CK17, CA 125. Substantial immunophenotypic overlap exists between pancreatobiliary and gastric adenocarcinomas. CK17 is expressed in as many as 88% of pancreatobiliary cancers but is only positive in 28% of gastric cancers.175 CA-125 is often present in less than 10% of cells in gastric carcinoma, and a tumor with more than 50% of cells staining would support a pancreatobiliary adenocarcinoma.176 Cytokeratins 7 and 20 do not play a role in the differential diagnosis, because the staining patterns are similar in both tumors.132 Lung Versus Gastric Adenocarcinoma. Antibodies: CK7, CK20, TTF-1, surfactant-A, Napsin-A, CDX-2 (Fig. 14-13). Primary pulmonary signet-ring cell carcinomas can be morphologically identical to gastric signet-ring cell adenocarcinoma.177 Cytokeratins 7 and 20 are only useful in differentiating between these entities when a neoplasm expresses the CK7negative/CK20-positive pattern seen in approximately 40% of gastric adenocarcinomas, compared with 0% of primary lung adenocarcinomas with signet-ring differentiation.122,124,125,127,132,170,177-180 TTF-1, surfactant-A, and napsin-A are positive in the majority of pulmonary 100 80 60 40 20 0 TTF-1 Lung carcinoma
CDX-2
KEY DIAGNOSTIC POINTS
H
Breast carcinoma
C
r1
0 K2
o
C
am
m
FP D C G
M
1 C U M
ER
0
CK7–/CK20+ Gastric carcinoma
Figure 14-13 Lung adenocarcinoma versus gastric signet-ring cell carcinoma. CK, Cytokeratin; TTF-1, thyroid transcription factor 1.
SMALL INTESTINE Celiac Disease
Celiac disease is one of the diseases that show increased numbers of IELs as a key histologic feature. In celiac disease, most IELs are activated T cells that include both alpha/beta and delta/gamma types. The dominant IEL T-cell immunophenotype in celiac disease is CD3 and CD8 positive.186 In general, staining for CD3 is not indicated for routine evaluation of duodenal biopsies for celiac disease. However, when the numbers of IELs are equivocally increased, or if a section is difficult to evaluate for technical reasons, CD3 IHC may be helpful. IHC for CD3 and CD8 is useful for risk stratification in patients with refractory celiac disease (for further discussion see “Theranostic Applications” below). Adenocarcinoma of the Small Intestine
Small intestinal adenocarcinomas stain diffusely and strongly with CK18 and CK19.120 In contrast to normal small intestinal epithelium, nonampullary small intestinal adenocarcinomas tend to develop CK7 expression and lose CK20 expression.120,187 Approximately two thirds of small intestinal adenocarcinomas coexpress both CK7 and CK20, and the remaining 33% are CK7 positive and CK20 negative; in one study, no tumors were CK7 negative and CK20 positive or CK7 and CK20 negative.187 They also stain with villin and CDX-2 in 60% to 70% of cases, a slightly lower rate than that of colorectal adenocarcinomas (Fig. 14-14).56,188 Approximately 50% stain with MUC1 or MUC2, and 38% stain with MUC5A.120,188 Alpha-methylacyl-Co-A racemase (AMACR) is rarely expressed in small intestinal adenocarcinomas, but it
520
Immunohistology of the Gastrointestinal Tract
A
B
C
D
Figure 14-14 A, Small intestinal adenocarcinoma (hematoxylin and eosin stain). This example strongly expresses cytokeratins 7 (B) and 20 (C) in addition to CDX-2 (D).
stains 62% of colorectal adenocarcinomas.189 The presence of CK7 staining and lack of AMACR staining can help differentiate small intestinal from colorectal adenocarcinoma. Similar to colon cancers, a minority of small bowel adenocarcinomas arise via the MSI pathway, but immunohistochemically, the vast majority of cases show loss of nuclear staining of the MMR protein MLH1 and preserved (intact) staining with MSH2.190,191 Adenocarcinomas that arise in the ampulla of Vater are discussed in Chapter 15. KEY DIAGNOSTIC POINTS Small Intestinal Adenocarcinomas • The majority (66%) of nonampullary small bowel adenocarcinomas are positive for CK7/20; this differs from colorectal adenocarcinomas, which tend to be CK7 negative and CK20 positive. • Fewer small intestinal adenocarcinomas (60% to 70%) express CDX-2 and villin, compared with colorectal adenocarcinomas. • CK7 and AMACR can help differentiate small intestinal adenocarcinomas (CK7+/AMACR−) from colorectal adenocarcinomas (CK7−/AMACR+).
APPENDIX, COLON, AND RECTUM Appendix
Some appendiceal epithelial neoplasms are similar to their colonic counterparts, whereas others are unique to the appendix. Similar to the colon, typical hyperplastic polyps and classic colonic-type adenomas do exist, but they are rare. Lesions with serrated, mucinous, or villous features, including sessile serrated adenomas with or without definite epithelial dysplasia, are more common. Some propose the use of the term low-grade appendiceal mucinous neoplasm to encompass sessile villous adenomas, cystadenomas, and serrated mucinous lesions, especially when these are associated with intraluminal mucin accumulation that may spread into the peritoneal cavity and cause the clinically recognized entity pseudomyxoma peritonei. These low-grade appendiceal mucinous neoplasms, as well as low-grade mucinous adenocarcinomas, have some different IHC staining characteristics compared with colorectal mucinous adenocarcinomas (Fig. 14-15).120,192-197 In addition to expressing CK20, approximately one third of mucinous lesions of the appendix coexpress CK7, with approximately 25% to 75% of cells staining; CDX-2 is also strongly reactive in the cell nuclei (Figs. 14-16 through
Diagnostic Immunohistochemistry
521
100 80 60 40 20 0 Appendix CK7
Colorectum CK20
CDX-2
Ovary MUC5AC
Pancreas MUC2
Figure 14-15 Immunophenotypes of mucinous adenocarcinoma. CK, Cytokeratin.
14-18). The proportion of CK7-positive cells in this group of lesions is substantially greater than the pattern of rare cells and the occasional cluster of positive cells that is typical of colorectal adenocarcinomas. More than 80% of appendiceal mucinous adenocarcinomas are MUC5A positive, which is similar to the proportion stained in gastric, pancreatic, and ovarian primary mucinous tumors (see Fig. 14-15). In contrast, as many as 33% of colorectal mucinous adenocarcinomas are MUC5A positive.194 Similar to colorectal mucinous adenocarcinomas, appendiceal mucinous tumors diffusely and strongly stain with cytokeratins 8, 13, 18, 19, and 20 and with MUC2, CDX-2, and DPC4.120,194,198,199 Nonmucinous, intestinal-type adenocarcinomas of the appendix are rare and are immunophenotypically similar to colonic adenocarcinomas. The IHC distinction of primary and metastatic mucinous adenocarcinomas can be problematic, especially when they involve the ovary.200,201 There can be overlap of the CK7/CK20 IHC profile of appendiceal and ovarian tumors.190,195 An antibody panel of CK7, CK20, CDX-2, MUC2, and MUC5A yields the most informative results (see Fig. 14-15). Lack of CK7 and diffuse, strong CK20 staining is supportive of a colorectal adenocarcinoma, whereas diffuse CK7 staining and nonreactive CK20 is supportive of a primary ovarian neoplasm.
Figure 14-16 Appendiceal mucinous neoplasm; low-grade, noninvasive. At higher magnification, the neoplastic cells are morphologically similar to those of so-called ovarian-primary mucinous borderline neoplasms.
Figure 14-17 Most appendiceal-primary mucinous neoplasms are cytokeratin 20 (CK20) positive (left) and are either negative or focally reactive with CK7 (right).
Even in cases in which focal or patchy staining of CK7 or CK20 is noted, the pattern of staining can be helpful. Ovarian tumors tend to show diffuse CK7 and patchy CK20 staining, whereas colorectal and appendiceal tumors tend to show patchy CK7 and diffuse CK20 staining.194 Strong and diffuse nuclear CDX-2 staining is supportive of a primary colorectal or appendiceal mucinous adenocarcinoma; CDX-2 reactivity in ovarian and pancreatic mucinous neoplasms is characteristically less intense and extensive.56,174,199,202 KEY DIAGNOSTIC POINTS Mucinous Appendiceal Neoplasms • Appendiceal mucinous neoplasms can be distinguished from colonic mucinous adenocarcinomas by their comparatively greater CK7 staining and MUC5A reactivity, whereas colonic neoplasms tend to be nonreactive. • An antibody panel of CK7, CK20, CDX-2, MUC2, and MUC5A can aid in the distinction between primary and metastatic mucinous adenocarcinomas.
Figure 14-18 CDX-2 is typically strongly reactive in the cell nuclei of appendiceal mucinous neoplasms.
Immunohistology of the Gastrointestinal Tract
There was little need to determine protein expression in colorectal polyps until the sessile serrated adenoma (SSA) was described as a new entity. Although SSAs have morphologic features similar to those of hyperplastic polyps (HPs), SSAs appear to arise via distinct genetic pathways, and they have different risks of progression to malignancy. Because our understanding of both SSAs and HPs is still evolving, the following summary of the immunophenotypic features of these polyps is based on a review of the current literature and is also likely to evolve over a short period of time. In one study, compared with typical (e.g., tubular or tubulovillous) adenomas, HPs, SSAs, and traditional serrated adenomas overexpressed MUC5AC, trefoil factor 1 (TFF1), and PDX-1. These lesions also showed preserved staining with MUC2 and had decreased expression of TFF3. CDX-2 was downregulated in HPs and SSAs.207 SSA-associated cancers may arise via the MSI pathway, a theory supported by the finding of loss of MLH1 and PMS2 immunoexpression in areas of dysplasia and cancer but not in adjacent SSA or normal mucosa.208 COX-2 is expressed in adenomas (typical adenomas and traditional serrated adenomas) but not in SSAs or HPs.209 Currently, no IHC marker can distinguish SSAs from HPs. However, one study found distinct patterns of CK7, CK20, and Ki-67 staining that may help distinguish SSAs from HPs,210 although this finding must be verified in larger studies before being used routinely. Dysplasia in Inflammatory Bowel Disease
Patients with long-standing inflammatory bowel disease (IBD) are at an increased risk for developing dysplasia and colorectal carcinoma. Surveillance colonoscopy with mucosal biopsies is currently the best and most widely used method to detect dysplasia and cancer in patients with IBD. However, for the pathologist, the assessment of dysplasia, especially in inflamed mucosa, can be challenging. IHC for p53 and Ki-67 can help confirm a histologic impression of dysplasia. Both p53 and Ki-67 tend to be overexpressed in areas of dysplasia compared with reactive and nonneoplastic epithelium.211-216 Similar to the assessment of dysplasia
Colorectal adenocarcinomas arise through different genetic pathways and should no longer be considered one disease. The majority of colorectal cancers arise via the chromosomal instability pathway with dysfunction of the adenomatous polyposis coli (APC)/βcatenin/Wnt signaling pathway. However, a subset of colorectal cancers arises via the MSI pathway, resulting from either a germline mutation (Lynch syndrome) or epigenetic gene silencing secondary to hypermethylation. Because all older studies on colorectal cancer viewed this cancer as one disease, the majority of this section describes the immunophenotype of colon cancer in general. But evidence is now emerging that the tumors that arise through these two main pathways have different immunophenotypic features. These differences are highlighted at the end of this section. Colorectal cancer cells contain mostly LMW cytokeratins, predominantly cytokeratins 8, 18, 19, and 20 (Fig. 14-19). They also stain with a broad molecularweight spectrum of antibodies that includes AE1/AE3 and CAM5.2.120,219-221 Cytokeratins 7 and 20. Most adenocarcinomas (80% to 100%) are diffusely and strongly positive for CK20, but some tumors will show only focal positivity or none at all (Fig. 14-20).120,127,219,222-224 Decreased CK20 staining occurs in microsatellite unstable adenocarcinomas. In general, colorectal adenocarcinomas infrequently express CK7 (~13% of cases); this frequency is independent of site (primary vs. metastatic) and mucinous subtype.120,124,125,129-131,222,224,225 On the basis of these staining patterns, colorectal adenocarcinoma is the major neoplasm that can be diffusely and strongly CK20 positive and completely CK7 negative. CDX-2. CDX-2 is a marker of intestinal differentiation and stains the nuclei of about 90% of colorectal adenocarcinomas, but the range of tumor positivity in the literature is variable, from 72% to 100%.56,174,226-228 100 80 60 40 20 0
C K K7 C 7 K – C /C 20 K7 K2 C +/C 0+ K7 K – 20 C /CK + K7 +/ 20– C K2 0– C K1 34 7 βE 1 C 2 K5 /6
Colonic Polyps
Colorectal Adenocarcinoma
C
The diagnosis of Hirschsprung disease relies on the histopathologic assessment of rectal biopsies. Acetylcholinesterase histochemistry can be used to highlight increased nerve fibers, but this method requires special tissue handling. Routine IHC can also aid in the diagnosis. Neuron-specific enolase (NSE), cathepsin D, Bcl-2, and calretinin, which normally stain ganglion cells, can be used to identify ganglion cells or the lack thereof.203-205 S-100 protein and NSE can be used to highlight hypertrophic nerve fibers within an aganglionic segment.206 In addition to staining ganglion cells, calretinin stains normal nerve fibers. The presence of calretinin-positive nerve fibers in the muscularis mucosae and superficial submucosa correlates strongly with the presence of ganglion cells and may be superior to acetylcholinesterase histochemistry.203
in BE, there can be overlap of the patterns of p53 and Ki-67 expression in reactive or inflamed epithelium. Surface involvement above the basal third of the crypts by these antibodies and strong p53 positivity support the presence of dysplasia.211,215,216 Increased AMACR expression also appears to be emerging as a useful marker of dysplasia and cancer in IBD.214,217,218
K1 C 8 K 35 19 βH C 11 AM AE 5. 1/ 2 AE 3
Hirschsprung Disease
C
522
Figure 14-19 Cytokeratin adenocarcinoma.
(CK)
expression
in
colorectal
Diagnostic Immunohistochemistry
Figure 14-20 The most common pattern of cytokeratin 20 staining in colorectal adenocarcinomas.
Similar to CK7, fewer poorly differentiated and mucinous colorectal adenocarcinomas are positive for CDX2,133 which may be related to MSI. In addition, CDX-2 is not specific for colorectal adenocarcinomas. Staining can be seen in adenocarcinomas of pancreatobiliary, gastric, small bowel, lung, ovarian (mucinous and endometrioid), and bladder origin, especially if they show intestinal differentiation.227-230 Villin. Villin stains the brush border of the intestines and is thus commonly positive in colorectal adenocarcinoma. The staining pattern is diffusely cytoplasmic with brush border accentuation and is seen in approximately 92% of cases (Fig. 14-21); however, villin expression is not as specific as CDX-2.30,31,33,56,228 It also stains other intestinal-type tumors, such as lung and bladder adenocarcinoma.30,33,228 Other Antibodies. Several other antibodies are typically positive in colorectal adenocarcinoma—such as MOC-31, mCEA, and COX-2—and these are shown in Table 14-3.20,169,222,231,232 CA 19-9 is positive in colorectal adenocarcinoma.225,233 Other antibodies such as CK5/6, MUC5AC, and CA-125 are typically negative (see Table 14-3).63,144,169,176 Colorectal Adenocarcinoma with Microsatellite Instability. Approximately 15% to 20% of colorectal
523
adenocarcinomas arise from deficiencies in the MMR complex function, resulting in a high level of microsatellite instability (MSI-H).234,235 Four main proteins— MLH1, MSH2, MSH6, and PMS2—comprise the DNA MMR complex. Carcinomas that arise via the MSI pathway tend to have characteristic pathologic features that include a right-sided location, patient age younger than 50 years, tumor-infiltrating lymphocytes, a lack of “dirty necrosis,” presence of peritumoral lymphoid aggregates (“Crohn-like reaction”), mucinous differentiation, medullary features, and/or a neoplasm that is well differentiated.234 In nonhereditary, sporadic MSI-H adenocarcinomas, hypermethylation of the MLH1 MMR promoter gene leads to deficiencies in MLH1 protein expression, resulting in loss of nuclear protein expression in the tumor cells. In hereditary adenocarcinomas (Lynch syndrome), germline mutations most commonly involve the MSH2 gene but can also involve the MLH1, MSH6, and PMS2 genes, resulting in loss of nuclear staining of the particular protein (Figs. 14-22 and 14-23).235,236 Antibodies to MLH1, MSH2, MSH6, and PMS2 are increasingly used to screen for Lynch syndrome and sporadic MSI, because detection of sporadic MSI-H tumors carries therapeutic and prognostic implications (see below).235,237,238 When interpreting these stains, it is important to note that MLH1 exists as a heterodimer with PMS2, and MSH2 heterodimerizes with MSH6. Thus if there is a defect in MLH1, PMS2 loss will be detected as well. However, if PMS2 protein expression is compromised, MLH1 will remain intact. The MSH2/ MSH6 pair shows a similar pattern of reactivity: an MSH6 gene defect causes MSH6 loss only, and MSH2 compromise causes loss of both MSH2 and MSH6.239 It is for this reason that some authors have proposed restricting the four-antibody panel to a two-antibody panel consisting of only PMS2 and MSH6.240,241 Complete loss of staining in tumor nuclei is required to report a complete loss of protein expression. Low or patchy levels of expression in tumor nuclei can be seen, depending on variable protein expression and levels of tissue fixation; and reduction, loss, or nucleolar expression of MSH6 has been noted in tumors that have been exposed to chemotherapy and/or radiation.242,243 Hence, if a two-antibody panel is used, any abnormal or equivocal staining should be confirmed by adding the
Figure 14-21 High and low magnifications of a mucinous colorectal adenocarcinoma stained with villin shows diffuse, homogeneous cytoplasmic immunoreactivity with a luminal, membranous, microvillous brush border accentuation. Although noncolonic adenocarcinomas can stain with villin, the microvillous brush border pattern is characteristic of colorectal adenocarcinoma.
524
Immunohistology of the Gastrointestinal Tract
TABLE 14-3 Expression of Various Antibodies (Excluding Cytokeratins) in Colorectal Adenocarcinomas Antibody
Reactivity
Comments
References
BerEP4
+
CA125
R
CA 19-9
S
169, 225, 233
Calretinin
R
460
CDX-2
+
56, 226-228, 303
COX-2
+
232
ER and PR
−
141, 144, 264
GCDFP-15
−
126, 169, 170
mCEA
+
60, 144, 169, 222, 232
MOC-31
+
20, 231
MUC1
S
120
MUC2
+
120, 144
MUC5AC
R
120, 144
S-100
R
Positive tumors usually show focal staining
463
Synaptophysin and chromogranin
R
Focal positivity can be seen in colonic adenocarcinomas without neuroendocrine features
8, 165, 315
TTF-1
−
144
Villin
+
30, 31, 33, 56, 228
117 Positive tumors usually show focal staining
118, 176
Reactivity: −, negative; R, rarely positive; S, sometimes positive; +, positive. COX-2, Cyclooxygenase 2; ER, estrogen receptor; GCDFP-15, gross cystic disease fluid protein 15; mCEA, monoclonal carcinoembryonic antigen; PR, progesterone receptor; TTF-1, thyroid transcription factor 1.
respective paired antibody and/or by confirming the MSI-H status by polymerase chain reaction (PCR). Another screening method is the use of molecular testing to detect MSI244; this is discussed further in the section “Beyond Immunohistochemistry.” Such screening methods can identify patients who should have additional genetic testing and counseling. Compared with typical glandular adenocarcinomas, signet-ring cell and mucinous adenocarcinomas are more often MSI-H and will have absent nuclear immunoreactivity with one of the MMR proteins. MSI-H
adenocarcinomas can aberrantly express CDX-2 and CK20. CK20 can be negative in as many as 32% of MSI-H colon cancers,132,245-247 whereas CDX-2 has been reported to be negative in 22% of MSI-H tumors.247 Interestingly, reduced or absent CDX-2 expression increases dramatically, to more than 85% of cases, when medullary-type MSI-H tumors are analyzed.245 As mentioned earlier, CK7 can be expressed in a minority of colon cancers, and this includes both MSI-H and microsatellite-stable tumors.247 To avoid potential erroneous diagnoses, especially when assessing small biopsy
Figure 14-22 High and low magnification of an invasive cecal adenocarcinoma from a 46-year-old patient with hereditary nonpolyposis colon cancer syndrome (Lynch syndrome). Neoplastic cell nuclei are completely devoid of MLH2 immunoreactivity, whereas cell nuclei of the surrounding stromal cells stain strongly. Only the complete absence of nuclear staining should be interpreted as a marker of a mismatch repair enzyme defect.
Diagnostic Immunohistochemistry
525
100 80 60 40 20 0 CK7
CK20 Colon
CDX-2
TTF1
Lung
Figure 14-24 Colorectal adenocarcinoma versus lung adenocarcinoma. CK, Cytokeratin; TTF1, thyroid transcription factor 1. Figure 14-23 Characteristic intact nuclear staining with MLH1 in a patient with a microsatellite-stable cancer. This pattern of staining would be present for all four mismatch repair protein antibodies: MLH1, MSH2, MSH6, and PMS2.
specimens and metastases of unknown origin, it is important to be aware that some cases of colorectal cancer can aberrantly express CK20, CDX-2, and/or CK7. Assessment of MSI can be helpful in such challenging cases. Adenocarcinoma Variants and Subtypes
Signet-Ring Cell Adenocarcinoma. In general, colonic signet-ring cell adenocarcinomas show similar CK7, CK20, and CDX-2 staining patterns compared with glandular colon adenocarcinomas.118,135 However, a higher percentage of signet-ring cell carcinomas are positive for MUC2 (100%) and MUC5AC (89%), and a lower percentage are positive for E-cadherin (56%).135 As noted earlier, microsatellite-unstable cancers with signet-ring cell morphology may show atypical expression of CK7, CK20, and CDX-2. Clear Cell Adenocarcinoma. Most clear cell adenocarcinomas are immunophenotypically identical to usualtype colorectal adenocarcinomas.248 Rare cases of primary colonic, clear cell carcinoma stain with AFP. Undifferentiated Neoplasms and Carcinomas with Rhabdoid Differentiation. These neoplasms are immunohistochemically similar to poorly differentiated carcinomas with spindle cell differentiation.249 The two most common GI locations for rhabdoid carcinomas are the colon and stomach.250 This variant most often occurs as a component within typical adenocarcinoma. KEY DIAGNOSTIC POINTS Colorectal Carcinomas • Colorectal adenocarcinomas are typically positive for CK20, CDX-2, and villin and are negative for CK7. • Stains for the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 can be used to screen for neoplasms with a high level of microsatellite instability (MSI-H). • MSI-H colonic adenocarcinomas can show decreased or absent CDX-2 and/or CK20 staining.
Key Diagnostic Panels
Colon Versus Lung Adenocarcinoma with a Microsatellite-Stable Colorectal Adenocarcinoma. Antibodies: CK7, CK20, CDX-2, TTF-1, and napsin-A (Fig. 14-24). Diffuse CK20 staining is strongly supportive of a colorectal adenocarcinoma, whereas diffuse, strong CK7 negative staining is strongly supportive of a lung adenocarcinoma.69 Focal staining with either antibody should be considered noncontributory, because it can be seen in either lung or colorectal adenocarcinomas.69,127,132,179 TTF-1, as well as surfactant-A, stains almost all low-grade nonmucinous pulmonary adenocarcinomas, compared with 0% of colon adenocarcinomas.181,251-257 Additionally, a relatively new antibody, napsin-A, has been shown to be very useful in this differential diagnosis, because it is expressed in only 2% of colorectal carcinomamas compared with approximately 87% of pulmonary adenocarcinomas in one study.183 Colorectal adenocarcinomas are CDX-2 positive, whereas nonmucinous pulmonary adenocarcinomas are CDX-2 negative.56,174 Primary pulmonary mucinous bronchioloalveolar and goblet cell adenocarcinomas are immunophenotypically different from usual-type pulmonary adenocarcinoma.32,258,259 This subset of adenocarcinomas can stain with CDX-2 and CK20, simulating metastatic colorectal adenocarcinoma.260,261 Importantly, the intensity and extent of CDX-2 staining in pulmonary mucinous adenocarcinomas is moderate and focal, unlike the diffuse, strong immunoreactivity seen in colorectal adenocarcinomas. Colorectal Adenocarcinoma Versus Müllerian Endometrioid Adenocarcinoma. Antibodies: CK7, CK20, CEA, CDX-2, ER, and Pax-8 (Fig. 14-25). CK7 is a useful initial antibody, because it is positive in more than 95% of endometrioid adenocarcinomas and is minimally reactive in colorectal adenocarcinomas. Conversely, CK20 is nonreactive in endometrioid adenocarcinomas and positive in colorectal adenocarcinomas.124,127,128,132,219,222, 262,263 An antibody panel that also includes mCEA,117,169,222,225 CDX-2,56,174 CA-125,169,176,225,263 and ER144,264 can aid in the distinction between these two lesions. Pax-8 is being increasingly used to distinguish endometrial from colorectal carcinoma, because it is positive in approximately 95% of müllerian endometrioid adenocarcinomas and is uniformly negative in colorectal adenocarcinomas.265
526
Immunohistology of the Gastrointestinal Tract
100
100
80
80
60
60
40
40
20
20
0
0 CK20
CDX-2
CEA Colon
CK7
CA125
ER
CDX-2
CA 19-9
Endometrioid
CEA Colon
CK20
PSA
Prostate
Figure 14-25 Colorectal adenocarcinoma versus müllerian endometrioid adenocarcinoma. CEA, Carcinoembryonic antigen; CK, cytokeratin; ER, estrogen receptor.
Figure 14-27 Colorectal adenocarcinoma vs. prostatic adenocarcinoma. CA, Carbohydrate antigen; CEA, carcinoembryonic antigen; CK, cytokeratin; PSA, prostate-specific antigen.
Colorectal Adenocarcinoma Versus Urothelial Carcinoma. Antibodies: CK7, CDX-2, p63, and thrombomodulin (Fig. 14-26). In this differential diagnosis, CDX-2 is currently the only antibody that definitively allows for the positive identification of colorectal adenocarcinoma, because it has been consistently negative in transitional cell carcinomas. Diffuse, strong CK7 and thrombomodulin staining are characteristic of many transitional cell carcinomas, and either is useful for supporting a transitional cell carcinoma diagnosis if positive.266 Many poorly differentiated transitional cell carcinomas undergo squamous differentiation and stain with CK5/6, 34βE12, and p63. Extensive staining with p63 in particular is supportive of a urothelial adenocarcinoma.267,268 The lack of staining should be interpreted as a noncontributory finding rather than supportive of a primary colonic neoplasm.
ANUS
Colorectal Adenocarcinoma Versus Prostate Adenocarcinoma. Antibodies: CDX-2, CA 19-9, CEA, CK20, and PSA (Fig. 14-27). CDX-2 and CA 19-9 stain colorectal adenocarcinomas and are nonreactive in prostate adenocarcinomas. CK20 and CEA support a diagnosis of colorectal adenocarcinoma only when they are diffusely and strongly reactive; both can focally stain high-grade prostatic adenocarcinomas. Prostate-specific antigen (PSA) is positive in prostatic adenocarcinoma and nonreactive in colorectal adenocarcinoma. Similar to other antibodies, the lack of staining with PSA should not be used to support the diagnosis of nonprostatic adenocarcinoma; many high-grade prostatic adenocarcinomas are PSA negative.127,132,233,269-271 100 80 60
Squamous Cell Carcinoma
Incidence of anal SCC is increasing in the United States, and these tumors share some similarities with their uterine cervical counterparts, including association with high-risk human papilloma virus (HPV) infection.272 Similar to other sites such as the head, neck, lung, and uterine cervix, CK5/6 and p63 are expressed in anal SCCs (Fig. 14-28, A-C).65,273 CK7 is usually negative, unless the carcinoma is a BSCC with an adenoid cystic pattern.274 Emerging evidence indicates two different histopathologic types of squamous cell in situ lesions/ invasive carcinomas of the anus.275 A bowenoid morphology of anal intraepithelial neoplasia (AIN) or invasive cancer is associated with positive p16 staining and lack of p53 staining (see Fig. 14-28, D-E), whereas a differentiated morphology that maintains squamous maturation is associated with positive p53 staining and lack of p16 staining. IHC for p16 is increasingly used to aid in the diagnosis of high-grade intraepithelial lesions associated with HPV. Full-thickness “blocklike” p16 expression in the context of suspicious cytologic features can be helpful in adjudicating lesions that can be difficult to categorize as low- or high-grade squamous intraepithelial lesions.276 Additionally, p16 can be useful when assessing resection margins that may be involved by cautery artifact. Anal Gland Adenocarcinoma
Anal gland adenocarcinoma is typically composed of small glands with scant mucin production. It invades the anorectal wall, has no identifiable intraluminal component, and has no association with a fistula.277 Anal gland adenocarcinoma is diffusely positive for CK7 and negative for CK20, CDX-2, p63, and CK5/6.277,278 Anal Paget Disease
40 20 0 CDX-2
p63 Colon
CK7
Thrombomodulin
Urothelial
Figure 14-26 Colorectal adenocarcinoma vs. urothelial carcinoma. CK, Cytokeratin.
The intraepidermal adenocarcinoma cells of anal Paget disease stain diffusely with AE1/AE3, CAM5.2, and CK7.279-281 Cytokeratin 7 is useful because it diffusely stains the neoplastic cells, whereas the surrounding normal squamous epithelium is nonreactive. GCDFP15 is also expressed in cases of primary anal Paget disease. When associated with an underlying rectal or urothelial adenocarcinoma, the pagetoid cells tend to
Diagnostic Immunohistochemistry
A
B
C
D
Figure 14-28 A, Anal squamous cell carcinoma (SCC) invading colonic mucosa (hematoxylin and eosin stain). As with SCCs at other sites, anal tumors are also strongly positive for cytokeratin 5/6 (B) and p63 (C). This example shows strong staining for p16 (D) and lack of overexpression of p53 (E).
coexpress CK20 and lack GCDFP-15 expression.282-284 MUC5AC and MUC1 stain most cases of anal Paget disease, whereas MUC2 is positive in cases with associated colorectal adenocarcinoma.285,286 CEA and BerEP4 are also expressed in the tumor cells.281-285
Neuroendocrine Lesions of the Gastrointestinal Tract Neuroendocrine tumors (NETs) arise from different types of neuroendocrine (NE) cells that are present
527
E
throughout the GI tract. At least 15 distinct NE cell types have been described. The biology and behavior of GI NETs is quite variable and is associated, in part, with the cell and site of origin. Because of these regional differences, GI NETs can be divided into foregut (stomach, duodenum, upper jejunum, and pancreas), midgut (lower jejunum, ileum, appendix, and cecum), and hindgut (colon and rectum) tumors.287 Because of the fact that small, benign-appearing NETs can metastasize, all NETs of the GI tract should be considered potentially malignant. The older term for these tumors is
528
Immunohistology of the Gastrointestinal Tract
KEY DIAGNOSTIC POINTS
KEY DIAGNOSTIC POINTS
Anal Malignancies
Esophageal Neuroendocrine Tumors
• Anal squamous cell carcinomas express CK5/6 and p63. • Immunohistochemisty for p16 can be useful in distinguishing low- from high-grade intraepithelial lesions associated with HPV infection. • Anal gland adenocarcinomas are CK7 positive and are negative for CK20, CDX-2, p63, and CK5/6. • Paget cells in primary anal disease are CK7 and GCDFP-15 positive and CK20 negative, whereas Paget cells associated with an underlying malignancy tend to be GCDFP-15 negative and positive for CK7, CK20, and MUC2.
• Synaptophysin and chromogranin are the immunohistochemistry mainstays of diagnosis. • Cytokeratin CAM5.2 is preferred over other cytokeratins.
carcinoid tumor, but this term is no longer commonly used and has been replaced by well-differentiated NET. In this chapter, the term neuroendocrine tumor represents a well-differentiated lesion; all poorly differentiated, high-grade lesions are noted as NECs. The general immunophenotype of GI NETs is similar to NETs elsewhere in the body. They are positive for LMWCKs (CAM5.2) and often, but not always, HMWCKs (AE1/AE3). CK20 is positive in as many as 25% of GI NETs, and CK7 is positive in only 11% of cases.288 The most commonly used antibodies to detect NE differentiation are synaptophysin and chromogranin A. Other useful, positively staining antibodies include CD56 (neural cell-adhesion molecule [N-CAM]), CD57 (Leu-7), and NSE. NESP-55, a member of the chromogranin family, is a promising marker of pancreatic neuroendocrine tumors (PanNETs), but it is typically negative in GI NETs.289 Some grading schemes, including that of the World Health Organization (WHO), use Ki-67 to categorize GI NETs.290 NETs at each specific site in the GI tract are now discussed along with sitespecific IHC stains that can be used to detect them. NEUROENDOCRINE TUMORS OF THE ESOPHAGUS
Most esophageal NE lesions are high-grade, poorly differentiated NECs that are often the large cell, rather than the small cell, type and may have a component of adenocarcinoma.291 Well-differentiated NETs often occur as small polypoid lesions in association with BE and may be found incidentally.292,293 Primary high-grade small cell NECs are rare and occur mainly as a component of an SCC.294-296 Immunophenotypically, NETs stain diffusely and strongly with synaptophysin and chromogranin, whereas high-grade, large cell carcinomas may only be positive for synaptophysin.293 Small cell carcinomas that arise in association with SCC may express CEA,296 and as many as half of esophageal small cell carcinomas may be immunoreactive with TTF-1.297,298 NEUROENDOCRINE TUMORS OF THE STOMACH
Three types of gastric NETs have been described: 1) those that are physiologic and arise in the setting of
autoimmune gastritis (type I), 2) those that arise in association with multiple endocrine neoplasia type I (MEN 1) or Zollinger-Ellison syndrome (type II); and 3) those that are sporadic (type III). It is important to distinguish type I from type III NETs, because type I tumors have nearly no metastatic risk, whereas type III tumors are much more aggressive.293 Poorly differentiated NECs are rare but could be considered the fourth type of gastric NET, and some may arise as a component of gastric adenocarcinoma. The cell of origin for type I, type II, and the vast majority of type III NETs are the enterochromaffin-like (ECL) cells that reside within oxyntic mucosa. These cells stain strongly for synaptophysin and chromogranin and will stain for vesicular monoamine transporter 2 (VMAT2), which recognizes these histamine-producing cells.293,299 Synaptophysin and chromogranin will also detect ECL-cell hyperplasia in the setting of AIG (see Fig. 14-7). Rare, poorly differentiated carcinomas are positive for synaptophysin but may be negative for chromogranin. In general, gastric carcinoids are positive for AE1/AE3 and CAM5.2, approximately 10% are positive for CK7, and they are negative for CDX-2 and CK20.226,288,300
KEY DIAGNOSTIC POINTS Gastric Neuroendocrine Tumors • Synaptophysin and chromogranin A stain the ECL cells that give rise to the majority of gastric NETs. • Synaptophysin and chromogranin A also highlight the proliferation of ECL cells in the oxyntic (fundic/body) mucosa in hypergastrinemia, hypochlorhydria, and ZollingerEllison syndrome. • The positive staining for AE1/AE3 and CAM5.2 seen in gastric carcinoids can help distinguish them from secondary involvement of the gastrointestinal tract by other NETs such as pheochromocytoma and retroperitoneal paraganglioma.
NEUROENDOCRINE TUMORS OF THE SMALL BOWEL
NETs of the small bowel have different etiologies and prognoses, depending on the site, and are thus typically divided into two groups by location: duodenal/proximal jejunal and distal jejunal/ileal NETs. Both groups of neoplasms are usually well differentiated and stain with synaptophysin and chromogranin. They tend to be CK7 and TTF-1 negative, and approximately 20% are CK20 positive.288,301
Diagnostic Immunohistochemistry
Most duodenal NETs are gastrin positive (gastrinomas), and a subset are functionally active and result in the Zollinger-Ellison syndrome (Fig. 14-29). The most common site of gastrinomas is the duodenal bulb. Approximately 20% of duodenal NETs produce, and stain for, somatostatin. Approximately one half of these tumors lack chromogranin A positivity but do stain with synaptophysin. Somatostatin-producing tumors tend to arise near the papilla of Vater. Duodenal NETs are positive for the pancreatic-duodenal homeobox 1 transcription factor PDX-1302 and tend to be negative (or weakly staining) for CDX-2 (see Fig. 14-29).302,303 Gangliocytic paragangliomas are a rare form of NET that tend to arise in the second portion of the
A
C
529
duodenum. Cytokeratins (CAM5.2), synaptophysin, and chromogranin will stain the nests of epithelial/ endocrine cells, S-100 protein will stain the neurally derived spindle cells and the ganglion cells, and neurofilament protein (NFP) would also stain the ganglion cells, if needed. Poorly differentiated NETs do exist and tend to involve the papilla of Vater. Most NETs of the distal small bowel occur in the terminal ileum and arise from serotonin-producing enterochromaffin (EC) cells. Because CDX-2 is positive in EC cells, nearly all terminal ileal NETs express it (see Fig. 14-29).303-305 These distal small bowel NETs are negative for PDX-1.302 Even at small sizes (1 to 2 cm), these are aggressive tumors often seen initially with
B
D
Figure 14-29 Gastrointestinal well-differentiated neuroendocrine tumors (NETs). A, Duodenal NETs are usually positive for gastrin. B, They lack staining for CDX-2. Terminal ileal NETs (C, hematoxylin and eosin [H&E] stain) are positive for CDX-2 (D). Continued
530
Immunohistology of the Gastrointestinal Tract
E
F Figure 14-29, cont’d Rectal NETs (E, H&E stain) are negative for CDX-2 (F).
lymph node and hepatic metastases. When these tumors metastasize to the liver, carcinoid syndrome can develop.
KEY DIAGNOSTIC POINTS Small Bowel Neuroendocrine Tumors • Proximal small bowel NETs that arise in the duodenum and proximal jejunum tend to behave less aggressively and have different immunophenotypes than distal small bowel tumors that arise in the terminal ileum. • Duodenal tumors often produce gastrin, are positive for PDX-1, and are negative for CDX-2; somatostatin-producing tumors are often found at the papilla of Vater. • Terminal ileal NETs are positive for CDX-2, and about two thirds are positive for CEA; they are negative for PDX-1.
NEUROENDOCRINE TUMORS OF THE APPENDIX
Well-differentiated NETs of the appendix tend to arise in the tip, are usually small (<1 cm) and found incidentally, and have little to no chance of metastasizing. Occasionally, well-differentiated NETs can occur toward the base of the appendix and grow to a larger size; these tumors should be considered potentially malignant, as is the case with most other NETs of the GI tract. Similar to ileal NETs, appendiceal NETs typically arise from EC cells and, as such, they are positive for CDX-2.302-304 These well-differentiated NETs tend to be negative for both CK7 and CK20,306 but they often have S-100
protein–positive sustentacular cells around the cell nests.306-308 Appendiceal goblet cell carcinoids have a mixed phenotype that shows both NE and glandular differentiation. Although these tumors stain for the NE markers synaptophysin and chromogranin, the pattern is more focal: only 5% to 25% of the cells stain, as compared with the more diffuse staining of well-differentiated NETs.309 Most goblet cell carcinoid tumors are positive for CK20, and as many as 70% are positive for CK7 (Fig. 14-30).306 They show preserved staining with β-catenin and E-cadherin, are positive for MUC2, and are negative for MUC1 and p53.309 Although typical appendiceal NETs are generally negative for CEA, goblet cell carcinoid tumors are positive.307 When the goblet cell carcinoid tumors are associated with a poorly differentiated adenocarcinoma component, known as mixed adenoneuroendocrine carcinoma (MANEC), the poorly differentiated cells stain for p53 and MUC1 and lose MUC2 staining.309 KEY DIAGNOSTIC POINTS Appendiceal Neuroendocrine Tumors • Typical well-differentiated appendiceal NETs (carcinoid tumors) are CDX-2 positive and negative with CK7/20. • Goblet cell carcinoids differ from typical appendiceal NETs and show positive staining for CK7, CK20, MUC2, and CEA and only focal staining with synaptophysin and chromogranin A. • Both goblet cell and typical appendiceal carcinoids stain with E-cadherin and β-catenin, unlike the more aggressive colorectal signet-ring cell carcinomas.
Diagnostic Immunohistochemistry
A
B
C
D
531
Figure 14-30 Appendiceal goblet cell carcinoid. A, Neoplastic goblet-shaped cells are present beneath two normal appendiceal crypts (hematoxylin and eosin stain). B, Goblet cell carcinoids are typically positive for cytokeratin 20 (CK20). C, Left: CK7 staining can be variable, note scattered cells positive for CK7 and a separate cluster of tumor cells that are completely negative. Right: CDX-2 is positive. D, Staining with synaptophysin (shown) and chromogranin (not shown) can be variably positive.
NEUROENDOCRINE TUMORS OF THE COLON AND RECTUM
Most well-differentiated NETs of the colorectum occur in the rectum. Rectal (hindgut) NETs are positive for synaptophysin and prostatic acid phosphatase and are usually negative for chromogranin.310-313 Unlike adenocarcinomas of the prostate, hindgut carcinoids do not stain with PSA or P504S.270 CEA weakly stains approximately 25% of these tumors, glucagon is positive in
KEY DIAGNOSTIC POINTS
approximately 10%,311 and CDX-2 is negative or shows only weak staining in a small percentage of cells (see Fig. 14-29).302-304 NETs can occur in the colon, and when they do, they tend to be right sided. The rightsided tumors are midgut tumors that have similar immunophenotypic profiles as ileal NETs. KEY DIAGNOSTIC PANEL FOR METASTATIC WELL-DIFFERENTIATED NEUROENDOCRINE TUMORS OF UNKNOWN PRIMARY
An IHC panel with CDX-2, PDX-1, NESP-55, and TTF-1 can be used to distinguish GI well-differentiated NETs from pancreatic endocrine and pulmonary carcinoid tumors (Fig. 14-31).302
Colorectal Neuroendocrine Tumors • Hindgut (rectal) NETs are positive for synaptophysin and prostatic acid phosphatase and typically lack staining for chromogranin and CDX-2. • Cecal and other right-sided NETs have a similar immunophenotypic profile to other midgut NETs such as ileal NETs.
HIGH-GRADE (POORLY DIFFERENTIATED) NEUROENDOCRINE CARCINOMAS
High-grade NECs of the GI tract are rare. Those that arise in the esophagus are described above under “Neuroendocrine Tumors of the Esophagus,” because most NETs of the esophagus are high-grade tumors.
532
Immunohistology of the Gastrointestinal Tract
Mesenchymal Lesions of the Gastrointestinal Tract
100 80 60 40
MESENCHYMAL LESIONS PRESENTING AS MURAL MASSES
20 0
CDX-2
PDX1
ng Lu
as nc Pa
R
ec
re
tu m
ix Ap
pe
nd
um Ile
nu de uo
D
St om
ac
h
m
Gastrointestinal Stromal Tumor
NESP-55
TTF1
Figure 14-31 Well-differentiated neuroendocrine tumors. TTF1, Thyroid transcription factor 1. (Modified from Srivastava A, Hornick JL: Immunohistochemical staining for CDX-2, PDX-1, NESP-55, and TTF-1 can help distinguish gastrointestinal carcinoid tumors from pancreatic endocrine and pulmonary carcinoid tumors. Am J Surg Pathol 2009;33:626-632.)
Primary high-grade NECs of the stomach are positive for synaptophysin and may be focally positive for chromogranin. High-grade NECs of the stomach can also occur in conjunction with, and as a component of, typical gastric adenocarcinoma. The morphologic features of a NEC, often the small cell type, must be present, in contrast to usual-type gastric adenocarcinomas with IHC features of NE differentiation, discussed earlier. It is best to require synaptophysin and/or chromogranin immunoreactivity in such a neoplasm to avoid confusing crushed, usual-type adenocarcinoma cells with true NE differentiation. These NE neoplasms are CK20 and CK5/6 negative, and they rarely stain with TTF-1.63,298,314 High-grade, poorly differentiated NECs of the duodenum occur primarily in the region of the papilla of Vater and are covered in detail in Chapter 15. High-grade NECs in the colon and rectum most commonly occur as a component of poorly differentiated adenocarcinomas; more than 50% of the tumor should show NE differentiation to be called NEC.315 Both small cell and non–small cell types exist, and both tend to stain with LMWCK (CAM5.2) in a perinuclear dot pattern, as well as with synaptophysin, chromogranin, and/or NSE (Fig. 14-32).315,316 CD117 stains a substantial minority of high-grade NECs; however, immunoreactivity has not been linked to KIT juxtamembrane (exon 11) mutations.317,318
CD117 and KIT Gene. The classification and diagnosis of GISTs radically changed with the recognition of the KIT protooncogene mutation as the key molecular event in gastric GISTs. In more than 85% of GISTs, KIT or platelet-derived growth factor alpha (PDGFRA) mutations are detected.319-325 The KIT mutation, most often in exon 11, results in constitutive activation of the KIT receptor, which is thought to promote proliferation and/or decrease apoptosis. The KIT protein can be immunohistochemically detected with antibodies to KIT which has been assigned to the 117 cluster designation (CD) antigen group. Activating mutations of KIT and CD117 immunostaining have been identified in neoplasms previously classified as leiomyomas, leiomyosarcomas, gastric autonomic nerve tumors, and some schwannomas; this has resulted in unification and substantial simplification of this group of neoplasms.326-329 CD117 also stains interstitial cells of Cajal, which are involved in the regulation of gut motility, leading to the suggestion that GISTs are derived from this cell type.330,331 Although KIT gene mutations almost always result in CD117 immunoreactivity, CD117-positive lesions do not invariably indicate a KIT mutation. For example, approximately 25% of neurofibromatosis type I (NF1) patients develop GISTs that stain with CD117 and CD34 but lack KIT gene mutations. Instead, these lesions have the typical NF1 (17q11.2) gene mutations found in NF1-associated neurofibromas.327 CD117 Staining in Gastrointestinal Stromal Tumors. CD117 stains approximately 95% of GISTs, and its immunoreactivity in GISTs is typically strongly and uniformly positive in a cytoplasmic pattern (Fig. 14-33), although a membranous pattern of staining can be seen. Additionally, approximately 50% of GISTs can show a
KEY DIAGNOSTIC POINTS High-Grade (Poorly Differentiated) Neuroendocrine Carcinomas • High-grade (poorly differentiated) small cell and non–small cell NECs can occur throughout the gastrointestinal tract but are rare. • In the stomach, they can occur as a primary neoplasm or in conjunction with an adenocarcinoma; in the colon, they tend to occur in association with an adenocarcinoma. • By definition, they must have morphologic features of a NEC and must stain with neuroendocrine markers, such as synaptophysin or chromogranin.
Figure 14-32 High-grade colonic neuroendocrine carcinoma with focal, moderate synaptophysin reactivity.
Diagnostic Immunohistochemistry
A
B
C
D
533
Figure 14-33 A, Gastric gastrointestinal stromal tumor (hematoxylin and eosin stain). B, The neoplastic cells stain strongly and diffusely with CD117. Inset: The perivascular connective tissue stroma is negative. C, Mast cells also stain strongly for CD117 and provide a positive internal control. D, Most GISTs (90%) are negative for desmin, whereas the fibers of the muscularis mucosae are strongly positive.
dotlike pattern of expression; these tumors are more likely to be extraintestinal in location and may show an epithelioid pattern of growth.332 The absence of CD117 staining in GISTs occurs in approximately 5% of cases and is strongly associated with a mutated PDGFRA gene.333,334 These CD117-negative GISTs typically have an epithelioid morphology and occur in the omentum or peritoneum.327,335-337 CD117 in Other Lesions. CD117 immunoreactivity is not restricted to GISTs and has been reported in a number of other neoplasms including melanoma, renal cell carcinoma, and seminoma.338-340 Sarcomas that have been reported to stain with KIT include angiosarcoma, clear cell sarcoma, Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), neuroblastoma, and Kaposi sarcoma.341-343 In some series, intraabdominal desmoidtype fibromatosis was reported to stain with KIT in as many as 75% of cases; typically this pattern of expression was noted to be weak and granular.332,344 More recently, however, it has been shown that the use of optimal primary antibody dilution and lack of the antigen retrieval step resulted in only minimal cytoplasmic staining in 5% of intraabdominal desmoid tumors.341,345,346
DOG-1 Staining in Gastrointestinal Stromal Tumors. Originally appreciated by using DNA microarray studies, DOG-1 (Discovered On GIST-1) was noted to be highly expressed in GISTs by using IHC.347 Studies have shown that this antibody is highly specific for GISTs and stains approximately half of CD117-negative GISTs.16 DOG-1 is uncommonly expressed in synovial sarcoma and benign smooth muscle neoplasms, both of which are in the differential diagnosis of GIST. Interestingly, DOG-1 is not expressed in many of the lesions that are CD117 positive, such as seminoma and melanoma.348 Other Antibodies. GISTs have variable degrees of neural or smooth muscle differentiation.327,331,349-365 CD34 strongly and diffusely stains approximately 70% of GISTs; the percentage of CD34-positive tumors varies by location, and 47% of small intestinal GISTs, 96% of rectal GISTs, and 100% of esophageal GISTs show CD34 expression.332,355,366 In GISTs with neural differentiation, fewer cells stain positively for CD34, and the intensity of staining is lower, compared with GISTs with smooth muscle differentiation. CD34 is not specific for GISTs; other CD34-positive neoplasms
534
Immunohistology of the Gastrointestinal Tract
include solitary fibrous tumors (SFTs), inflammatory fibroid polyps, and dedifferentiated liposarcoma. CD99 is also diffusely and strongly positive in most GISTs. Muscle-specific actin (HHF-35) and smooth muscle actins stain strongly in 10% to 47% of cases. As with CD34, the likelihood of actin expression in GISTs varies by location; as many as 47% of small intestinal and 10% to 13% of rectal tumors are positive for actin.355,367 As many as 10% of GISTs stain with desmin, and desminpositive GISTs are more likely to show an epithelioid morphology compared with the typical spindle cell pattern of growth.346,367-369 Additionally, other markers of smooth muscle differentiation, such as h-caldesmon, commonly stain GISTs.370 Cytoplasmic and nuclear expression of S-100 protein is restricted to 5% to 10% of GISTs.355,367,368,371 Typically, those GISTs that are S-100 positive are present in the small intestine.355 Cytokeratins, most commonly CAM5.2 and 35βH11, stain GISTs in a patchy pattern, which occasionally can be of strong intensity. Cytokeratin AE1/AE3 is usually less intense and stains rare individual cells and small clusters of cells. Signet-ring GISTs are negative with cytokeratin antibodies. Synaptophysin can be strongly and diffusely positive in gastric GISTs, but chromogranin is nonreactive.
KEY DIAGNOSTIC POINTS Gastrointestinal Stromal Tumors • CD117 is typically expressed when mutation of the KIT gene results in elevated KIT protein, a sensitive marker for GISTs. • CD117 may be negative in GISTs, owing to either limited tumor sampling or, rarely (~5%), a unique subset of CD117-negative GISTs that typically show an epithelioid morphology and are more likely to harbor a PDGFRA mutation. • Although CD117 is the mainstay of immunohistochemistry for diagnosis of GISTs, these tumors may also stain for other antigens, including DOG1, CD34, CD99, HHF-35, smooth muscle actins (SMAs), S-100 protein, and lowmolecular-weight cytokeratins.
A
Other Mesenchymal Lesions Presenting as Mural Masses
Schwannoma. Schwannomas of the GI tract are uncommon neoplasms that share morphologic features with GISTs.356,359,372,373 Schwannomas are uniformly diffusely and strongly positive in both a nuclear and cytoplasmic pattern for S-100 protein. Other positive markers in schwannomas include CD57 (Leu-7) and GFAP. They do not stain with CD117, CD34, or smooth muscle actin (SMA). Granular Cell Tumor. Granular cell tumors of the GI tract typically occur as mucosal or submucosal masses and are most common within the esophagus. These tumors have an immunophenotype identical to granular cell tumors of other sites. S-100 protein, CD57, and vimentin are usually strongly and diffusely reactive. The pattern of desmin staining in granular cell tumors can be useful in distinguishing these neoplasms from smooth muscle tumors with granular cytoplasmic change. Desmin is usually negative or weakly positive in rare cells in granular cell tumors but is strongly and diffusely positive in leiomyomas. Cytokeratin, EMA, and mCEA are negative.374,375 Desmoid-Type Fibromatosis. Desmoid-type fibromatosis is rarely CD117 positive.371 In difficult cases, nuclear staining for β-catenin can be useful in separating desmoidtype fibromatosis from other tumors, because β-catenin shows nuclear expression in approximately 75% of desmoid tumors (Fig. 14-34).376-379 Of note, many tumors in the differential diagnosis of desmoid-type fibromatosis may show cytoplasmic staining for β-catenin, thus only a nuclear pattern of staining should be considered a significant finding. CD34 can stain lesional cells weakly and in a patchy distribution, and it is typically less intense than CD34 staining seen in SFTs. Most desmoid tumors are reactive with SMA and desmin.355 Solitary Fibrous Tumor and Pseudotumor. Solitary fibrous tumors infrequently arise in the upper abdomen and mesentery.355,380-382 They are positive with CD34, CD99, and SMA but do not stain with CD117.355,373,383
B Figure 14-34 A, Desmoid-type fibromatosis (hematoxylin and eosin stain). B, The nuclei are positive for β-catenin.
Diagnostic Immunohistochemistry
Also, approximately 25% of SFTs stain with β-catenin in a nuclear pattern.378 Nodular fibrous pseudotumors are rare lesions in this location. They share strong CD117 reactivity with GISTs although they are morphologically distinct from GISTs. Smooth Muscle Neoplasms. The distal third of the esophagus is a common site for leiomyomas; these neoplasms stain diffusely and strongly with desmin and actins, but they are negative for CD34 and CD117.353,357 Most smooth muscle neoplasms that arise as mural lesions outside of the esophagus are classified as leiomyosarcomas. These tumors stain with SMA in 63% to 100% of cases151,332,384,385 and with desmin in 33% to 100% of cases.332,386-388 The degree of desmin and SMA expression decreases with increased grade of the tumor. Leiomyosarcomas are negative for KIT, CD34, β-catenin, and S-100 protein. Melanoma. The small intestine is a common metastatic site of cutaneous melanomas; primary melanomas rarely arise in the GI tract and are most common in the anorectum and esophagus. Their immunophenotype is identical to their cutaneous counterparts; they stain with vimentin, S-100 protein, human melanoma black 45 (HMB-45), melan-A, and tyrosinase (Fig. 14-35).389-391 As noted earlier, KIT may be positive in a subset of melanomas.
535
Differential Diagnosis
The IHC differential diagnosis of a mesenchymal neoplasm in the upper abdomen includes GIST, schwannoma, SFT, desmoid-type fibromatosis, and leiomyosarcoma.355,392 These lesions can share many morphologic features, especially in fine-needle core biopsy specimens. The antibodies CD117, CD34, S-100, GFAP, CD99, β-catenin, and desmin provide a useful diagnostic antibody panel (Fig. 14-36).
KEY DIAGNOSTIC POINTS Mural Mesenchymal Lesions • Immunohistochemistry aids in separating many of the lesions that may show morphologic overlap with GIST, including schwannoma, desmoid-type fibromatosis, solitary fibrous tumor, and leiomyosarcoma. • A panel of antibodies that includes CD117, CD34, S-100 protein, GFAP, CD99, β-catenin, and desmin is useful in the diagnosis of these mesenchymal lesions. • SMA expression decreases with increased grade of tumor. • Leiomyosarcomas are negative for KIT, CD34, β-catenin, and S-100 protein.
A
B Figure 14-35 A, Esophageal melanoma. Subjacent to a normal squamous mucosa, undifferentiated, dyshesive neoplastic cells expand the submucosa (inset, higher magnification). B, Esophageal melanoma. Tyrosinase diffusely stains the neoplastic cells with moderate intensity. Many cells have a perinuclear dot, Golgi zone accentuation in addition to homogeneous cytoplasmic staining (right panel, higher magnification of left panel).
536
Immunohistology of the Gastrointestinal Tract
Fibromatosis GIST
β-catenin (+ nuclear) SMA +/− Desmin +/−
+
Start
CD117
(−) or weak (+)
−
S-100
CD34++ β-catenin (+/− nuclear)
SMA + Desmin +
++
Solitary fibrous tumor
Smooth muscle tumor
Schwannoma
Figure 14-36 Immunohistochemical staining of mesenchymal neoplasms. GIST, Gastrointestinal stromal tumor; SMA, smooth muscle actin.
MESENCHYMAL LESIONS PRESENTING AS POLYPOID LESIONS
Spindle cell lesions uncommonly manifest as endoscopically biopsied or resected polyps of the luminal GI tract. The differential diagnosis of these lesions includes neurofibroma, ganglioneuroma, perineurioma, inflammatory fibroid polyp, and leiomyoma of the muscularis mucosae. Importantly, in the evaluation of these lesions, a GIST that occurs as a polypoid lesion should always be kept in the differential diagnosis; as such, CD 117 IHC should always be ordered. Neural Lesions
Neural lesions are common polypoid lesions of the GI tract and are most often present in the colon. These lesions typically show a spindle cell morphology with interdigitation of the neoplastic cells between colonic crypts; in the case of ganglioneuroma, intermixed ganglion-type cells are
A
seen (Fig. 14-37). Both neurofibroma and ganglioneuroma stain with S-100 protein, and the ganglion cells stain with neurofilament and synaptophysin.393-395 Of note, both neurofibromas and ganglioneuromas are associated with NF1 gene mutations.327,396-398 Although most ganglioneuromas are solitary, there is an additional association with MEN 2B and various polyposis syndromes, including Cowden syndrome.398 Perineurioma of the GI tract is a relatively recently described entity that rarely presents as a polypoid lesion. These lesions are composed of a bland spindle cell proliferation with delicate cytoplasmic protrusions that emanate from either side of an elongated nucleus. Similar to neurofibroma, this lesion grows around and entraps adjacent intestinal crypts. Analogous to perineuriomas of the soft tissues, intestinal perineuriomas almost uniformly stain with EMA; a subset of these lesions expresses claudin-1. These lesions are negative for smooth muscle markers, S-100 protein, and KIT.
B
Figure 14-37 Mucosal ganglioneuroma. A, Hematoxylin and eosin stain shows spindled stroma and one ganglion cell (arrow); eosinophils are prominent in this example. B, S-100 (red chromogen) strongly stains the stromal spindle cells, which wrap around negatively staining colonic crypts. The ganglion cells stain for neurofilament and synaptophysin (not shown).
Theranostic Applications
The lesion known as benign fibroblastic polyp is a recently described entity in the colon and is histologically similar to perineurioma; it is typically negative for EMA.399-402 However, a recent study has shown that a vast majority of these lesions stains with other perineurial markers, such as glucose transporter 1 (GLUT-1) and claudin-1.400 Thus it is likely that lesions formerly classified as benign fibroblastic polyps represent colonic perineuriomas. Fibroblastic Lesions
Inflammatory fibroid polyp is a lesion that most commonly presents in the gastric antrum and the distal small intestine.403 This lesion is composed of bland spindle cells set in a vascularized and loosely hyalinized stroma with abundant inflammatory cells that contain a large number of eosinophils. These tumors are thought to be derived from fibroblasts or dendritic cells and stain with CD34, fascin, CD35, calponin, and, less likely, SMA.404,405 Expression of KIT in these tumors has been reported but is rare.406 Smooth Muscle Lesions
Leiomyoma of the muscularis mucosae is the most common mucosal-based mesenchymal lesion of the GI tract that may present as a polypoid mass.407,408 These tumors often arise in the colon, but leiomyomas are also likely to occur within the wall of the esophagus.387 Unlike many of the previously mentioned lesions, this tumor displaces the colonic crypts and does not grow between crypts. As with leiomyomas elsewhere, these lesions stain strongly with SMA and desmin and are negative for KIT.
Genomic Applications Epithelial Lesions COLORECTAL ADENOCARCINOMA
Most colorectal adenocarcinomas arise through mutations of the β-catenin or APC genes or other genes in the Wnt signaling pathway. Dysfunction of this pathway can lead to translocation of β-catenin protein into the nucleus, where it can act as a transcriptional activator of other genes such as c-myc and cyclin D1. Nuclear β-catenin expression is highly associated with progression of colorectal tissue from normal epithelial tissue to polyps and from adenoma to carcinoma.409,410 A subset of colorectal cancers arises via the MSI pathway owing to mutations or alterations in specific DNA MMR proteins. This pathway is discussed further later in this chapter.
Neuroendocrine Lesions Approximately 5% to 10% of GI NETs are associated with a hereditary disease. The inherited syndromes and their associated genes include multiple endocrine neoplasia type I (MEN1 gene), neurofibromatosis type 1 (NF1 gene), von Hippel-Lindau disease (VHL gene), and the tuberous sclerosis complex (TSC1 or TSC2 gene).411
537
Mesenchymal Lesions GASTROINTESTINAL STROMAL TUMORS
As noted earlier, the classification of spindle cell lesions of the GI tract was immeasurably aided by the discovery of activating mutations in the KIT gene in approximately 80% of cases. Additionally, 5% to 7% of cases show a mutated PDGFRA gene, and the remaining 15% of cases show a lack of mutated KIT and PDGFRA proteins.333,412,413 A vast majority of these tumors show expression of the KIT protein by using IHC. DESMOID-TYPE FIBROMATOSIS
As many as 60% of desmoid-type fibromatosis harbors mutations in the β-catenin gene, and an additional 10% of these tumors show mutations in the APC gene.414-419 Mutations in either of these two genes result in abnormal accumulation of the β-catenin protein in the nucleus.416,420 This abnormal accumulation can be detected by IHC, as noted earlier, and has utility in the diagnosis of desmoid-type fibromatosis.
Theranostic Applications Epithelial Lesions REFRACTORY CELIAC DISEASE
IHC has been shown to have utility in the evaluation of patients with refractory celiac disease. These patients continue to suffer from symptoms despite strict adherence to a gluten-free diet and are more likely to develop further complications, such as ulcerative jejunoileitis and enteropathy-associated T-cell lymphoma. Patients with refractory sprue and loss of CD8 expression in more than 50% of the CD3-positive intraepithelial lymphocytes are more likely to develop enteropathyassociated T-cell lymphoma.421 Patients with this abnormal T-cell phenotype are more likely to receive more aggressive immunosuppressive therapy. BARRETT ESOPHAGUS
Currently, high-grade dysplasia remains the most reliable predictor of progression to adenocarcinoma in BE. However, it would be more helpful to be able to predict progression in earlier lesions. One study found that p53 protein overexpression in low-grade dysplasia, as assessed by IHC, is predictive of progression (Fig. 14-38).422 Of the 21% of cases of low-grade dysplasia that overexpressed p53 in their study, 60% progressed to high-grade dysplasia or carcinoma on follow-up biopsies, 25% had persistent low-grade dysplasia, and 13% “regressed.” Although the results of this study are promising, they have not yet been incorporated into routine clinical use. GASTRIC ADENOCARCINOMA
Nuclear regulatory and cell-cycle molecules E-cadherin, p16, and CDX-2 have been studied as markers of poor
538
Immunohistology of the Gastrointestinal Tract
A
B
Figure 14-38 A, Low-grade dysplasia in Barrett esophagus shows overexpression of p53. B, Low-grade dysplasia without p53 expression.
prognosis.423,424 Rb, cyclin D1, p53, and Ki-67 (MIB-1) are useful adjunctive tests for distinguishing between intestinal metaplasia and well-differentiated adenocarcinoma. Combinations of MUC1, MUC5A, and MUC5B staining, reflecting so-called intestinal or gastric type differentiation, appear to be prognostically significant.425-428 MUC antibodies do not appear to be useful in distinguishing among adenocarcinomas of different sites.120,429 Her2 IMMUNOHISTOCHEMISTRY ON GASTRIC AND GASTROESOPHAGEAL ADENOCARCINOMA
With the recent description of the effectiveness of trastuzumab therapy in patients with Her2-positive gastric and gastroesophageal adenocarcinomas, testing of these tumors has become the standard of care, especially in patients with widespread disease.430 Her2 overexpression is seen in approximately 30% of gland-forming adenocarcinomas and in 5% of diffuse-type cancers, compared with approximately 20% of breast carcinomas.431,432 Whereas the technical aspects of the Her2 ICH assay are identical to those of breast carcinoma, the interpretation differs in several ways. First, 10% of tumor cells, rather than 30% in breast carcinoma, must express Her2 to qualify as positive staining; the interpretation score is based on intensity of Her2 expression. Second, basolateral staining, rather than the complete, circumferential tumor cell staining seen in breast cancer, is permissible. Third, the interpretation guidelines differ depending on specimen type, with 10% the cutoff for resection specimens; a cluster of five or more positive-staining tumor cells qualifies as positive when
interpreting biopsy specimens.433 Additionally, intratumoral heterogeneity is more common in gastric and gastroesophageal junction adenocarcinomas compared with breast cancers.434 As with breast carcinomas, tumors scored as equivocal (2+) by using IHC should be tested by using Her2 in situ hybridization (ISH). COLORECTAL ADENOCARCINOMA
Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor. Humanized monoclonal antibody chemotherapy to EGFR blocks its functional activation. IHC documentation of EGFR reactivity within colorectal cancers was once necessary to initiate anti-EGFR therapy. Currently, the initiation of therapy relies on documentation of a normal (wild-type) KRAS gene, because tumors with KRAS mutations do not respond to anti-EGFR therapy. MSI-H/DNA mismatch repair protein–deficient colorectal cancers, evaluated by either MSI testing or IHC, may have a better survival in subsets of colon cancer patients.435 This effect may be related to the CpG island methylator phenotype (widespread promoter methylation).436 MSI-H tumors may also respond differently to 5-fluorouracil–based chemotherapy.437
Mesenchymal Lesions In the appropriate histologic context, documentation of either KIT protein expression by IHC or a mutation in the KIT/PDGFRA genes is among the criteria for a diagnosis of GIST. The primary modality of medical
Summary
treatment of these tumors is administration of the KIT/ PDGFRA inhibitors imatinib mesylate and sunitinib malate. Thus expression of KIT protein by using IHC is an important predictive marker in mesenchymal tumors of the GI tract.
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications Epithelial Lesions EPSTEIN-BARR VIRUS–ASSOCIATED GASTRIC CARCINOMAS
Approximately 4% of gastric carcinomas are associated with Epstein-Barr virus (EBV) infection (see Fig. 14-11, D).438,439 These tumors have a typical, lymphocyte-rich stroma that is a clue to the proper diagnosis,147 and they show EBV-encoded RNA by ISH440,441 and have a better prognosis compared with typical gastric adenocarcinoma.147,442-444 BARRETT ESOPHAGUS
As mentioned earlier, histomorphology is the best tool to predict behavior in BE. Promising biomarkers on the horizon use various techniques, but they need to be validated. Two techniques that have been used to stratify risk in BE are DNA content analysis (by flow cytometry) and loss of heterozygosity (LOH) as measured by PCR. In one study, patients with no DNA content abnormalities at baseline biopsy had 0% incidence of adenocarcinoma at 5 years, whereas patients with aneuploidy or increased tetraploidy (4N) at baseline biopsy had a 28% incidence of adenocarcinoma at 5 years.445 In another study, of patients with baseline 17p LOH (p53 locus), 37% (20/54) progressed to adenocarcinoma; in patients with no loss at 17p, 3% (6/202) progressed to adenocarcinoma.446 COLORECTAL ADENOCARCINOMA
As mentioned earlier, a subset of colorectal cancers arise via the MSI pathway. Molecular testing for MSI by using a panel of microsatellite markers can detect microsatelliteunstable (MSI-H) tumors.244 Most MSI-H colorectal cancers are sporadic and arise from epigenetic gene silencing (hypermethylation) of one of the MMR protein genes, most commonly MLH1.447 Hence, molecular testing for MLH1 promoter methylation can help distinguish sporadic from heritable MSI-H colorectal cancer, because this phenomenon is absent in the inherited form (Lynch syndrome). Mutational analysis for the V600E mutation in the BRAF gene can also help in this distinction, because this mutation is absent in Lynch syndrome cancers and is present in approximately 50% of sporadic colon carcinomas.448,449 The minority of MSI-H colorectal cancers that are inherited arise from germline mutations of one of the MMR protein genes MLH1, MSH2, MSH6, or PMS2, with MSH2 the most commonly involved gene.235 As mentioned earlier,
539
defects in the various MMR proteins can be detected by IHC. Zhang discusses the utility of MSI testing in colorectal cancer patients at risk for hereditary non polyposis colorectal cancer (Lynch) syndrome.239,244 Whereas a discussion of reflex testing in all or a subset of colorectal cancers is beyond the scope of this text, the Evaluation of Genomic Applications in Practice and Prevention Working Group recommends offering genetic testing for Lynch syndrome to individuals with newly diagnosed colorectal cancer to reduce morbidity and mortality in relatives,448 and the most recent National Comprehensive Cancer Network (NCCN) Guidelines state that “many NCCN institutions and other comprehensive cancer centers now perform IHC and sometimes MSI testing on all colorectal cancer regardless of family history to determine which patients should have genetic testing for Lynch syndrome.”450,451 KRAS MUTATIONAL ANALYSIS
Cetuximab is a monoclonal antibody that binds EGFR and competitively inhibits ligand binding. In the past, cetuximab was approved for irinotecan-resistant metastatic colorectal cancers that expressed EGFR by IHC. However, only a minority of these patients respond to anti-EGFR therapy.452 More recently, it was discovered that tumors with KRAS gene mutations were resistant to cetuximab and had an overall poorer survival.453,454 Hence KRAS mutational analysis, performed on either the primary or metastatic tumor, is becoming a routine test for patients with metastatic colorectal cancers, and cetuximab is given to those with no evidence of KRAS mutations (KRAS wild-type status).
Mesenchymal Lesions KIT MUTATION STATUS AND CHEMOTHERAPY SUSCEPTIBILITY
Imatinib mesylate and sunitinib malate chemotherapy inhibits the tyrosine kinase activity of the KIT and PDGFRA proteins. This drug has proven itself to be a potent inhibitor of GIST growth in most patients.455 However, the efficacy of these agents does vary with the particular KIT or PDGFRA mutation. For example, GISTs with exon 11 KIT mutations are more sensitive to tyrosine kinase inhibitors than those tumors with either wild-type KIT or exon 9 mutated GISTs.412,456,457 As a result of this information, many centers perform KIT and/or PDGFRA mutation analysis to determine potential chemotherapeutic sensitivity.
Summary IHC is a powerful tool in the lumenal GI tract, and it has essential theranostic and genomic applications in addition to crucial diagnostic applications. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Gown AM: Immunohistochemical determination of primary sites of carcinomas. J Histotechnology. 22:209–215, 1999. 2. Bracke ME, Van Roy FM, Mareel MM: The E-cadherin/catenin complex in invasion and metastasis. Curr Top Microbiol Immunol. 213(Pt 1):123–161, 1996. 3. Willert K, Nusse R: Beta-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev. 8:95–102, 1998. 4. Zhou Q, Toivola DM, Feng N, et al: Keratin 20 helps maintain intermediate filament organization in intestinal epithelia. Mol Biol Cell. 14:2959–2971, 2003. 5. Hinoi T, Lucas PC, Kuick R, et al: CDX2 regulates liver intestinecadherin expression in normal and malignant colon epithelium and intestinal metaplasia. Gastroenterology. 123:1565–1577, 2002. 6. Bonhomme C, Duluc I, Martin E, et al: The Cdx2 homeobox gene has a tumour suppressor function in the distal colon in addition to a homeotic role during gut development. Gut. 52:1465–1471, 2003. 7. Capella C, Heitz PU, Hofler H, et al: Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virchows Arch. 425:547–560, 1995. 8. Mertz H, Vyberg M, Paulsen SM: Immunohistochemical detection of neuroendocrine markers in tumors of the lungs and gastrointestinal tract. Appl Immunohistochem. 6:175–180, 1998. 9. Portela-Gomes GM, Stridsberg M: Chromogranin A in the human gastrointestinal tract: an immunocytochemical study with region-specific antibodies. J Histochem Cytochem. 50:1487– 1492, 2002. 10. Hla T, Neilson K: Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A. 89:7384–7388, 1992. 11. Sano H, Kawahito Y, Wilder RL, et al: Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res. 55:3785–3789, 1995. 12. Sheehan KM, Sheahan K, O’Donoghue DP, et al: The relationship between cyclooxygenase-2 expression and colorectal cancer. JAMA. 282:1254–1257, 1999. 13. Lim HY, Joo HJ, Choi JH, et al: Increased expression of cyclooxygenase-2 protein in human gastric carcinoma. Clin Cancer Res. 6:519–525, 2000. 14. Okami J, Yamamoto H, Fujiwara Y, et al: Overexpression of cyclooxygenase-2 in carcinoma of the pancreas. Clin Cancer Res. 5:2018–2024, 1999. 15. Tucker ON, Dannenberg AJ, Yang EK, et al: Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Cancer Res. 59:987–990, 1999. 16. Espinosa I, Lee CH, Kim MK, et al: A novel monoclonal antibody against DOG1 is a sensitive and specific marker for gastrointestinal stromal tumors. Am J Surg Pathol. 32:210–218, 2008. 17. Chiaravalli AM, Furlan D, Facco C, et al: Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences. Virchows Arch. 438:39–48, 2001. 18. Myklebust AT, Beiske K, Pharo A, et al: Selection of anti-SCLC antibodies for diagnosis of bone marrow metastasis. Br J Cancer. 14(Suppl):49–53, 1991. 19. Winter MJ, Nagtegaal ID, van Krieken JH, et al: The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology. Am J Pathol. 163: 2139–2148, 2003. 20. Morrison C, Marsh W, Jr, Frankel WL: A comparison of CD10 to pCEA, MOC-31, and hepatocyte for the distinction of malignant tumors in the liver. Mod Pathol. 15:1279–1287, 2002. 21. Niemann TH, Hughes JH, De Young BR: MOC-31 aids in the differentiation of metastatic adenocarcinoma from hepatocellular carcinoma. Cancer. 87:295–298, 1999. 22. Van Klinken BJ, Dekker J, Buller HA, et al: Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the human gastrointestinal tract. Am J Physiol. 273:G296–G302, 1997. 23. Ischia R, Lovisetti-Scamihorn P, Hogue-Angeletti R, et al: Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem. 272:11657–11662, 1997.
539.e1
24. Leitner B, Lovisetti-Scamihorn P, Heilmann J, et al: Subcellular localization of chromogranins, calcium channels, amine carriers, and proteins of the exocytotic machinery in bovine splenic nerve. J Neurochem. 72:1110–1116, 1999. 25. Lovisetti-Scamihorn P, Fischer-Colbrie R, Leitner B, et al: Relative amounts and molecular forms of NESP55 in various bovine tissues. Brain Res. 829:99–106, 1999. 26. Ireland AP, Clark GW, DeMeester TR: Barrett’s esophagus. The significance of p53 in clinical practice. Ann Surg. 225:17–30, 1997. 27. Yang A, Kaghad M, Wang Y, et al: p63, a p53 homolog at 3q2729, encodes multiple products with transactivating, deathinducing, and dominant-negative activities. Mol Cell. 2:305–316, 1998. 28. Marin MC, Kaelin WG, Jr: p63 and p73: old members of a new family. Biochim Biophys Acta. 1470:M93–M100, 2000. 29. Daniely Y, Liao G, Dixon D, et al: Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium. Am J Physiol Cell Physiol. 287:C171–C181, 2004. 30. Bacchi CE, Gown AM: Distribution and pattern of expression of villin, a gastrointestinal-associated cytoskeletal protein, in human carcinomas: a study employing paraffin-embedded tissue. Lab Invest. 64:418–424, 1991. 31. Nishizuka S, Chen ST, Gwadry FG, et al: Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling. Cancer Res. 63:5243–5250, 2003. 32. Savera AT, Torres FX, Lindin MD, et al: Primary versus metastatic pulmonary adenocarcinoma: an immunohistochemical study using villin and cytokeratins 7 and 20. Appl Immunohistochem. 4:86–94, 1996. 33. Tan J, Sidhu G, Greco MA, et al: Villin, cytokeratin 7, and cytokeratin 20 expression in pulmonary adenocarcinoma with ultrastructural evidence of microvilli with rootlets. Hum Pathol. 29:390–396, 1998. 34. Odze RD: Update on the diagnosis and treatment of Barrett esophagus and related neoplastic precursor lesions. Arch Pathol Lab Med. 132:1577–1585, 2008. 35. Kilgore SP, Ormsby AH, Gramlich TL, et al: The gastric cardia: fact or fiction? Am J Gastroenterol. 95:921–924, 2000. 36. Flucke U, Steinborn E, Dries V, et al: Immunoreactivity of cytokeratins (CK7, CK20) and mucin peptide core antigens (MUC1, MUC2, MUC5AC) in adenocarcinomas, normal and metaplastic tissues of the distal oesophagus, oesophago-gastric junction and proximal stomach. Histopathology. 43:127–134, 2003. 37. Glickman JN, Wang H, Das KM, et al: Phenotype of Barrett’s esophagus and intestinal metaplasia of the distal esophagus and gastroesophageal junction: an immunohistochemical study of cytokeratins 7 and 20, Das-1 and 45 MI. Am J Surg Pathol. 25:87–94, 2001. 38. Jovanovic I, Tzardi M, Mouzas IA, et al: Changing pattern of cytokeratin 7 and 20 expression from normal epithelium to intestinal metaplasia of the gastric mucosa and gastroesophageal junction. Histol Histopathol. 17:445–454, 2002. 39. Kurtkaya-Yapicier O, Gencosmanoglu R, Avsar E, et al: The utility of cytokeratins 7 and 20 (CK7/20) immunohistochemistry in the distinction of short-segment Barrett esophagus from gastric intestinal metaplasia: is it reliable? BMC Clin Pathol. 3:5, 2003. 40. Latchford A, Eksteen B, Jankowski J: The continuing tale of cytokeratins in Barrett’s mucosa: as you like it. Gut. 49:746–747, 2001. 41. Mohammed IA, Streutker CJ, Riddell RH: Utilization of cytokeratins 7 and 20 does not differentiate between Barrett’s esophagus and gastric cardiac intestinal metaplasia. Mod Pathol. 15:611–616, 2002. 42. Odze R: Cytokeratin 7/20 immunostaining: Barrett’s oesophagus or gastric intestinal metaplasia? Lancet. 359:1711–1713, 2002. 43. Ormsby AH, Goldblum JR, Rice TW, et al: Cytokeratin subsets can reliably distinguish Barrett’s esophagus from intestinal metaplasia of the stomach. Hum Pathol. 30:288–294, 1999. 44. Faller G, Borchard F, Ell C, et al: Histopathological diagnosis of Barrett’s mucosa and associated neoplasias: results of a consensus conference of the Working Group for Gastroenterological
539.e2
Immunohistology of the Gastrointestinal Tract
Pathology of the German Society for Pathology on 22 September 2001 in Erlangen. Virchows Arch. 443:597–601, 2003. 45. Chatelain D, Flejou JF: High-grade dysplasia and superficial adenocarcinoma in Barrett’s esophagus: histological mapping and expression of p53, p21 and Bcl-2 oncoproteins. Virchows Arch. 442:18–24, 2003. 46. Copelli SB, Mazzeo C, Gimenez A, et al: Molecular analysis of p53 tumor-suppressor gene and microsatellites in preneoplastic and neoplastic lesions of the colon and esophagus. Oncol Rep. 8:923–929, 2001. 47. Croft J, Parry EM, Jenkins GJ, et al: Analysis of the premalignant stages of Barrett’s oesophagus through to adenocarcinoma by comparative genomic hybridization. Eur J Gastroenterol Hepatol. 14:1179–1186, 2002. 48. Raouf AA, Evoy DA, Carton E, et al: Loss of Bcl-2 expression in Barrett’s dysplasia and adenocarcinoma is associated with tumor progression and worse survival but not with response to neoadjuvant chemoradiation. Dis Esophagus. 16:17–23, 2003. 49. Reid BJ, Blount PL, Rabinovitch PS: Biomarkers in Barrett’s esophagus. Gastrointest Endosc Clin N Am. 13:369–397, 2003. 50. Taniere P, Borghi-Scoazec G, Saurin JC, et al: Cytokeratin expression in adenocarcinomas of the esophagogastric junction: a comparative study of adenocarcinomas of the distal esophagus and of the proximal stomach. Am J Surg Pathol. 26:1213–1221, 2002. 51. Weiss MM, Kuipers EJ, Hermsen MA, et al: Barrett’s adenocarcinomas resemble adenocarcinomas of the gastric cardia in terms of chromosomal copy number changes, but relate to squamous cell carcinomas of the distal oesophagus with respect to the presence of high-level amplifications. J Pathol. 199:157–165, 2003. 52. Symmans PJ, Linehan JM, Brito MJ, et al: p53 expression in Barrett’s oesophagus, dysplasia, and adenocarcinoma using antibody DO-7. J Pathol. 173:221–226, 1994. 53. Cawley HM, Meltzer SJ, De Benedetti VM, et al: Anti-p53 antibodies in patients with Barrett’s esophagus or esophageal carcinoma can predate cancer diagnosis. Gastroenterology. 115:19–27, 1998. 54. Ramel S, Reid BJ, Sanchez CA, et al: Evaluation of p53 protein expression in Barrett’s esophagus by two-parameter flow cytometry. Gastroenterology. 102:1220–1228, 1992. 55. Levine DS: Barrett’s oesophagus and p53. Lancet. 344:212–213, 1994. 56. Werling RW, Yaziji H, Bacchi CE, et al: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 27:303–310, 2003. 57. Deamant FD, Pombo MT, Battifora H: Estrogen receptor immunohistochemistry as a predictor of site of origin in metastatic breast cancer. Appl Immunohistochem. 1:188–192, 1993. 58. Driessen A, Nafteux P, Lerut T, et al: Identical cytokeratin expression pattern CK7+/CK20- in esophageal and cardiac cancer: etiopathological and clinical implications. Mod Pathol. 17:49–55, 2004. 59. Gulmann C, Counihan I, Grace A, et al: Cytokeratin 7/20 and mucin expression patterns in oesophageal, cardia and distal gastric adenocarcinomas. Histopathology. 43:453–461, 2003. 60. Sheahan K, O’Brien MJ, Burke B, et al: Differential reactivities of carcinoembryonic antigen (CEA) and CEA-related monoclonal and polyclonal antibodies in common epithelial malignancies. Am J Clin Pathol. 94:157–164, 1990. 61. Ormsby AH, Goldblum JR, Rice TW, et al: The utility of cytokeratin subsets in distinguishing Barrett’s-related oesophageal adenocarcinoma from gastric adenocarcinoma. Histopathology. 38:307–311, 2001. 62. Shen B, Ormsby AH, Shen C, et al: Cytokeratin expression patterns in noncardia, intestinal metaplasia-associated gastric adenocarcinoma: implication for the evaluation of intestinal metaplasia and tumors at the esophagogastric junction. Cancer. 94:820–831, 2002. 63. Chu PG, Weiss LM: Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod Pathol. 15:6–10, 2002. 64. Clover J, Oates J, Edwards C: Anti-cytokeratin 5/6: a positive marker for epithelioid mesothelioma. Histopathology. 31:140– 143, 1997.
65. Kaufmann O, Fietze E, Mengs J, et al: Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol. 116:823–830, 2001. 66. Lam KY, Loke SL, Shen XC, et al: Cytokeratin expression in non-neoplastic oesophageal epithelium and squamous cell carcinoma of the oesophagus. Virchows Arch. 426:345–349, 1995. 67. Moll R: Cytokeratins as markers of differentiation in the diagnosis of epithelial tumors. Subcell Biochem. 31:205–262, 1998. 68. Ordonez NG: Value of cytokeratin 5/6 immunostaining in distinguishing epithelial mesothelioma of the pleura from lung adenocarcinoma. Am J Surg Pathol. 22:1215–1221, 1998. 69. Scarpatetti M, Tsybrovsky O, Popper HH: Cytokeratin typing as an aid in the differential diagnosis of primary versus metastatic lung carcinomas, and comparison with normal lung. Virchows Arch. 440:70–76, 2002. 70. Shah KD, Tabibzadeh SS, Gerber MA: Comparison of cytokeratin expression in primary and metastatic carcinomas. Diagnostic application in surgical pathology. Am J Clin Pathol. 87:708–715, 1987. 71. Suo Z, Holm R, Nesland JM: Squamous cell carcinomas. An immunohistochemical study of cytokeratins and involucrin in primary and metastatic tumours. Histopathology. 23:45–54, 1993. 72. Di Como CJ, Urist MJ, Babayan I, et al: p63 expression profiles in human normal and tumor tissues. Clin Cancer Res. 8:494– 501, 2002. 73. Ikeda Y, Kuwano H, Ikebe M, et al: Immunohistochemical detection of CEA, CA19-9, and DF3 in esophageal carcinoma limited to the submucosal layer. J Surg Oncol. 56:7–12, 1994. 74. Lager DJ, Callaghan EJ, Worth SF, et al: Cellular localization of thrombomodulin in human epithelium and squamous malignancies. Am J Pathol. 146:933–943, 1995. 75. Moll R, Lowe A, Laufer J, et al: Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol. 140:427–447, 1992. 76. Rossen K, Thomsen HK: Ber-EP4 immunoreactivity depends on the germ layer origin and maturity of the squamous epithelium. Histopathology. 39:386–389, 2001. 77. Lau SK, Luthringer DJ, Eisen RN: Thyroid Transcription Factor—1: A review. Appl Immunohistochem Mol Morphol. 10:97–102, 2002. 78. Kornstein MJ, Rosai J: CD5 labeling of thymic carcinomas and other nonlymphoid neoplasms. Am J Clin Pathol. 109:722–726, 1998. 79. Tateyama H, Eimoto T, Tada T, et al: Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma. Am J Clin Pathol. 111:235–240, 1999. 80. Ordonez NG: Value of calretinin immunostaining in differentiating epithelial mesothelioma from lung adenocarcinoma. Mod Pathol. 11:929–933, 1998. 81. Owonikoko T, Loberg C, Gabbert HE, et al: Comparative analysis of basaloid and typical squamous cell carcinoma of the oesophagus: a molecular biological and immunohistochemical study. J Pathol. 193:155–161, 2001. 82. Sarbia M, Bittinger F, Porschen R, et al: bcl-2 expression and prognosis in squamous-cell carcinomas of the esophagus. Int J Cancer. 69:324–328, 1996. 83. Banks ER, Frierson HF, Jr, Mills SE, et al: Basaloid squamous cell carcinoma of the head and neck. A clinicopathologic and immunohistochemical study of 40 cases. Am J Surg Pathol. 16:939– 946, 1992. 84. Luna MA, el Naggar A, Parichatikanond P, et al: Basaloid squamous carcinoma of the upper aerodigestive tract. Clinicopathologic and DNA flow cytometric analysis. Cancer. 66:537–542, 1990. 85. Tsang WY, Chan JK, Lee KC, et al: Basaloid-squamous carcinoma of the upper aerodigestive tract and so-called adenoid cystic carcinoma of the oesophagus: the same tumour type? Histopathology. 19:35–46, 1991. 86. Tsubochi H, Suzuki T, Suzuki S, et al: Immunohistochemical study of basaloid squamous cell carcinoma, adenoid cystic and mucoepidermoid carcinoma in the upper aerodigestive tract. Anticancer Res. 20:1205–1211, 2000.
References 87. Li TJ, Zhang YX, Wen J, et al: Basaloid squamous cell carcinoma of the esophagus with or without adenoid cystic features. Arch Pathol Lab Med. 128:1124–1130, 2004. 88. Abe K, Sasano H, Itakura Y, et al: Basaloid-squamous carcinoma of the esophagus. A clinicopathologic, DNA ploidy, and immunohistochemical study of seven cases. Am J Surg Pathol. 20:453– 461, 1996. 89. Kawahara K, Makimoto K, Maekawa T, et al: An immunohistochemical examination of basaloid squamous cell carcinoma of the esophagus: report of a case. Surg Today. 31:655–659, 2001. 90. Popnikolov NK, Ayala AG, Graves K, et al: Benign myoepithelial tumors of the breast have immunophenotypic characteristics similar to metaplastic matrix-producing and spindle cell carcinomas. Am J Clin Pathol. 120:161–167, 2003. 91. Guarino M, Reale D, Micoli G, et al: Carcinosarcoma of the oesophagus with rhabdomyoblastic differentiation. Histopathology. 22:493–498, 1993. 92. Lauwers GY, Grant LD, Scott GV, et al: Spindle cell squamous carcinoma of the esophagus: analysis of ploidy and tumor proliferative activity in a series of 13 cases. Hum Pathol. 29:863–868, 1998. 93. Ooi A, Kawahara E, Okada Y, et al: Carcinosarcoma of the esophagus. An immunohistochemical and electron microscopic study. Acta Pathol Jpn. 36:151–159, 1986. 94. Rosty D, Prevot S, Tiret E: Adenocarcinosarcoma in Barrett’s esophagus: Report of a case. Int J Surg Pathol. 4:43, 1996. 95. Iezzoni JC, Mills SE: Sarcomatoid carcinomas (carcinosarcomas) of the gastrointestinal tract: a review. Semin Diagn Pathol. 10:176–187, 1993. 96. Amatya VJ, Takeshima Y, Kaneko M, et al: Esophageal carcinosarcoma with basaloid squamous carcinoma and rhabdomyosarcoma components with TP53 mutation. Pathol Int. 54:803–809, 2004. 97. Drut R, Drut RM: Lymphocytic gastritis in pediatric celiac disease—immunohistochemical study of the intraepithelial lymphocytic component. Med Sci Monit. 10:CR38–CR42, 2004. 98. Alsaigh N, Odze R, Goldman H, et al: Gastric and esophageal intraepithelial lymphocytes in pediatric celiac disease. Am J Surg Pathol. 20:865–870, 1996. 99. Andersen LP, Dorland A, Karacan H, et al: Possible clinical importance of the transformation of Helicobacter pylori into coccoid forms. Scand J Gastroenterol. 35:897–903, 2000. 100. Cao J, Li ZQ, Borch K, et al: Detection of spiral and coccoid forms of Helicobacter pylori using a murine monoclonal antibody. Clin Chim Acta. 267:183–196, 1997. 101. Goldstein NS: Chronic inactive gastritis and coccoid Helicobacter pylori in patients treated for gastroesophageal reflux disease or with H pylori eradication therapy. Am J Clin Pathol. 118:719–726, 2002. 102. Ashton-Key M, Diss TC, Isaacson PG: Detection of Helicobacter pylori in gastric biopsy and resection specimens. J Clin Pathol. 49:107–111, 1996. 103. Jonkers D, Houben G, de Bruine A, et al: Prevalence of gastric metaplasia in the duodenal bulb and distribution of Helicobacter pylori in the gastric mucosa. A clinical and histopathological study in 96 consecutive patients. Ital J Gastroenterol Hepatol. 30:481–483, 1998. 104. Rotimi O, Cairns A, Gray S, et al: Histological identification of Helicobacter pylori: comparison of staining methods. J Clin Pathol. 53:756–759, 2000. 105. van der Wouden EJ, Thijs JC, van Zwet AA, et al: Reliability of biopsy-based diagnostic tests for Helicobacter pylori after treatment aimed at its eradication. Eur J Gastroenterol Hepatol. 11:1255–1258, 1999. 106. Singhal AV, Sepulveda AR: Helicobacter heilmannii gastritis: a case study with review of literature. Am J Surg Pathol. 29:1537– 1539, 2005. 107. Bordi C, Annibale B, Azzoni C, et al: Endocrine cell growths in atrophic body gastritis. Critical evaluation of a histological classification. J Pathol. 182:339–346, 1997. 108. Solcia E, Rindi G, Fiocca R, et al: Distinct patterns of chronic gastritis associated with carcinoid and cancer and their role in tumorigenesis. Yale J Biol Med. 65:793–804; discussion 827–829, 1992. 109. Torbenson M, Abraham SC, Boitnott J, et al: Autoimmune gastritis: distinct histological and immunohistochemical findings
539.e3
before complete loss of oxyntic glands. Mod Pathol. 15:102–109, 2002. 110. Stolte M, Baumann K, Bethke B, et al: Active autoimmune gastritis without total atrophy of the glands. Z Gastroenterol. 30:729–735, 1992. 111. Ohchi T, Misumi A, Akagi M: A study on the distribution of G-cells in human gastric mucosa. Gastroenterol Jpn. 19:41–52, 1984. 112. Burt RW: Gastric fundic gland polyps. Gastroenterology. 125: 1462–1469, 2003. 113. Declich P, Isimbaldi G, Sironi M, et al: Sporadic fundic gland polyps: an immunohistochemical study of their antigenic profile. Pathol Res Pract. 192:808–815, 1996. 114. Sekine S, Shibata T, Yamauchi Y, et al: Beta-catenin mutations in sporadic fundic gland polyps. Virchows Arch. 440:381–386, 2002. 115. Hassan A, Yerian LM, Kuan SF, et al: Immunohistochemical evaluation of adenomatous polyposis coli, beta-catenin, c-Myc, cyclin D1, p53, and retinoblastoma protein expression in syndromic and sporadic fundic gland polyps. Hum Pathol. 35:328– 334, 2004. 116. Lauren P: The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. an attempt at a histo-clinical classification. Acta Pathol Microbiol Scand. 64:31– 49, 1965. 117. Ma CK, Zarbo RJ, Frierson HF, Jr, et al: Comparative immunohistochemical study of primary and metastatic carcinomas of the liver. Am J Clin Pathol. 99:551–557, 1993. 118. Goldstein NS, Long A, Kuan SF, et al: Colon signet ring cell adenocarcinoma: immunohistochemical characterization and comparison with gastric and typical colon adenocarcinomas. Appl Immunohistochem Mol Morphol. 8:183–188, 2000. 119. Kim MA, Lee HS, Yang HK, et al: Cytokeratin expression profile in gastric carcinomas. Hum Pathol. 35:576–581, 2004. 120. Lee MJ, Lee HS, Kim WH, et al: Expression of mucins and cytokeratins in primary carcinomas of the digestive system. Mod Pathol. 16:403–410, 2003. 121. McKinley M, Listrom MB, Fenoglio-Preiser C: Cytokeratin 19: a potential marker of colonic differentiation. Surg Pathol. 3:107– 113, 1990. 122. Baars JH, De Ruijter JL, Smedts F, et al: The applicability of a keratin 7 monoclonal antibody in routinely Papanicolaou-stained cytologic specimens for the differential diagnosis of carcinomas. Am J Clin Pathol. 101:257–261, 1994. 123. Ramaekers F, van Niekerk C, Poels L, et al: Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocarcinomas. Am J Pathol. 136:641–655, 1990. 124. Tot T: Adenocarcinomas metastatic to the liver: the value of cytokeratins 20 and 7 in the search for unknown primary tumors. Cancer. 85:171–177, 1999. 125. Wauters CC, Smedts F, Gerrits LG, et al: Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary. Hum Pathol. 26:852–855, 1995. 126. Kaufmann O, Deidesheimer T, Muehlenberg M, et al: Immunohistochemical differentiation of metastatic breast carcinomas from metastatic adenocarcinomas of other common primary sites. Histopathology. 29:233–240, 1996. 127. Miettinen M: Keratin 20: immunohistochemical marker for gastrointestinal, urothelial, and Merkel cell carcinomas. Mod Pathol. 8:384–388, 1995. 128. Cathro HP, Stoler MH: Expression of cytokeratins 7 and 20 in ovarian neoplasia. Am J Clin Pathol. 117:944–951, 2002. 129. Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 13:962–972, 2000. 130. Kende AI, Carr NJ, Sobin LH: Expression of cytokeratins 7 and 20 in carcinomas of the gastrointestinal tract. Histopathology. 42:137–140, 2003. 131. Park SY, Kim HS, Hong EK, et al: Expression of cytokeratins 7 and 20 in primary carcinomas of the stomach and colorectum and their value in the differential diagnosis of metastatic carcinomas to the ovary. Hum Pathol. 33:1078–1085, 2002. 132. Wang NP, Zee S, Zarbo RJ: Coordinate expression of cytokeratins 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem. 3:99–107, 1995.
539.e4
Immunohistology of the Gastrointestinal Tract
133. Kaimaktchiev V, Terracciano L, Tornillo L, et al: The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas. Mod Pathol. 17:1392–1399, 2004. 134. Liu Q, Teh M, Ito K, et al: CDX2 expression is progressively decreased in human gastric intestinal metaplasia, dysplasia and cancer. Mod Pathol. 20:1286–1297, 2007. 135. Chu PG, Weiss LM: Immunohistochemical characterization of signet-ring cell carcinomas of the stomach, breast, and colon. Am J Clin Pathol. 121:884–892, 2004. 136. Tian MM, Zhao AL, Li ZW, et al: Phenotypic classification of gastric signet ring cell carcinoma and its relationship with clinicopathologic parameters and prognosis. World J Gastroenterol. 13:3189–3198, 2007. 137. Nguyen MD, Plasil B, Wen P, et al: Mucin profiles in signet-ring cell carcinoma. Arch Pathol Lab Med. 130:799–804, 2006. 138. O’Connell FP, Wang HH, Odze RD: Utility of immunohistochemistry in distinguishing primary adenocarcinomas from metastatic breast carcinomas in the gastrointestinal tract. Arch Pathol Lab Med. 129:338–347, 2005. 139. Llinares K, Escande F, Aubert S, et al: Diagnostic value of MUC4 immunostaining in distinguishing epithelial mesothelioma and lung adenocarcinoma. Mod Pathol. 17:150–157, 2004. 140. Cameron BL, Butler JA, Rutgers J, et al: Immunohistochemical determination of the estrogen receptor content of gastrointestinal adenocarcinomas. Am Surg. 58:758–760, 1992. 141. Nash JW, Morrison C, Frankel WL: The utility of estrogen receptor and progesterone receptor immunohistochemistry in the distinction of metastatic breast carcinoma from other tumors in the liver. Arch Pathol Lab Med. 127:1591–1595, 2003. 142. Vang R, Gown AM, Barry TS, et al: Immunohistochemistry for estrogen and progesterone receptors in the distinction of primary and metastatic mucinous tumors in the ovary: an analysis of 124 cases. Mod Pathol. 19:97–105, 2006. 143. Lee BH, Hecht JL, Pinkus JL, et al: WT1, estrogen receptor, and progesterone receptor as markers for breast or ovarian primary sites in metastatic adenocarcinoma to body fluids. Am J Clin Pathol. 117:745–750, 2002. 144. Park SY, Kim BH, Kim JH, et al: Panels of immunohistochemical markers help determine primary sites of metastatic adenocarcinoma. Arch Pathol Lab Med. 131:1561–1567, 2007. 145. Theodoropoulos GE, Lazaris AC, Panoussopoulos D, et al: Significance of estrogen receptors and cathepsin D tissue detection in gastric adenocarcinoma. J Surg Oncol. 58:176–183, 1995. 146. van Velthuysen ML, Taal BG, van der Hoeven JJ, et al: Expression of oestrogen receptor and loss of E-cadherin are diagnostic for gastric metastasis of breast carcinoma. Histopathology. 46:153–157, 2005. 147. Grogg KL, Lohse CM, Pankratz VS, et al: Lymphocyte-rich gastric cancer: associations with Epstein-Barr virus, microsatellite instability, histology, and survival. Mod Pathol. 16:641–651, 2003. 148. Jung HY, Jung KC, Shim YH, et al: Methylation of the hMLH1 promoter in multiple gastric carcinomas with microsatellite instability. Pathol Int. 51:445–451, 2001. 149. Endoh Y, Tamura G, Ajioka Y, et al: Frequent hypermethylation of the hMLH1 gene promoter in differentiated-type tumors of the stomach with the gastric foveolar phenotype. Am J Pathol. 157:717–722, 2000. 150. Ohmura K, Tamura G, Endoh Y, et al: Microsatellite alterations in differentiated-type adenocarcinomas and precancerous lesions of the stomach with special reference to cellular phenotype. Hum Pathol. 31:1031–1035, 2000. 151. Azumi N, Ben-Ezra J, Battifora H: Immunophenotypic diagnosis of leiomyosarcomas and rhabdomyosarcomas with monoclonal antibodies to muscle-specific actin and desmin in formalin-fixed tissue. Mod Pathol. 1:469–474, 1988. 152. Ueyama T, Nagai E, Yao T, et al: Vimentin-positive gastric carcinomas with rhabdoid features. A clinicopathologic and immunohistochemical study. Am J Surg Pathol. 17:813–819, 1993. 153. Nakayama Y, Murayama H, Iwasaki H, et al: Gastric carcinosarcoma (sarcomatoid carcinoma) with rhabdomyoblastic and osteoblastic differentiation. Pathol Int. 47:557–563, 1997.
154. Randjelovic T, Filipovic B, Babic D, et al: Carcinosarcoma of the stomach: a case report and review of the literature. World J Gastroenterol. 13:5533–5536, 2007. 155. Maitra A, Murakata LA, Albores-Saavedra J: Immunoreactivity for hepatocyte paraffin 1 antibody in hepatoid adenocarcinomas of the gastrointestinal tract. Am J Clin Pathol. 115:689–694, 2001. 156. Plaza JA, Vitellas K, Frankel WL: Hepatoid adenocarcinoma of the stomach. Ann Diagn Pathol. 8:137–141, 2004. 157. Roberts CC, Colby TV, Batts KP: Carcinoma of the stomach with hepatocyte differentiation (hepatoid adenocarcinoma). Mayo Clin Proc. 72:1154–1160, 1997. 158. Terracciano LM, Glatz K, Mhawech P, et al: Hepatoid adenocarcinoma with liver metastasis mimicking hepatocellular carcinoma: an immunohistochemical and molecular study of eight cases. Am J Surg Pathol. 27:1302–1312, 2003. 159. Villari D, Caruso R, Grosso M, et al: Hep Par 1 in gastric and bowel carcinomas: an immunohistochemical study. Pathology. 34:423–426, 2002. 160. Kumashiro Y, Yao T, Aishima S, et al: Hepatoid adenocarcinoma of the stomach: histogenesis and progression in association with intestinal phenotype. Hum Pathol. 38:857–863, 2007. 161. Jan YJ, Chen JT, Ho WL, et al: Primary coexistent adenocarcinoma and choriocarcinoma of the stomach. A case report and review of the literature. J Clin Gastroenterol. 25:550–554, 1997. 162. Saigo PE, Brigati DJ, Sternberg SS, et al: Primary gastric choriocarcinoma. An immunohistological study. Am J Surg Pathol. 5:333–342, 1981. 163. Hirano Y, Hara T, Nozawa H, et al: Combined choriocarcinoma, neuroendocrine cell carcinoma and tubular adenocarcinoma in the stomach. World J Gastroenterol. 14:3269–3272, 2008. 164. Blumenfeld W, Chandhoke DK, Sagerman P, et al: Neuroendocrine differentiation in gastric adenocarcinomas. An immunohistochemical study. Arch Pathol Lab Med. 120:478–481, 1996. 165. Park JG, Choe GY, Helman LJ, et al: Chromogranin-A expression in gastric and colon cancer tissues. Int J Cancer. 51:189–194, 1992. 166. Qvigstad G, Sandvik AK, Brenna E, et al: Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique. Histochem J. 32:551–556, 2000. 167. Bhargava R, Beriwal S, Dabbs DJ: Mammaglobin vs GCDFP-15: an immunohistologic validation survey for sensitivity and specificity. Am J Clin Pathol. 127:103–113, 2007. 168. Battifora H: Metastatic breast carcinoma to the stomach simulating linitis plastica. Appl Immunohistochem. 2:225–228, 1994. 169. Brown RW, Campagna LB, Dunn JK, et al: Immunohistochemical identification of tumor markers in metastatic adenocarcinoma. A diagnostic adjunct in the determination of primary site. Am J Clin Pathol. 107:12–19, 1997. 170. Perry A, Parisi JE, Kurtin PJ: Metastatic adenocarcinoma to the brain: an immunohistochemical approach. Hum Pathol. 28:938– 943, 1997. 171. Raju U, Ma CK, Shaw A: Signet ring variant of lobular carcinoma of the breast: a clinicopathologic and immunohistochemical study. Mod Pathol. 6:516–520, 1993. 172. Sentani K, Oue N, Tashiro T, et al: Immunohistochemical staining of Reg IV and claudin-18 is useful in the diagnosis of gastrointestinal signet ring cell carcinoma. Am J Surg Pathol. 32:1182–1189, 2008. 173. Drier JK, Swanson PE, Cherwitz DL, et al: S100 protein immunoreactivity in poorly differentiated carcinomas. Immunohistochemical comparison with malignant melanoma. Arch Pathol Lab Med. 111:447–452, 1987. 174. Barbareschi M, Murer B, Colby TV, et al: CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs. Am J Surg Pathol. 27:141–149, 2003. 175. Sarbia M, Fritze F, Geddert H, et al: Differentiation between pancreaticobiliary and upper gastrointestinal adenocarcinomas: Is analysis of cytokeratin 17 expression helpful? Am J Clin Pathol. 128:255–259, 2007. 176. Loy TS, Quesenberry JT, Sharp SC: Distribution of CA 125 in adenocarcinomas. An immunohistochemical study of 481 cases. Am J Clin Pathol. 98:175–179, 1992.
References 177. Merchant SH, Amin MB, Tamboli P, et al: Primary signet-ring cell carcinoma of lung: immunohistochemical study and comparison with non-pulmonary signet-ring cell carcinomas. Am J Surg Pathol. 25:1515–1519, 2001. 178. Harlamert HA, Mira J, Bejarano PA, et al: Thyroid transcription factor-1 and cytokeratins 7 and 20 in pulmonary and breast carcinoma. Acta Cytol. 42:1382–1388, 1998. 179. Loy TS, Calaluce RD: Utility of cytokeratin immunostaining in separating pulmonary adenocarcinomas from colonic adenocarcinomas. Am J Clin Pathol. 102:764–767, 1994. 180. Moch H, Oberholzer M, Dalquen P, et al: Diagnostic tools for differentiating between pleural mesothelioma and lung adenocarcinoma in paraffin embedded tissue. Part I: Immunohistochemical findings. Virchows Arch A Pathol Anat Histopathol. 423:19–27, 1993. 181. Bejarano PA, Baughman RP, Biddinger PW, et al: Surfactant proteins and thyroid transcription factor-1 in pulmonary and breast carcinomas. Mod Pathol. 9:445–452, 1996. 182. Di Loreto C, Puglisi F, Di Lauro V, et al: TTF-1 protein expression in pleural malignant mesotheliomas and adenocarcinomas of the lung. Cancer Lett. 124:73–78, 1998. 183. Turner BM, Cagle PT, Sainz IM, et al: Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med. 136:163–171, 2012. 184. Rossi G, Marchioni A, Romagnani E, et al: Primary lung cancer presenting with gastrointestinal tract involvement: clinicopathologic and immunohistochemical features in a series of 18 consecutive cases. J Thorac Oncol. 2:115–120, 2007. 185. Jagirdar J: Application of immunohistochemistry to the diagnosis of primary and metastatic carcinoma to the lung. Arch Pathol Lab Med. 132:384–396, 2008. 186. Ferguson A, Arranz E, O’Mahony S: Clinical and pathological spectrum of coeliac disease—active, silent, latent, potential. Gut. 34:150–151, 1993. 187. Chen ZM, Wang HL: Alteration of cytokeratin 7 and cytokeratin 20 expression profile is uniquely associated with tumorigenesis of primary adenocarcinoma of the small intestine. Am J Surg Pathol. 28:1352–1359, 2004. 188. Zhang MQ, Lin F, Hui P, et al: Expression of mucins, SIMA, villin, and CDX2 in small-intestinal adenocarcinoma. Am J Clin Pathol. 128:808–816, 2007. 189. Chen ZM, Ritter JH, Wang HL: Differential expression of alphamethylacyl coenzyme A racemase in adenocarcinomas of the small and large intestines. Am J Surg Pathol. 29:890–896, 2005. 190. Planck M, Ericson K, Piotrowska Z, et al: Microsatellite instability and expression of MLH1 and MSH2 in carcinomas of the small intestine. Cancer. 97:1551–1557, 2003. 191. Svrcek M, Jourdan F, Sebbagh N, et al: Immunohistochemical analysis of adenocarcinoma of the small intestine: a tissue microarray study. J Clin Pathol. 56:898–903, 2003. 192. Guerrieri C, Franlund B, Boeryd B: Expression of cytokeratin 7 in simultaneous mucinous tumors of the ovary and appendix. Mod Pathol. 8:573–576, 1995. 193. Guerrieri C, Franlund B, Fristedt S, et al: Mucinous tumors of the vermiform appendix and ovary, and pseudomyxoma peritonei: histogenetic implications of cytokeratin 7 expression. Hum Pathol. 28:1039–1045, 1997. 194. Ji H, Isacson C, Seidman JD, et al: Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 21:391–400, 2002. 195. Ronnett BM, Kurman RJ, Shmookler BM, et al: The morphologic spectrum of ovarian metastases of appendiceal adenocarcinomas: a clinicopathologic and immunohistochemical analysis of tumors often misinterpreted as primary ovarian tumors or metastatic tumors from other gastrointestinal sites. Am J Surg Pathol. 21:1144–1155, 1997. 196. Ronnett BM, Shmookler BM, Diener-West M, et al: Immunohistochemical evidence supporting the appendiceal origin of pseudomyxoma peritonei in women. Int J Gynecol Pathol. 16:1– 9, 1997.
539.e5
197. Seidman JD, Elsayed AM, Sobin LH, et al: Association of mucinous tumors of the ovary and appendix. A clinicopathologic study of 25 cases. Am J Surg Pathol. 17:22–34, 1993. 198. O’Connell JT, Hacker CM, Barsky SH: MUC2 is a molecular marker for pseudomyxoma peritonei. Mod Pathol. 15:958–972, 2002. 199. Tornillo L, Moch H, Diener PA, et al: CDX-2 immunostaining in primary and secondary ovarian carcinomas. J Clin Pathol. 57:641–643, 2004. 200. Lee KR, Young RH: The distinction between primary and metastatic mucinous carcinomas of the ovary: gross and histologic findings in 50 cases. Am J Surg Pathol. 27:281–292, 2003. 201. Seidman JD, Kurman RJ, Ronnett BM: Primary and metastatic mucinous adenocarcinomas in the ovaries: incidence in routine practice with a new approach to improve intraoperative diagnosis. Am J Surg Pathol. 27:985–993, 2003. 202. Fraggetta F, Pelosi G, Cafici A, et al: CDX2 immunoreactivity in primary and metastatic ovarian mucinous tumours. Virchows Arch. 443:782–786, 2003. 203. Kapur RP, Reed RC, Finn L, et al: Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung disease. Pediatr Dev Pathol 2008:1. 204. MacKenzie JM, Dixon MF: An immunohistochemical study of the enteric neural plexi in Hirschsprung’s disease. Histopathology. 11:1055–1066, 1987. 205. Wester T, Olsson Y, Olsen L: Expression of bcl-2 in enteric neurons in normal human bowel and Hirschsprung disease. Arch Pathol Lab Med. 123:1264–1268, 1999. 206. Monforte-Munoz H, Gonzalez-Gomez I, Rowland JM, et al: Increased submucosal nerve trunk caliber in aganglionosis: a “positive” and objective finding in suction biopsies and segmental resections in Hirschsprung’s disease. Arch Pathol Lab Med. 122:721–725, 1998. 207. Mochizuka A, Uehara T, Nakamura T, et al: Hyperplastic polyps and sessile serrated ‘adenomas’ of the colon and rectum display gastric pyloric differentiation. Histochem Cell Biol. 128:445–455, 2007. 208. Sheridan TB, Fenton H, Lewin MR, et al: Sessile serrated adenomas with low- and high-grade dysplasia and early carcinomas: an immunohistochemical study of serrated lesions “caught in the act.” Am J Clin Pathol. 126:564–571, 2006. 209. Kawasaki T, Nosho K, Ohnishi M, et al: Cyclooxygenase-2 overexpression is common in serrated and non-serrated colorectal adenoma, but uncommon in hyperplastic polyp and sessile serrated polyp/adenoma. BMC Cancer. 8:33, 2008. 210. Torlakovic EE, Gomez JD, Driman DK, et al: Sessile serrated adenoma (SSA) vs. traditional serrated adenoma (TSA). Am J Surg Pathol. 32:21–29, 2008. 211. Wong NA, Mayer NJ, MacKell S, et al: Immunohistochemical assessment of Ki67 and p53 expression assists the diagnosis and grading of ulcerative colitis-related dysplasia. Histopathology. 37:108–114, 2000. 212. Harpaz N, Peck AL, Yin J, et al: p53 protein expression in ulcerative colitis-associated colorectal dysplasia and carcinoma. Hum Pathol. 25:1069–1074, 1994. 213. Bruwer M, Schmid KW, Senninger N, et al: Immunohistochemical expression of P53 and oncogenes in ulcerative colitisassociated colorectal carcinoma. World J Surg. 26:390–396, 2002. 214. Marx A, Wandrey T, Simon P, et al: Combined alpha-methylacyl coenzyme A racemase/p53 analysis to identify dysplasia in inflammatory bowel disease. Hum Pathol. 40:166–173, 2009. 215. Andersen SN, Rognum TO, Bakka A, et al: Ki-67: a useful marker for the evaluation of dysplasia in ulcerative colitis. Mol Pathol. 51:327–332, 1998. 216. Sjoqvist U, Ost A, Lofberg R: Increased expression of proliferative Ki-67 nuclear antigen is correlated with dysplastic colorectal epithelium in ulcerative colitis. Int J Colorectal Dis. 14:107–113, 1999. 217. Dorer R, Odze RD: AMACR immunostaining is useful in detecting dysplastic epithelium in Barrett’s esophagus, ulcerative colitis, and Crohn’s disease. Am J Surg Pathol. 30:871–877, 2006. 218. Strater J, Wiesmuller C, Perner S, et al: Alpha-methylacyl-CoA racemase (AMACR) immunohistochemistry in Barrett’s and
539.e6
Immunohistology of the Gastrointestinal Tract
colorectal mucosa: only significant overexpression favours a diagnosis of intraepithelial neoplasia. Histopathology. 52:399– 402, 2008. 219. Maeda T, Kajiyama K, Adachi E, et al: The expression of cytokeratins 7, 19, and 20 in primary and metastatic carcinomas of the liver. Mod Pathol. 9:901–909, 1996. 220. O’Hara BJ, Paetau A, Miettinen M: Keratin subsets and monoclonal antibody HBME-1 in chordoma: immunohistochemical differential diagnosis between tumors simulating chordoma. Hum Pathol. 29:119–126, 1998. 221. Thomas P, Battifora H: Keratins versus epithelial membrane antigen in tumor diagnosis: an immunohistochemical comparison of five monoclonal antibodies. Hum Pathol. 18:728–734, 1987. 222. Berezowski K, Stastny JF, Kornstein MJ: Cytokeratins 7 and 20 and carcinoembryonic antigen in ovarian and colonic carcinoma. Mod Pathol. 9:426–429, 1996. 223. Tot T: Cytokeratins 20 and 7 as biomarkers: usefulness in discriminating primary from metastatic adenocarcinoma. Eur J Cancer. 38:758–763, 2002. 224. Vang R, Gown AM, Barry TS, et al: Cytokeratins 7 and 20 in primary and secondary mucinous tumors of the ovary: analysis of coordinate immunohistochemical expression profiles and staining distribution in 179 cases. Am J Surg Pathol. 30:1130– 1139, 2006. 225. Lagendijk JH, Mullink H, Van Diest PJ, et al: Tracing the origin of adenocarcinomas with unknown primary using immunohistochemistry: differential diagnosis between colonic and ovarian carcinomas as primary sites. Hum Pathol. 29:491–497, 1998. 226. Moskaluk CA, Zhang H, Powell SM, et al: Cdx2 protein expression in normal and malignant human tissues: an immunohistochemical survey using tissue microarrays. Mod Pathol. 16: 913–919, 2003. 227. De Lott LB, Morrison C, Suster S, et al: CDX2 is a useful marker of intestinal-type differentiation: a tissue microarray-based study of 629 tumors from various sites. Arch Pathol Lab Med. 129:1100–1105, 2005. 228. Suh N, Yang XJ, Tretiakova MS, et al: Value of CDX2, villin, and alpha-methylacyl coenzyme A racemase immunostains in the distinction between primary adenocarcinoma of the bladder and secondary colorectal adenocarcinoma. Mod Pathol. 18:1217– 1222, 2005. 229. Vang R, Gown AM, Wu LS, et al: Immunohistochemical expression of CDX2 in primary ovarian mucinous tumors and metastatic mucinous carcinomas involving the ovary: comparison with CK20 and correlation with coordinate expression of CK7. Mod Pathol. 19:1421–1428, 2006. 230. Inamura K, Satoh Y, Okumura S, et al: Pulmonary adenocarcinomas with enteric differentiation: histologic and immunohistochemical characteristics compared with metastatic colorectal cancers and usual pulmonary adenocarcinomas. Am J Surg Pathol. 29:660–665, 2005. 231. Proca DM, Niemann TH, Porcell AI, et al: MOC31 immunoreactivity in primary and metastatic carcinoma of the liver. Report of findings and review of other utilized markers. Appl Immunohistochem Mol Morphol. 8:120–125, 2000. 232. Wendum D, Svrcek M, Rigau V, et al: COX-2, inflammatory secreted PLA2, and cytoplasmic PLA2 protein expression in small bowel adenocarcinomas compared with colorectal adenocarcinomas. Mod Pathol. 16:130–136, 2003. 233. Loy TS, Sharp SC, Andershock CJ, et al: Distribution of CA 19-9 in adenocarcinomas and transitional cell carcinomas. An immunohistochemical study of 527 cases. Am J Clin Pathol. 99:726– 728, 1993. 234. Greenson JK, Huang SC, Herron C, et al: Pathologic predictors of microsatellite instability in colorectal cancer. Am J Surg Pathol. 33:126–133, 2009. 235. Hampel H, Frankel WL, Martin E, et al: Feasibility of screening for lynch syndrome among patients with colorectal cancer. J Clin Oncol. 26:5783–5788, 2008. 236. Wright CL, Stewart ID: Histopathology and mismatch repair status of 458 consecutive colorectal carcinomas. Am J Surg Pathol. 27:1393–1406, 2003. 237. Jover R, Paya A, Alenda C, et al: Defective mismatchrepair colorectal cancer: clinicopathologic characteristics and
usefulness of immunohistochemical analysis for diagnosis. Am J Clin Pathol. 122:389–394, 2004. 238. Shia J: Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn. 10:293–300, 2008. 239. Geiersbach KB, Samowitz WS: Microsatellite instability and colorectal cancer. Arch Pathol Lab Med. 135:1269–1277, 2011. 240. Shia J, Tang LH, Vakiani E, et al: Immunohistochemistry as firstline screening for detecting colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome: a 2-antibody panel may be as predictive as a 4-antibody panel. Am J Surg Pathol. 33:1639–1645, 200. 241. Mojtahed A, Schrijver I, Ford JM, et al: A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas, skin sebaceous tumors, and gynecologic tract carcinomas. Mod Pathol. 24:1004–1014, 2011. 242. Bao F, Panarelli NC, Rennert H, et al: Neoadjuvant therapy induces loss of MSH6 expression in colorectal carcinoma. Am J Surg Pathol. 34:1798–1804, 2010. 243. Radu OM, Nikiforova MN, Farkas LM, et al: Challenging cases encountered in colorectal cancer screening for Lynch syndrome reveal novel findings: nucleolar MSH6 staining and impact of prior chemoradiation therapy. Hum Pathol. 42:1247–1258, 2011. 244. Zhang L: Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part II. The utility of microsatellite instability testing. J Mol Diagn. 10:301–307, 2008. 245. Hinoi T, Tani M, Lucas PC, et al: Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon. Am J Pathol. 159:2239–2248, 2001. 246. McGregor DK, Wu TT, Rashid A, et al: Reduced expression of cytokeratin 20 in colorectal carcinomas with high levels of microsatellite instability. Am J Surg Pathol. 28:712–718, 2004. 247. Lugli A, Tzankov A, Zlobec I, et al: Differential diagnostic and functional role of the multi-marker phenotype CDX2/CK20/ CK7 in colorectal cancer stratified by mismatch repair status. Mod Pathol. 21:1403–1412, 2008. 248. Jewell LD, Barr JR, McCaughey WT, et al: Clear-cell epithelial neoplasms of the large intestine. Arch Pathol Lab Med. 112:197– 199, 1988. 249. Yang AH, Chen WY, Chiang H: Malignant rhabdoid tumour of colon. Histopathology. 24:89–91, 1994. 250. Amrikachi M, Ro JY, Ordonez NG, et al: Adenocarcinomas of the gastrointestinal tract with prominent rhabdoid features. Ann Diagn Pathol. 6:357–363, 2002. 251. Chang YL, Lee YC, Liao WY, et al: The utility and limitation of thyroid transcription factor-1 protein in primary and metastatic pulmonary neoplasms. Lung Cancer. 44:149–157, 2004. 252. Kaufmann O, Dietel M: Thyroid transcription factor-1 is the superior immunohistochemical marker for pulmonary adenocarcinomas and large cell carcinomas compared to surfactant proteins A and B. Histopathology. 36:8–16, 2000. 253. Moldvay J, Jackel M, Bogos K, et al: The role of TTF-1 in differentiating primary and metastatic lung adenocarcinomas. Pathol Oncol Res. 10:85–88, 2004. 254. Nicholson AG, McCormick CJ, Shimosato Y, et al: The value of PE-10, a monoclonal antibody against pulmonary surfactant, in distinguishing primary and metastatic lung tumours. Histopathology. 27:57–60, 1995. 255. Reis-Filho JS, Carrilho C, Valenti C, et al: Is TTF1 a good immunohistochemical marker to distinguish primary from metastatic lung adenocarcinomas? Pathol Res Pract. 196:835–840, 2000. 256. Srodon M, Westra WH: Immunohistochemical staining for thyroid transcription factor-1: a helpful aid in discerning primary site of tumor origin in patients with brain metastases. Hum Pathol. 33:642–645, 2002. 257. Zamecnik J, Kodet R: Value of thyroid transcription factor-1 and surfactant apoprotein A in the differential diagnosis of pulmonary carcinomas: a study of 109 cases. Virchows Arch. 440:353– 361, 2002.
References 258. Goldstein NS, Thomas M: Mucinous and nonmucinous bronchioloalveolar adenocarcinomas have distinct staining patterns with thyroid transcription factor and cytokeratin 20 antibodies. Am J Clin Pathol. 116:319–325, 2001. 259. Shah RN, Badve S, Papreddy K, et al: Expression of cytokeratin 20 in mucinous bronchioloalveolar carcinoma. Hum Pathol. 33:915–920, 2002. 260. Saad RS, Cho P, Silverman JF, et al: Usefulness of Cdx2 in separating mucinous bronchioloalveolar adenocarcinoma of the lung from metastatic mucinous colorectal adenocarcinoma. Am J Clin Pathol. 122:421–427, 2004. 261. Yatabe Y, Koga T, Mitsudomi T, et al: CK20 expression, CDX2 expression, K-ras mutation, and goblet cell morphology in a subset of lung adenocarcinomas. J Pathol. 203:645–652, 2004. 262. Loy TS, Calaluce RD, Keeney GL: Cytokeratin immunostaining in differentiating primary ovarian carcinoma from metastatic colonic adenocarcinoma. Mod Pathol. 9:1040–1044, 1996. 263. Young RH, Hart WR: Metastatic intestinal carcinomas simulating primary ovarian clear cell carcinoma and secretory endometrioid carcinoma: a clinicopathologic and immunohistochemical study of five cases. Am J Surg Pathol. 22:805–815, 1998. 264. Slattery ML, Samowitz WS, Holden JA: Estrogen and progesterone receptors in colon tumors. Am J Clin Pathol. 113:364–368, 2000. 265. Laury AR, Perets R, Piao H, et al: A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 35:816–826, 2011. 266. Ordonez NG: Value of thrombomodulin immunostaining in the diagnosis of mesothelioma. Histopathology. 31:25–30, 1997. 267. Tamboli P, Mohsin SK, Hailemariam S, et al: Colonic adenocarcinoma metastatic to the urinary tract versus primary tumors of the urinary tract with glandular differentiation: a report of 7 cases and investigation using a limited immunohistochemical panel. Arch Pathol Lab Med. 126:1057–1063, 2002. 268. Wang HL, Lu DW, Yerian LM, et al: Immunohistochemical distinction between primary adenocarcinoma of the bladder and secondary colorectal adenocarcinoma. Am J Surg Pathol. 25:1380–1387, 2001. 269. Goldstein NS: Immunophenotypic characterization of 225 prostate adenocarcinomas with intermediate or high Gleason scores. Am J Clin Pathol. 117:471–477, 2002. 270. Jiang Z, Fanger GR, Woda BA, et al: Expression of alphamethylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: a study of 761 cases. Hum Pathol. 34:792–796, 2003. 271. Torbenson M, Dhir R, Nangia A, et al: Prostatic carcinoma with signet ring cells: a clinicopathologic and immunohistochemical analysis of 12 cases, with review of the literature. Mod Pathol. 11:552–559, 1998. 272. Longacre TA, Kong CS, Welton ML: Diagnostic problems in anal pathology. Adv Anat Pathol. 15:263–278, 2008. 273. Owens SR, Greenson JK: Immunohistochemical staining for p63 is useful in the diagnosis of anal squamous cell carcinomas. Am J Surg Pathol. 31:285–290, 2007. 274. Chetty R, Serra S, Hsieh E: Basaloid squamous carcinoma of the anal canal with an adenoid cystic pattern: histologic and immunohistochemical reappraisal of an unusual variant. Am J Surg Pathol. 29:1668–1672, 2005. 275. Roma AA, Goldblum JR, Fazio V, et al: Expression of 14-33sigma, p16 and p53 proteins in anal squamous intraepithelial neoplasm and squamous cell carcinoma. Int J Clin Exp Pathol. 1:419–425, 2008. 276. Darragh TM, Colgan TJ, Cox JT, et al; Members of LAST Project Work Groups: The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med. 136:1266– 1297, 2012. 277. Hobbs CM, Lowry MA, Owen D, et al: Anal gland carcinoma. Cancer. 92:2045–2049, 2001. 278. Lisovsky M, Patel K, Cymes K, et al: Immunophenotypic characterization of anal gland carcinoma: loss of p63 and cytokeratin 5/6. Arch Pathol Lab Med. 131:1304–1311, 2007.
539.e7
279. Battles OE, Page DL, Johnson JE: Cytokeratins, CEA, and mucin histochemistry in the diagnosis and characterization of extramammary Paget’s disease. Am J Clin Pathol. 108:6–12, 1997. 280. Helm KF, Goellner JR, Peters MS: Immunohistochemical stains in extramammary Paget’s disease. Am J Dermatopathol. 14:402– 407, 1992. 281. Smith KJ, Tuur S, Corvette D, et al: Cytokeratin 7 staining in mammary and extramammary Paget’s disease. Mod Pathol. 10:1069–1074, 1997. 282. Nowak MA, Guerriere-Kovach P, Pathan A, et al: Perianal Paget’s disease: distinguishing primary and secondary lesions using immunohistochemical studies including gross cystic disease fluid protein-15 and cytokeratin 20 expression. Arch Pathol Lab Med. 122:1077–1081, 1998. 283. Ohnishi T, Watanabe S: The use of cytokeratins 7 and 20 in the diagnosis of primary and secondary extramammary Paget’s disease. Br J Dermatol. 142:243–247, 2000. 284. Ramalingam P, Hart WR, Goldblum JR: Cytokeratin subset immunostaining in rectal adenocarcinoma and normal anal glands. Arch Pathol Lab Med. 125:1074–1077, 2001. 285. Kondo Y, Kashima K, Daa T, et al: The ectopic expression of gastric mucin in extramammary and mammary Paget’s disease. Am J Surg Pathol. 26:617–623, 2002. 286. Kuan SF, Montag AG, Hart J, et al: Differential expression of mucin genes in mammary and extramammary Paget’s disease. Am J Surg Pathol. 25:1469–1477, 2001. 287. Williams ED, Sandler M: The classification of carcinoid tumours. Lancet. 1:238–239, 1963. 288. Cai YC, Banner B, Glickman J, et al: Cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors. Hum Pathol. 32:1087–1093, 2001. 289. Srivastava A, Padilla O, Fischer-Colbrie R, et al: Neuroendocrine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids. Am J Surg Pathol. 28:1371–1378, 2004. 290. Solcia E, Kloppel G, Sobin LH: World Health Organization International Histological Classification of Tumours: Histological Typing of Endocrine Tumours, ed 2, Berlin, 2000, Springer. 291. Capella C, Solcia E, Sobin LH, et al: Endocrine tumours of the oesophagus. In Hamilton SR, Aaltonen LA, editors: Pathology and genetics, tumours of the digestive system: WHO classification of tumours, Lyon, France, 2000, IARC, pp 26–27. 292. Hoang MP, Hobbs CM, Sobin LH, et al: Carcinoid tumor of the esophagus: a clinicopathologic study of four cases. Am J Surg Pathol. 26:517–522, 2002. 293. Kloppel G, Rindi G, Anlauf M, et al: Site-specific biology and pathology of gastroenteropancreatic neuroendocrine tumors. Virchows Arch. 451(Suppl 1):S9–27, 2007. 294. Osugi H, Takemura M, Morimura K, et al: Clinicopathologic and immunohistochemical features of surgically resected small cell carcinoma of the esophagus. Oncol Rep. 9:1245–1249, 2002. 295. Takubo K, Nakamura K, Sawabe M, et al: Primary undifferentiated small cell carcinoma of the esophagus. Hum Pathol. 30:216– 221, 1999. 296. Yamamoto J, Ohshima K, Ikeda S, et al: Primary esophageal small cell carcinoma with concomitant invasive squamous cell carcinoma or carcinoma in situ. Hum Pathol. 34:1108–1115, 2003. 297. Cheuk W, Chan JK: Thyroid transcription factor-1 is of limited value in practical distinction between pulmonary and extrapulmonary small cell carcinomas. Am J Surg Pathol. 25:545–546, 2001. 298. Cheuk W, Kwan MY, Suster S, et al: Immunostaining for thyroid transcription factor 1 and cytokeratin 20 aids the distinction of small cell carcinoma from Merkel cell carcinoma, but not pulmonary from extrapulmonary small cell carcinomas. Arch Pathol Lab Med. 125:228–231, 2001. 299. Rindi G, Paolotti D, Fiocca R, et al: Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors. Virchows Arch. 436:217–223, 2000. 300. Saqi A, Alexis D, Remotti F, et al: Usefulness of CDX2 and TTF-1 in differentiating gastrointestinal from pulmonary carcinoids. Am J Clin Pathol. 123:394–404, 2005.
539.e8
Immunohistology of the Gastrointestinal Tract
301. Oliveira AM, Tazelaar HD, Myers JL, et al: Thyroid transcription factor-1 distinguishes metastatic pulmonary from welldifferentiated neuroendocrine tumors of other sites. Am J Surg Pathol. 25:815–819, 2001. 302. Srivastava A, Hornick JL: Immunohistochemical staining for CDX-2, PDX-1, NESP-55, and TTF-1 can help distinguish gastrointestinal carcinoid tumors from pancreatic endocrine and pulmonary carcinoid tumors. Am J Surg Pathol. 33:626–632, 2009. 303. Barbareschi M, Roldo C, Zamboni G, et al: CDX-2 homeobox gene product expression in neuroendocrine tumors: its role as a marker of intestinal neuroendocrine tumors. Am J Surg Pathol. 28:1169–1176, 2004. 304. Jaffee IM, Rahmani M, Singhal MG, et al: Expression of the intestinal transcription factor CDX2 in carcinoid tumors is a marker of midgut origin. Arch Pathol Lab Med. 130:1522–1526, 2006. 305. La Rosa S, Rigoli E, Uccella S, et al: CDX2 as a marker of intestinal EC-cells and related well-differentiated endocrine tumors. Virchows Arch. 445:248–254, 2004. 306. Alsaad KO, Serra S, Schmitt A, et al: Cytokeratins 7 and 20 immunoexpression profile in goblet cell and classical carcinoids of appendix. Endocr Pathol. 18:16–22, 2007. 307. Burke AP, Sobin LH, Federspiel BH, et al: Appendiceal carcinoids: correlation of histology and immunohistochemistry. Mod Pathol. 2:630–637, 1989. 308. Moyana TN, Satkunam N: A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis. Cancer. 70:1081–1088, 1992. 309. Tang LH, Shia J, Soslow RA, et al: Pathologic classification and clinical behavior of the spectrum of goblet cell carcinoid tumors of the appendix. Am J Surg Pathol. 32:1429–1443, 2008. 310. Azumi N, Battifora H: The distribution of vimentin and keratin in epithelial and nonepithelial neoplasms. A comprehensive immunohistochemical study on formalin- and alcohol-fixed tumors. Am J Clin Pathol. 88:286–296, 1987. 311. Federspiel BH, Burke AP, Sobin LH, et al: Rectal and colonic carcinoids. A clinicopathologic study of 84 cases. Cancer. 65:135–140, 1990. 312. Fahrenkamp AG, Wibbeke C, Winde G, et al: Immunohistochemical distribution of chromogranins A and B and secretogranin II in neuroendocrine tumours of the gastrointestinal tract. Virchows Arch. 426:361–367, 1995. 313. Nash SV, Said JW: Gastroenteropancreatic neuroendocrine tumors. A histochemical and immunohistochemical study of epithelial (keratin proteins, carcinoembryonic antigen) and neuroendocrine (neuron-specific enolase, bombesin and chromogranin) markers in foregut, midgut, and hindgut tumors. Am J Clin Pathol. 86:415–422, 1986. 314. Kaufmann O, Dietel M: Expression of thyroid transcription factor-1 in pulmonary and extrapulmonary small cell carcinomas and other neuroendocrine carcinomas of various primary sites. Histopathology. 36:415–420, 2000. 315. Bernick PE, Klimstra DS, Shia J, et al: Neuroendocrine carcinomas of the colon and rectum. Dis Colon Rectum. 47:163–169, 2004. 316. Sarsfield P, Anthony PP: Small cell undifferentiated (‘neuroendocrine’) carcinoma of the colon. Histopathology. 16:357–363, 1990. 317. Akintola-Ogunremi O, Pfeifer JD, Tan BR, et al: Analysis of protein expression and gene mutation of c-kit in colorectal neuroendocrine carcinomas. Am J Surg Pathol. 27:1551–1558, 2003. 318. Ishikubo T, Akagi K, Kurosumi M, et al: Immunohistochemical and mutational analysis of c-kit in gastrointestinal neuroendocrine cell carcinoma. Jpn J Clin Oncol. 36:494–498, 2006. 319. Allander SV, Nupponen NN, Ringner M, et al: Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res. 61:8624–8628, 2001. 320. Bernet L, Zuniga A, Cano R: Characterization of GIST/GIPACT tumors by inmunohistochemistry and exon 11 analysis of c-kit by PCR. Rev Esp Enferm Dig. 95:688–691:683–687, 2003. 321. Kitamura Y, Hirota S, Nishida T: Molecular pathology of c-kit proto-oncogene and development of gastrointestinal stromal tumors. Ann Chir Gynaecol. 87:282–286, 1998.
322. Miettinen M, Lasota J: Gastrointestinal stromal tumors (GISTs): definition, occurrence, pathology, differential diagnosis and molecular genetics. Pol J Pathol. 54:3–24, 2003. 323. Rubin BP, Singer S, Tsao C, et al: KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res. 61:8118– 8121, 2001. 324. Taniguchi M, Nishida T, Hirota S, et al: Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors. Cancer Res. 59:4297–4300, 1999. 325. Miettinen M, Lasota J: Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med. 130:1466–1478, 2006. 326. Antonioli DA: Gastrointestinal autonomic nerve tumors. Expanding the spectrum of gastrointestinal stromal tumors. Arch Pathol Lab Med. 113:831–833, 1989. 327. Fuller CE, Williams GT: Gastrointestinal manifestations of type 1 neurofibromatosis (von Recklinghausen’s disease). Histopathology. 19:1–11, 1991. 328. Lee JR, Joshi V, Griffin JW, Jr, et al: Gastrointestinal autonomic nerve tumor: immunohistochemical and molecular identity with gastrointestinal stromal tumor. Am J Surg Pathol. 25:979–987, 2001. 329. Miettinen M, El-Rifai W, Sobin L, et al: Evaluation of malignancy and prognosis of gastrointestinal stromal tumors: a review. Hum Pathol. 33:478–483, 2002. 330. Chan JK: Mesenchymal tumors of the gastrointestinal tract: a paradise for acronyms (STUMP, GIST, GANT, and now GIPACT), implication of c-kit in genesis, and yet another of the many emerging roles of the interstitial cell of Cajal in the pathogenesis of gastrointestinal diseases? Adv Anat Pathol. 6:19–40, 1999. 331. Sircar K, Hewlett BR, Huizinga JD, et al: Interstitial cells of Cajal as precursors of gastrointestinal stromal tumors. Am J Surg Pathol. 23:377–389, 1999. 332. Yamaguchi U, Hasegawa T, Masuda T, et al: Differential diagnosis of gastrointestinal stromal tumor and other spindle cell tumors in the gastrointestinal tract based on immunohistochemical analysis. Virchows Arch. 445:142–150, 2004. 333. Corless CL, Schroeder A, Griffith D, et al: PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol. 23:5357–5364, 2005. 334. Lasota J, Dansonka-Mieszkowska A, Sobin LH, et al: A great majority of GISTs with PDGFRA mutations represent gastric tumors of low or no malignant potential. Lab Invest. 84:874– 883, 2004. 335. Duensing A, Heinrich MC, Fletcher CD, et al: Biology of gastrointestinal stromal tumors: KIT mutations and beyond. Cancer Invest. 22:106–116, 2004. 336. Duensing A, Medeiros F, McConarty B, et al: Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs). Oncogene. 23:3999–4006, 2004. 337. Hornick JL, Fletcher CD: The significance of KIT (CD117) in gastrointestinal stromal tumors. Int J Surg Pathol. 12:93–97, 2004. 338. Guler ML, Daniels JA, Abraham SC, et al: Expression of melanoma antigens in epithelioid gastrointestinal stromal tumors: a potential diagnostic pitfall. Arch Pathol Lab Med. 132:1302– 1306, 2008. 339. Memeo L, Jhang J, Assaad AM, et al: Immunohistochemical analysis for cytokeratin 7, KIT, and PAX2: value in the differential diagnosis of chromophobe cell carcinoma. Am J Clin Pathol. 127:225–229, 2007. 340. Nikolaou M, Valavanis C, Aravantinos G, et al: Kit expression in male germ cell tumors. Anticancer Res. 27:1685–1688, 2007. 341. Hornick JL, Fletcher CD: Immunohistochemical staining for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Am J Clin Pathol. 117:188–193, 2002. 342. Miettinen M, Lasota J: KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol. 13:205–220, 2005. 343. Parfitt JR, Rodriguez-Justo M, Feakins R, et al: Gastrointestinal Kaposi’s sarcoma: CD117 expression and the potential for misdiagnosis as gastrointestinal stromal tumour. Histopathology. 52:816–823, 2008.
References 344. Yantiss RK, Spiro IJ, Compton CC, et al: Gastrointestinal stromal tumor versus intra-abdominal fibromatosis of the bowel wall: a clinically important differential diagnosis. Am J Surg Pathol. 24:947–957, 2000. 345. Lucas DR, al-Abbadi M, Tabaczka P, et al: c-Kit expression in desmoid fibromatosis. Comparative immunohistochemical evaluation of two commercial antibodies. Am J Clin Pathol. 119:339– 345, 2003. 346. Miettinen M: Are desmoid tumors kit positive? Am J Surg Pathol. 25:549–550, 2001. 347. West RB, Corless CL, Chen X, et al: The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status. Am J Pathol. 165:107–113, 2004. 348. Miettinen M, Wang ZF, Lasota J: DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study of 1840 cases. Am J Surg Pathol. 33:1401–1408, 2009. 349. Dhimes P, Lopez-Carreira M, Ortega-Serrano MP, et al: Gastrointestinal autonomic nerve tumours and their separation from other gastrointestinal stromal tumours: an ultrastructural and immunohistochemical study of seven cases. Virchows Arch. 426:27–35, 1995. 350. Franquemont DW, Frierson HF, Jr: Muscle differentiation and clinicopathologic features of gastrointestinal stromal tumors. Am J Surg Pathol. 16:947–954, 1992. 351. Greenson JK: Gastrointestinal stromal tumors and other mesenchymal lesions of the gut. Mod Pathol. 16:366–375, 2003. 352. Herrera GA, Cerezo L, Jones JE, et al: Gastrointestinal autonomic nerve tumors. ‘Plexosarcomas’. Arch Pathol Lab Med. 113:846–853, 1989. 353. Miettinen M: Gastrointestinal stromal tumors. An immunohistochemical study of cellular differentiation. Am J Clin Pathol. 89:601–610, 1988. 354. Miettinen M, Monihan JM, Sarlomo-Rikala M, et al: Gastrointestinal stromal tumors/smooth muscle tumors (GISTs) primary in the omentum and mesentery: clinicopathologic and immunohistochemical study of 26 cases. Am J Surg Pathol. 23:1109– 1118, 1999. 355. Miettinen M, Sobin LH, Sarlomo-Rikala M: Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with a reference to CD117 (KIT). Mod Pathol. 13:1134–1142, 2000. 356. Miettinen M, Virolainen M, Maarit Sarlomo R: Gastrointestinal stromal tumors—value of CD34 antigen in their identification and separation from true leiomyomas and schwannomas. Am J Surg Pathol. 19:207–216, 1995. 357. Saul SH, Rast ML, Brooks JJ: The immunohistochemistry of gastrointestinal stromal tumors. Evidence supporting an origin from smooth muscle. Am J Surg Pathol. 11:464–473, 1987. 358. Shek TW, Luk IS, Loong F, et al: Inflammatory cell-rich gastrointestinal autonomic nerve tumor. An expansion of its histologic spectrum. Am J Surg Pathol. 20:325–331, 1996. 359. Suster S: Gastrointestinal stromal tumors. Semin Diagn Pathol. 13:297–313, 1996. 360. Suster S, Fletcher CD: Gastrointestinal stromal tumors with prominent signet-ring cell features. Mod Pathol. 9:609–613, 1996. 361. Tazawa K, Tsukada K, Makuuchi H, et al: An immunohistochemical and clinicopathological study of gastrointestinal stromal tumors. Pathol Int. 49:786–798, 1999. 362. Tworek JA, Goldblum JR, Weiss SW, et al: Stromal tumors of the anorectum: a clinicopathologic study of 22 cases. Am J Surg Pathol. 23:946–954, 1999. 363. Tworek JA, Goldblum JR, Weiss SW, et al: Stromal tumors of the abdominal colon: a clinicopathologic study of 20 cases. Am J Surg Pathol. 23:937–945, 1999. 364. Wong NA, Young R, Malcomson RD, et al: Prognostic indicators for gastrointestinal stromal tumours: a clinicopathological and immunohistochemical study of 108 resected cases of the stomach. Histopathology. 43:118–126, 2003. 365. Yao T, Aoyagi K, Hizawa K: Gastric epithelioid tumor (leiomyoma) with granular changes. Int J Surg Pathol. 4:37–42, 1996. 366. Natkunam Y, Rouse RV, Zhu S, et al: Immunoblot analysis of CD34 expression in histologically diverse neoplasms. Am J Pathol. 156:21–27, 2000.
539.e9
367. Miettinen M, Furlong M, Sarlomo-Rikala M, et al: Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the rectum and anus: a clinicopathologic, immunohistochemical, and molecular genetic study of 144 cases. Am J Surg Pathol. 25:1121–1133, 2001. 368. Miettinen M, Lasota J: Gastrointestinal stromal tumors— definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch. 438:1–12, 2001. 369. Zhu X, Zhang XQ, Li BM, et al: Esophageal mesenchymal tumors: endoscopy, pathology and immunohistochemistry. World J Gastroenterol. 13:768–773, 2007. 370. Miettinen MM, Sarlomo-Rikala M, Kovatich AJ, et al: Calponin and h-caldesmon in soft tissue tumors: consistent h-caldesmon immunoreactivity in gastrointestinal stromal tumors indicates traits of smooth muscle differentiation. Mod Pathol. 12:756–762, 1999. 371. Fletcher CD, Berman JJ, Corless C, et al: Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol. 33:459–465, 2002. 372. Prevot S, Bienvenu L, Vaillant JC, et al: Benign schwannoma of the digestive tract: a clinicopathologic and immunohistochemical study of five cases, including a case of esophageal tumor. Am J Surg Pathol. 23:431–436, 1999. 373. Sarlomo-Rikala M, Kovatich AJ, Barusevicius A, et al: CD117: a sensitive marker for gastrointestinal stromal tumors that is more specific than CD34. Mod Pathol. 11:728–734, 1998. 374. Fanburg-Smith JC, Meis-Kindblom JM, Fante R, et al: Malignant granular cell tumor of soft tissue: diagnostic criteria and clinicopathologic correlation. Am J Surg Pathol. 22:779–794, 1998. 375. Goldblum JR, Rice TW, Zuccaro G, et al: Granular cell tumors of the esophagus: a clinical and pathologic study of 13 cases. Ann Thorac Surg. 62:860–865, 1996. 376. Montgomery E, Torbenson MS, Kaushal M, et al: Beta-catenin immunohistochemistry separates mesenteric fibromatosis from gastrointestinal stromal tumor and sclerosing mesenteritis. Am J Surg Pathol. 26:1296–1301, 2002. 377. Bhattacharya B, Dilworth HP, Iacobuzio-Donahue C, et al: Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions. Am J Surg Pathol. 29:653–659, 2005. 378. Carlson JW, Fletcher CD: Immunohistochemistry for betacatenin in the differential diagnosis of spindle cell lesions: analysis of a series and review of the literature. Histopathology. 51:509–514, 2007. 379. Ng TL, Gown AM, Barry TS, et al: Nuclear beta-catenin in mesenchymal tumors. Mod Pathol. 18:68–74, 2005. 380. Fukunaga M, Naganuma H, Nikaido T, et al: Extrapleural solitary fibrous tumor: a report of seven cases. Mod Pathol. 10:443–450, 1997. 381. Fukunaga M, Naganuma H, Ushigome S, et al: Malignant solitary fibrous tumour of the peritoneum. Histopathology. 28:463–466, 1996. 382. Young RH, Clement PB, McCaughey WT: Solitary fibrous tumors (‘fibrous mesotheliomas’) of the peritoneum. A report of three cases and a review of the literature. Arch Pathol Lab Med. 114:493–495, 1990. 383. Shidham VB, Chivukula M, Gupta D, et al: Immunohistochemical comparison of gastrointestinal stromal tumor and solitary fibrous tumor. Arch Pathol Lab Med. 126:1189–1192, 2002. 384. Perez-Montiel MD, Plaza JA, Dominguez-Malagon H, et al: Differential expression of smooth muscle myosin, smooth muscle actin, h-caldesmon, and calponin in the diagnosis of myofibroblastic and smooth muscle lesions of skin and soft tissue. Am J Dermatopathol. 28:105–111, 2006. 385. Rangdaeng S, Truong LD: Comparative immunohistochemical staining for desmin and muscle-specific actin. A study of 576 cases. Am J Clin Pathol. 96:32–45, 1991. 386. Miettinen M, Kopczynski J, Makhlouf HR, et al: Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic, immunohistochemical, and molecular genetic study of 167 cases. Am J Surg Pathol. 27:625–641, 2003. 387. Miettinen M, Sarlomo-Rikala M, Sobin LH, et al: Esophageal stromal tumors: a clinicopathologic, immunohistochemical, and
539.e10
Immunohistology of the Gastrointestinal Tract
molecular genetic study of 17 cases and comparison with esophageal leiomyomas and leiomyosarcomas. Am J Surg Pathol. 24:211–222, 2000. 388. Miettinen M, Sarlomo-Rikala M, Sobin LH, et al: Gastrointestinal stromal tumors and leiomyosarcomas in the colon: a clinicopathologic, immunohistochemical, and molecular genetic study of 44 cases. Am J Surg Pathol. 24:1339–1352, 2000. 389. Chute DJ, Cousar JB, Mills SE: Anorectal malignant melanoma: morphologic and immunohistochemical features. Am J Clin Pathol. 126:93–100, 2006. 390. Fetsch PA, Marincola FM, Filie A, et al: Melanoma-associated antigen recognized by T cells (MART-1): the advent of a preferred immunocytochemical antibody for the diagnosis of metastatic malignant melanoma with fine-needle aspiration. Cancer. 87:37–42, 1999. 391. Lohmann CM, Hwu WJ, Iversen K, et al: Primary malignant melanoma of the oesophagus: a clinical and pathological study with emphasis on the immunophenotype of the tumours for melanocyte differentiation markers and cancer/testis antigens. Melanoma Res. 13:595–601, 2003. 392. Graadt van Roggen JF, van Velthuysen ML, Hogendoorn PC: The histopathological differential diagnosis of gastrointestinal stromal tumours. J Clin Pathol. 54:96–102, 2001. 393. Gould VE, Wiedenmann B, Lee I, et al: Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry. Am J Pathol. 126:243–257, 1987. 394. Marchevsky AM: Mediastinal tumors of peripheral nervous system origin. Semin Diagn Pathol. 16:65–78, 1999. 395. Witkin GB, Rosai J: Solitary fibrous tumor of the mediastinum. A report of 14 cases. Am J Surg Pathol. 13:547–557, 1989. 396. Davis GB, Berk RN: Intestinal neurofibromas in von Recklinghausen’s disease. Am J Gastroenterol. 60:410–414, 1973. 397. Hochberg FH, Dasilva AB, Galdabini J, et al: Gastrointestinal involvement in von Recklinghausen’s neurofibromatosis. Neurology. 24:1144–1151, 1974. 398. Shekitka KM, Sobin LH: Ganglioneuromas of the gastrointestinal tract. Relation to Von Recklinghausen disease and other multiple tumor syndromes. Am J Surg Pathol. 18:250–257, 1994. 399. Hornick JL, Fletcher CD: Intestinal perineuriomas: clinicopathologic definition of a new anatomic subset in a series of 10 cases. Am J Surg Pathol. 29:859–865, 2005. 400. Groisman GM, Polak-Charcon S: Fibroblastic polyp of the colon and colonic perineurioma: 2 names for a single entity? Am J Surg Pathol. 32:1088–1094, 2008. 401. Groisman GM, Amar M, Meir A: Expression of the intestinal marker Cdx2 in the columnar-lined esophagus with and without intestinal (Barrett’s) metaplasia. Mod Pathol. 17:1282–1288, 2004. 402. Kalof AN, Pritt B, Cooper K, et al: Benign fibroblastic polyp of the colorectum. J Clin Gastroenterol. 39:778–781, 2005. 403. Appelman HD: Mesenchymal tumors of the gastrointestinal tract. In Ming SC, Goldman H, editors: Pathology of the Gastrointestinal Tract, ed 2, Baltimore, 1998, Williams and Wilkins, pp 361–398. 404. Pantanowitz L, Antonioli DA, Pinkus GS, et al: Inflammatory fibroid polyps of the gastrointestinal tract: evidence for a dendritic cell origin. Am J Surg Pathol. 28:107–114, 2004. 405. Hasegawa T, Yang P, Kagawa N, et al: CD34 expression by inflammatory fibroid polyps of the stomach. Mod Pathol. 10:451–456, 1997. 406. Makhlouf HR, Sobin LH: Inflammatory myofibroblastic tumors (inflammatory pseudotumors) of the gastrointestinal tract: how closely are they related to inflammatory fibroid polyps? Hum Pathol. 33:307–315, 2002. 407. Miettinen M, Sarlomo-Rikala M, Sobin LH: Mesenchymal tumors of muscularis mucosae of colon and rectum are benign leiomyomas that should be separated from gastrointestinal stromal tumors—a clinicopathologic and immunohistochemical study of eighty-eight cases. Mod Pathol. 14:950–956, 2001. 408. Moyana TN, Friesen R, Tan LK: Colorectal smooth-muscle tumors. A pathobiologic study with immunohistochemistry and histomorphometry. Arch Pathol Lab Med. 115:1016–1021, 1991. 409. Chen RH, Ding WV, McCormick F: Wnt signaling to betacatenin involves two interactive components. Glycogen synthase
kinase-3beta inhibition and activation of protein kinase C. J Biol Chem. 275:17894–17899, 2000. 410. He TC, Sparks AB, Rago C, et al: Identification of c-MYC as a target of the APC pathway. Science. 281:1509–1512, 1998. 411. Anlauf M, Garbrecht N, Bauersfeld J, et al: Hereditary neuroendocrine tumors of the gastroenteropancreatic system. Virchows Arch. 451(Suppl 1):S29–S38, 2007. 412. Debiec-Rychter M, Sciot R, Le Cesne A, et al: KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours. Eur J Cancer. 42:1093–1103, 2006. 413. Heinrich MC, Corless CL, Duensing A, et al: PDGFRA activating mutations in gastrointestinal stromal tumors. Science. 299:708–710, 2003. 414. Alman BA, Li C, Pajerski ME, et al: Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors). Am J Pathol. 151:329–334, 1997. 415. Miyoshi Y, Iwao K, Nawa G, et al: Frequent mutations in the beta-catenin gene in desmoid tumors from patients without familial adenomatous polyposis. Oncol Res. 10:591– 594, 1998. 416. Munemitsu S, Albert I, Souza B, et al: Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Natl Acad Sci U S A. 92:3046– 3050, 1995. 417. Saito T, Oda Y, Tanaka K, et al: beta-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours. J Pathol. 195:222–228, 2001. 418. Tejpar S, Li C, Yu C, et al: Tcf-3 expression and beta-catenin mediated transcriptional activation in aggressive fibromatosis (desmoid tumour). Br J Cancer. 85:98–101, 2001. 419. Tejpar S, Nollet F, Li C, et al: Predominance of beta-catenin mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor). Oncogene. 18:6615–6620, 1999. 420. Rubinfeld B, Albert I, Porfiri E, et al: Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 272:1023–1026, 1996. 421. Cellier C, Delabesse E, Helmer C, et al: Refractory sprue, coeliac disease, and enteropathy-associated T-cell lymphoma. French Coeliac Disease Study Group. Lancet. 356:203–208, 2000. 422. Weston AP, Banerjee SK, Sharma P, et al: p53 protein overexpression in low grade dysplasia (LGD) in Barrett’s esophagus: immunohistochemical marker predictive of progression. Am J Gastroenterol. 96:1355–1362, 2001. 423. Chen HC, Chu RY, Hsu PN, et al: Loss of E-cadherin expression correlates with poor differentiation and invasion into adjacent organs in gastric adenocarcinomas. Cancer Lett. 201:97–106, 2003. 424. Mizoshita T, Tsukamoto T, Nakanishi H, et al: Expression of Cdx2 and the phenotype of advanced gastric cancers: relationship with prognosis. J Cancer Res Clin Oncol. 129:727–734, 2003. 425. Lee HS, Lee HK, Kim HS, et al: MUC1, MUC2, MUC5AC, and MUC6 expressions in gastric carcinomas: their roles as prognostic indicators. Cancer. 92:1427–1434, 2001. 426. Wang JY, Chang CT, Hsieh JS, et al: Role of MUC1 and MUC5AC expressions as prognostic indicators in gastric carcinomas. J Surg Oncol. 83:253–260, 2003. 427. Gurbuz Y, Kahlke V, Kloppel G: How do gastric carcinoma classification systems relate to mucin expression patterns? An immunohistochemical analysis in a series of advanced gastric carcinomas. Virchows Arch. 440:505–511, 2002. 428. Pinto-de-Sousa J, David L, Reis CA, et al: Mucins MUC1, MUC2, MUC5AC and MUC6 expression in the evaluation of differentiation and clinico-biological behaviour of gastric carcinoma. Virchows Arch. 440:304–310, 2002. 429. Wang RQ, Fang DC: Alterations of MUC1 and MUC3 expression in gastric carcinoma: relevance to patient clinicopathological features. J Clin Pathol. 56:378–384, 2003. 430. Bang YJ, Van Cutsem E, Feyereislova A, et al; ToGA Trial Investigators: Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet. 376:687–697, 2010.
References 431. Park DI, Yun JW, Park JH, et al: HER-2/neu amplification is an independent prognostic factor in gastric cancer. Dig Dis Sci. 51:1371–1379, 2006. 432. Brien TP, Depowski PL, Sheehan CE, et al: Prognostic factors in gastric cancer. Mod Pathol. 11:870–877, 1998. 433. Rüschoff J, Hanna W, Bilous M, et al: HER2 testing in gastric cancer: a practical approach. Mod Pathol. 25:637–650, 2012. 434. Hofmann M, Stoss O, Shi D, et al: Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology. 52:797–805, 2008. 435. Samowitz WS, Curtin K, Ma KN, et al: Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level. Cancer Epidemiol Biomarkers Prev. 10:917–923, 2001. 436. Ogino S, Nosho K, Kirkner GJ, et al: CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 58:90–96, 2009. 437. Carethers JM, Smith EJ, Behling CA, et al: Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer. Gastroenterology. 126:394–401, 2004. 438. Corvalan A, Koriyama C, Akiba S, et al: Epstein-Barr virus in gastric carcinoma is associated with location in the cardia and with a diffuse histology: a study in one area of Chile. Int J Cancer. 94:527–530, 2001. 439. Horiuchi K, Mishima K, Ohsawa M, et al: Carcinoma of stomach and breast with lymphoid stroma: localisation of Epstein-Barr virus. J Clin Pathol. 47:538–540, 1994. 440. Selves J, Bibeau F, Brousset P, et al: Epstein-Barr virus latent and replicative gene expression in gastric carcinoma. Histopathology. 28:121–127, 1996. 441. Tokunaga M, Land CE, Uemura Y, et al: Epstein-Barr virus in gastric carcinoma. Am J Pathol. 143:1250–1254, 1993. 442. Kijima Y, Ishigami S, Hokita S, et al: The comparison of the prognosis between Epstein-Barr virus (EBV)-positive gastric carcinomas and EBV-negative ones. Cancer Lett. 200:33–40, 2003. 443. Nakamura S, Ueki T, Yao T, et al: Epstein-Barr virus in gastric carcinoma with lymphoid stroma. Special reference to its detection by the polymerase chain reaction and in situ hybridization in 99 tumors, including a morphologic analysis. Cancer. 73:2239– 2249, 1994. 444. van Beek J, zur Hausen A, Klein Kranenbarg E, et al: EBVpositive gastric adenocarcinomas: a distinct clinicopathologic entity with a low frequency of lymph node involvement. J Clin Oncol. 22:664–670, 2004. 445. Reid BJ, Levine DS, Longton G, et al: Predictors of progression to cancer in Barrett’s esophagus: baseline histology and flow cytometry identify low- and high-risk patient subsets. Am J Gastroenterol. 95:1669–1676, 2000. 446. Reid BJ, Prevo LJ, Galipeau PC, et al: Predictors of progression in Barrett’s esophagus II: baseline 17p (p53) loss of heterozygosity identifies a patient subset at increased risk for neoplastic progression. Am J Gastroenterol. 96:2839–2848, 2001. 447. Herman JG, Umar A, Polyak K, et al: Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci USA. 95:6870–6875, 1998.
539.e11
448. Deng G, Bell I, Crawley S, et al: BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res. 10:191–195, 2004. 449. Domingo E, Laiho P, Ollikainen M, et al: BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet. 41:664–668, 2004. 450. Berg AO: Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med. 11:35–41, 2009. 451. NCCN Guidelines v 2.2012 CRC Screening. Available at www .nccn.org/professionals/physician_gls/pdf/colorectal_screening .pdf. 452. Cunningham D, Humblet Y, Siena S, et al: Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 351:337–345, 2004. 453. De Roock W, Piessevaux H, De Schutter J, et al: KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol. 19:508–515, 2008. 454. Lievre A, Bachet JB, Boige V, et al: KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 26:374–379, 2008. 455. Singer S, Rubin BP, Lux ML, et al: Prognostic value of KIT mutation type, mitotic activity, and histologic subtype in gastrointestinal stromal tumors. J Clin Oncol. 20:3898–3905, 2002. 456. Debiec-Rychter M, Dumez H, Judson I, et al: Use of c-KIT/ PDGFRA mutational analysis to predict the clinical response to imatinib in patients with advanced gastrointestinal stromal tumours entered on phase I and II studies of the EORTC Soft Tissue and Bone Sarcoma Group. Eur J Cancer. 40:689–695, 2004. 457. Heinrich MC, Corless CL, Demetri GD, et al: Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol. 21:4342–4349, 2003. 458. Lee WA: Alpha-methylacyl-CoA-racemase expression in adenocarcinoma, dysplasia and non-neoplastic epithelium of the stomach. Oncology. 71:246–250, 2006. 459. Huang W, Zhao J, Li L, et al: a-Methylacyl coenzyme A racemase is highly expressed in the intestinal-type adenocarcinoma and high-grade dysplasia lesions of the stomach. Histol Histopathol. 23:1315–1320, 2008. 460. Doglioni C, Dei Tos AP, Laurino L, et al: A novel immunocytochemical marker for mesothelioma. Am J Surg Pathol. 20:1037– 1046, 1996. 461. Jung KC, Park WS, Bae YM, et al: Immunoreactivity of CD99 in stomach cancer. J Korean Med Sci. 17:483–489, 2002. 462. McCluggage WC, Maxwell P: Adenocarcinomas of various sites may exhibit immunoreactivity with anti-inhibin antibodies. Histopathology. 35:216–220, 1999. 463. Herrera GA, Turbat-Herrera EA, Lott RL: S-100 protein expression by primary and metastatic adenocarcinomas. Am J Clin Pathol. 89:168–176, 1988.
C H A P T E R 1 5
IMMUNOHISTOLOGY OF PANCREAS AND HEPATOBILIARY TRACT OLCA BASTURK, ALTON B. FARRIS III, N. VOLKAN ADSAY
Pancreas 540 Extrahepatic Biliary Tract (Gallbladder and Extrahepatic Bile Ducts) 559 Ampulla 563 Liver 568 Summary 583
to merge when IHC is needed the most. This should not come as a surprise. After all, basic morphology and IHC are two different facets of the same phenotypic process in pathologic conditions. If one is “unusual” or outside of the general realm, the other tends to be so also.1-3
Biology of Antigens EPITHELIAL MARKERS
Pancreas The pancreas is one of the most versatile organs in the types of neoplasia that it generates. This is partly because it is almost unique in harboring two entirely distinct functional components, exocrine and neuroendocrine, in an otherwise intimate mixture. Tumors that arise from these two components have vastly different pathologic and biologic characteristics. Pancreatic neoplasms are classified based on their cellular lineage; that is, which component of the organ they recapitulate: acinar, ductal, neuroendocrine, or others. However, crossdifferentiation also occurs. For example, acinar carcinomas often contain numerous neuroendocrine (NE) cells or a separate NE component. Additionally, the pancreas is one of a few organs, along with liver and kidney, that has an organ-specific blastic tumor of its own, pancreatoblastoma, characterized by differentiation along all components of the organ. Thus immunohistochemistry (IHC) plays a crucial role in delineating the differentiation of neoplasms that arise in this organ and is an invaluable adjunct in the often challenging differential diagnoses. IHC has been an important tool in unraveling the mechanisms of tumorigenesis as well. In this chapter, the cellular lineage markers and the application of IHC in the diagnosis and management of specific tumor types will be reviewed. Here, we feel obliged to make a cautionary statement: It is our strong bias that IHC is an extremely powerful tool, but only if it is used cautiously and in combination with morphology. It is our opinion, and experience, that no single IHC marker is diagnostic by itself. There are always exceptions and, unfortunately, these exceptions tend 540
As expected, so-called panepithelial markers such as CAM5.2 and cytokeratins (CKs) 8 and 18 are expressed in the pancreatic acini and ducts and in the extrahepatic and periampullary ducts;4 however, certain subsets show differential expression patterns. Acinar cells generally do not label with AE1/AE3, CK7, or CK19 (Fig. 15-1), whereas ductal cells are strongly positive for these markers. Both acini and ducts are typically negative for CK20, which is positive in the intestinal mucosa adjacent to the ampulla. In general, expression of CKs, even the wide-spectrum CKs (CAM5.2, AE1/AE3, CK8, and CK18), is typically less, if not absent, in islet cells compared with other elements. GLANDULAR/DUCTAL MARKERS Mucin-Related Glycoproteins and Oncoproteins
In the pancreas and ampulla, the glandular/ductal system is characterized by mucin production. With the exception of centroacinar cells/intercalated ducts in the pancreas and the serous cystadenomas that presumably recapitulate this earliest component of the ductal system, virtually all glandular elements and their neoplasms exhibit some degree and some type of mucin formation. The most widely expressed mucin-related glycoproteins and oncoproteins in the glandular/ductal neoplasms of this region are membrane-associated mucins, carbohydrate antigen (CA) 19-9, carcinoembryonic antigen (CEA), B72.3 (tumor-associated glycoprotein 72 [TAG-72]), and ductal of pancreas 2 (DUPAN-2).4 Most of these are well described elsewhere in this book. Below, the ones more pertinent to the pancreas will be discussed.
Pancreas
Figure 15-1 Pancreatic ductal cells are strongly positive for cytokeratin 7, whereas acinar cells generally do not label with this marker.
Mucins. Mucins are high-molecular-weight (HMW) glycoproteins produced by various epithelial cells. They are categorized into membrane-associated mucins (MUC1, MUC3 MUC4, MUC12, MUC16, and MUC17), gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6), and soluble mucin (MUC7).5,6 MUC1, panepithelial membrane mucin or the “mammary-type” mucin, is constitutively expressed in the cell apices of the centroacinar cells, intercalated ducts, intralobular ducts, and focally in the interlobular ducts but not in the main pancreatic ducts, acini, or islets. It is thought to have an inhibitory role in cell-tocell and cell-to-stroma interactions and in cytotoxic immunity.7 MUC1 also appears to function as a signal transducer, closely interacting with the epidermal growth factor receptor (EGFR) family and participating in the progression of carcinogenesis.7 It is expressed in almost all examples of pancreatobiliary-type adenocarcinomas: invasive ductal adenocarcinomas of the pancreas, cholangiocarcinomas of the bile duct, and a subset of ampullary adenocarcinomas that presumably arise from periampullary ductules. The expression is predominantly confined to the luminal membrane in the ductforming areas, but it is also intracytoplasmic in the poorly differentiated areas. Therefore MUC1 is considered a marker of aggressiveness.8,9 MUC2, intestinal-type secretory mucin, goblet-type mucin, or gel-forming mucin, is not constitutively expressed in the pancreas or ampullary ductules with the exception of the scattered goblet cells, where it functions as a protective barrier. It is a product of the MUC2 gene, which is known to have tumor suppressor properties, and as such it is considered to be responsible for the more indolent behavior of the tumors.8,10 Carcinomas with prominent intestinal differentiation, namely, intestinal type pancreatic intraductal papillary mucinous neoplasms (IPMNs), colloid carcinomas that often arise in association with IPMNs, and intestinal-type adenocarcinomas that arise from the ampulla/duodenum all typically show diffuse expression of MUC2. They
541
also show diffuse expression of CDX-2, a transcription factor responsible for intestinal programming and an important upstream regulator of MUC2. Whereas diffuse expression of MUC2 is mostly confined to tumors with intestinal differentiation, CDX-2 can be expressed to some degree in pancreatobiliary-type tumors as well. Similar to MUC1, MUC4 is a membrane-associated mucin; however, it is not expressed in normal pancreatic tissue. It is less well studied, but preliminary evidence suggests that, like MUC1, MUC4 may be a marker of ductal adenocarcinoma and a sign of aggressiveness.11 Gastric-type mucins, especially those that mark gastric surface-epithelial (foveolar) mucin, such as MUC5AC, are fairly ubiquitous in the gastrointestinal (GI) tract, including the ampulla, wherever there are gastric-like glands, and in tumors that arise from these sites, presumably because of the close embryologic foregut association with pancreatobiliary tissue. Normal pancreatic ducts, however, do not express this marker.6 The expression of MUC6, gastric pyloric glandular mucin or pyloric-type mucin, is somewhat more restricted. In addition to decorating Brunner glands, intercalated ducts of the pancreas, and the pyloric-like glands that occur in the walls of some preinvasive neoplasia, such as IPMNs and mucinous cystic neoplasms (MCNs), MUC6 is also expressed extensively in some subsets of intraductal neoplasia that show oncocytic phenotype (intraductal oncocytic papillary neoplasms),12 or those with nondescript morphology (intraductal tubulopapillary neoplasms).13 ACINAR (ENZYMATIC) MARKERS
It has long been known that trypsin, one of the best characterized serine proteinases, is produced as a zymogen (trypsinogen) in the acinar cells of the pancreas, secreted into the duodenum, activated into the mature form of trypsin by enterokinase, and functions as an essential food-digestive enzyme as well as a catalyst for the cleavage of the other pancreatic proenzymes (chymotrypsinogen, prophospholipase, procarboxypeptidase, proelastase, etc.) to their active forms.4,14 To date, four trypsin genes—PRSS1, 2, 3, and 4—have been characterized in humans, and the first three have been demonstrated as the zymogens in human pancreatic juice. With IHC methods, trypsin 1 can also be demonstrated in acinar cells, as are the other pancreatic enzymes such as chymotrypsin, lipase, amylase, and elastase. Ductal and NE cells are negative for these enzymes. More importantly, although studies have shown that trypsins or trypsin-like enzymes are produced by other human cancer cells such as stomach, ovary, lung, colon, and others, IHC identification of pancreatic enzyme production is helpful in confirming the diagnosis of pancreatic acinar cell carcinoma (ACC).14 Although this requires confirmation, it has been reported that the tumor-derived trypsin is likely to contribute to tumor invasion and metastasis by degrading extracellular matrix (ECM) proteins and by activating the latent forms of matrix metalloproteinases (MMPs).14
542
Immunohistology of Pancreas and Hepatobiliary Tract
NEUROENDOCRINE MARKERS
The IHC demonstration of the specific NE cell peptides allows classification of pancreatic neuroendocrine tumors (PanNETs); however, it is not always possible to demonstrate these in PanNETs. Therefore it is of diagnostic importance to use broad-spectrum NE cell markers for the general identification of the NE nature of islet cells and PanNETs. These protein markers, localized in the secretory granules in the cytosol or in the cellular membrane, are present in most (rarely in all) normal and neoplastic NE cells. The markers most commonly used in routine histopathology have been the secretory granule proteins chromogranin and synaptophysin and the cytosolic enzyme neuron-specific enolase (NSE).15 As is well known, chromogranin is the most specific of these, but unfortunately, its sensitivity is only approximately 80% to 90% (Fig. 15-2). Chromogranins are a family of glycoproteins that include chromogranin A, B, and C and the proteins derived from them. Among these, chromogranin A in particular has attracted great interest. Different chromogranin A antibodies are commercially available, and the staining results with these antibodies may vary. Also, the intensity of chromogranin varies with the amount of neurosecretory granules in the cytoplasm, which tend to be more abundant in the perivascular zones in normal islets.15 Synaptophysin, also known as protein p38, is a glycoprotein initially found in small vesicle membranes of neurons and of chromaffin cells in the adrenal medulla. It has been routinely used as a broad-spectrum marker in normal and neoplastic NE cells, including those of the pancreas; however, strong synaptophysin immunoreactivity has been well documented in tumors without any NE differentiation, including solid-pseudopapillary neoplasm (SPN) of the pancreas, which is one of the most important differential diagnoses of NE neoplasia of this organ.15 NSE is a cytosolic isoenzyme of the glycolytic enzyme enolase, which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. It has
been considered a marker for NE cells and tumors, including PanNETs. Its staining intensity is unrelated to the content of secretory granules and their peptide storage, therefore NSE can immunostain even degranulated tumor cells. It may also be demonstrated in some non-NE tumors. Thus the use of NSE as an NE marker requires careful evaluation, and it is important not to rely on NSE staining results alone but always to use them in combination with other markers of NE differentiation.15 Cell membrane–associated proteins, such as neural cell-adhesion molecule (NCAM1 [CD56]) and Leu-7 (CD57), have raised interest as NE cell markers, but they lack specificity, because they are also expressed in non-NE cells and tumors.15 Our experience and analysis of the literature also indicate a high incidence of “unexpected” or “unexplained” positivity reported with various antibodies in the islets.16 For most of the antibodies, the staining pattern is a faint blush and does not show any preferential staining pattern of the isletic hormones, suggesting that it is a cross-reaction with a cytosolic component.16 ADHESION MOLECULES AND OTHER MARKERS E-Cadherin
Cadherins are transmembrane glycoproteins that are prime mediators of cell-cell adhesion via calciumdependent interactions. Different members of the family are found in different locations: E-cadherin is found in epithelial tissue; N-cadherin is found in muscle and adult neural tissues, and P-cadherin is found in the placenta.17 E-cadherin plays a key role in the maintenance of epithelial integrity and polarity function.18,19 Normal E-cadherin is localized to the cell membrane with a crisp staining pattern. Decrease in membrane staining compared with normal or complete absence of staining (with the antibody raised against the extracellular domain of E-cadherin) and/or nuclear staining (with the antibody recognizing the cytoplasmic domain of E-cadherin), as seen in SPNs of the pancreas, is regarded as abnormal.19,20 Therefore, E-cadherin staining is of diagnostic use in the IHC workup of SPNs,20 because all cases will show either absence of membrane staining or nuclear positivity, depending on the antibody used.20 The exact mechanism by which E-cadherin enters the nucleus is not known, but it is considered to be closely related to several partner molecules such as β-catenin.19 The cytoplasmic domain of E-cadherin interacts with the catenin molecules that mediate its binding to the actin cytoskeleton.21 Cytoplasmic dotlike staining has also been described with the antibody to full-length E-cadherin.20 β-Catenin
Figure 15-2 Immunohistochemistry staining for chromogranin shows strong positivity in islets of Langerhans that is more prominent in the peripheral cells. Ducts and acini are negative.
β-catenin plays a key role in the Wnt signaling pathway as a transcriptional activator. Its normal expression shows distinct membrane decoration of the pancreatic acini and the ducts. Activating mutations in the β-catenin
Pancreas
543
1 9. U 9 C 5A B7 C 2. Lo ss P 3 of SC SM A AD C 4 D X2 C K2 M 0 U C 2
C
C
A1
K8
C
U M
M
M
es ot he
lin EM C AM A 5. 2 C AE K1 1/ 9 AE 3 C K1 8 C S- K7 10 0A pC 4 EA
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Figure 15-3 Immunohistogram of pancreatic ductal adenocarcinoma with selected antibodies. CK, Cytokeratin; EMA, epithelial membrane antigen; MUC, membrane-associated mucin; pCEA, polyclonal carcinoembryonic antigen; PSCA, prostate stem cell antigen.
gene CTNNB1 result in 1) dysregulation and redistribution of β-catenin protein that leads to characteristic strong cytoplasmic and nuclear immunoreactivity, which is seen in 100% of SPNs and in 50% to 80% of pancreatoblastomas, usually in the squamoid corpuscles; 2) overexpression of its target cyclin D1 gene, CCND1, which is variably reported in 74% to 100% of SPNs21 and in most pancreatoblastomas.
As mentioned previously, DAs express several mucinrelated glycoproteins, including MUC1 (Fig. 15-6), which is reported to be associated with a poorer prognosis,51-54 MUC3, MUC4, MUC5AC, and in a lesser percentage MUC633,51,52,55-61 but not MUC2, which is virtually nonexistent in DAs, unless focal mucinous differentiation or metaplastic goblet cells are present. The intestinal differentiation marker CDX-2 is also positive in only approximately 30% of DAs, and its
Exocrine Neoplasms DUCTAL ADENOCARCINOMA
Ductal adenocarcinoma (DA) is the most common tumor of the pancreas (>85% of pancreatic tumors).22 It often forms a solid mass, which can be closely mimicked by pancreatitis, and therefore most cases require fine needle aspiration (FNA) or core biopsy for diagnosis. Unfortunately, the tumor is usually scirrhous with an abundant stromal component and very low tumor cell yield, which makes the diagnosis of DA one of the most challenging in surgical pathology. Moreover, DA also has a very insidious growth pattern, and in fact, along with ovarian cancer, it is the most common cause of intraabdominal carcinomatosis and is one of the most common sources of carcinomas of unknown primary; therefore it is important to know its immunolabeling pattern (Fig. 15-3).3 DAs express cytokeratins and epithelial membrane antigen (EMA). The keratins expressed consistently are CK7, 8, 18, and 19 (as normal ductal cells), CK13,23-28 and in a lesser percentage CK4, 10, 17, and 20.23-46 Although CK7 is expressed relatively diffusely and strongly in the vast majority of cases (Fig. 15-4, A), CK20 expression is less common, detected in about a third of the cases, and is usually focal (see Fig. 15-4, B).28,31,32,35,38-40,44,47,48 This pattern of immunolabeling can be diagnostically useful, because most acinar and NE neoplasms of the pancreas do not express CK7, and most colorectal cancers express CK20 but not CK7.4 Areas of squamous differentiation in DA are also appropriately CK5/6 and p63 positive (Fig. 15-5).41,49,50
A
B Figure 15-4 A, Cytokeratin 7 is expressed diffusely and strongly in the vast majority of ductal adenocarcinomas. B, Cytokeratin 20 expression is less common and is usually focal.
544
Immunohistology of Pancreas and Hepatobiliary Tract
Figure 15-5 Among invasive carcinomas of the pancreas, p63 expression is detected only in areas with squamous differentiation.
labeling is typically fainter and variegated compared with colonic adenocarcinomas.62-65 Oncoproteins that are widely expressed in DA include CA 19-9, CEA, B72.3 (TAG-72), DUPAN-2, and CA 125.4 The expression of CEA (Fig. 15-7), B72.3, and CA 125 may be useful in distinguishing DA
Figure 15-6 Membrane-associated mucin 1 is expressed in all pancreatobiliary-type adenocarcinoma; invasive ductal adenocarcinoma of the pancreas is shown here. Expression is predominantly confined to the luminal membrane in the duct-forming areas, whereas it is also intracytoplasmic in the poorly differentiated areas.
Figure 15-7 Carcinoembryonic antigen labeling in ductal adenocarcinoma; areas of well-defined tubule formation show more luminal surface labeling, and poorly differentiated areas display more intense cytoplasmic expression. Nonneoplastic glands are often negative or only focally positive for this marker. However, it should be kept in mind that overlaps do happen.
or pancreatic intraepithelial neoplasm 3 (PanIN-3) from reactive glands, because nonneoplastic glands are often negative or only focally positive for these markers.4 However, caution must be exercised, because overlap is common. Moreover, even the lower grade PanINs, such as PanIN-1A, can express these markers. Recently another glycoprotein, CEACAM1, a member of the CEA family, was reported to be positive in DA and not in normal pancreas or chronic pancreatitis, and that serum levels of CEACAM1 might serve as a useful indicator for the presence of pancreatic cancer.66 DAs are typically negative for pancreatic enzymes such as trypsin, chymotrypsin, and lipase,67,68 unless there is a mixed acinar component, which is very uncommon. They also fail to label with NE markers; however, in 30% of DA, scattered, possibly nonneoplastic NE cells are found in close association with the neoplastic cells, which can be highlighted with IHC stains for chromogranin A, synaptophysin, and NSE.69-71 The latter two markers can occasionally show more diffuse expression, which should not be regarded as evidence of “neuroendocrine differentiation” if the tumor is an otherwise conventional adenocarcinoma. The desmoplastic stroma associated with DAs expresses a variety of inflammatory and stromal markers. The inflammatory cells are mostly T cells (CD3 positive).72 Scattered B cells (CD20 positive) and macrophages (MAC387 and KP1 positive) are also usually present.72 The spindle cells express α-smooth muscle actin (SMA), smooth muscle myosin heavy chain, and collagen IV—markers of myofibroblastic differentiation.73 They are also reported to be positive for heat shock protein 47 and fibronectin and for proteins associated with tissue remolding, such as urokinase-type plasminogen activator, the MMPs, and the tissue inhibitors of metalloproteinases.74-77
Pancreas
Genomic Applications of Immunohistochemistry
It has been recognized that DA, like other malignant processes, is a genetic disease produced by progressive mutations in cancer-related genes. Tumor Suppressor Genes. Numerous studies have shown that inactivation of the TP53 gene occurs in 50% to 80% of cases.78-89 As is well known, however, immunolabeling for the p53 protein is not entirely specific for a TP53 gene mutation.4 The SMAD4 (mothers against decapentaplegic homolog 4) gene is inactivated in approximately 55% of the DAs but practically never in benign conditions.79,90-93 IHC labeling for the SMAD4 (formerly DPC4) gene product has been shown to mirror SMAD4 gene status,94 therefore the IHC absence of its protein product in the ductal epithelium of a biopsy specimen is strongly suggestive of carcinoma,84,95 as long as the built-in controls are labeling properly. Also, inactivation of the SMAD4 gene is relatively uncommon in nonpancreatobiliary carcinomas, thus loss of SMAD4 may also serve as a marker of a pancreatic DA in small FNA samples and in metastatic sites.90,96 CDKN2A (formerly P16) is inactivated in approximately 95% of DAs (75% to 80% genetic inactivation plus 15% silencing by hypermethylation);79-82,85,97-99 however, because of the fact that a small minority of benign ductal epithelial cells also labels for its protein product, p16, loss of p16 not as strongly suggestive of carcinoma.84,85,87,100 Oncogenes. The oncogene most frequently activated in pancreatic cancer is the KRAS oncogene,101 which will be discussed below, but a number of other oncogenes can be activated in DA, including ERBB2 (formerly HER2/neu, overexpressed in approximately 70% of the cases),102-105 and AKT2 (amplified in 10% to 20% of cases).106,107 Novel Tumor Markers. Gene expression analyses of DA have identified a large number of genes that are differentially overexpressed in DA compared with normal pancreatic tissue.108-111 Among them, mesothelin is expressed in close to 100% of DAs,58,112-115 sea urchin fascin is expressed in 95%,75,115 a number of S-100 protein subtypes are expressed in 93%,116-118 14-3-3 sigma is found in 90%,108 and prostate stem cell antigen (PSCA) is found in 60%.119 The concurrent use of K homology domain containing (KOC) and S-100A4 proteins has been found in some studies to improve the diagnostic sensitivity of biliary brushings cytology, and it demonstrates specificity similar to cytology alone in the diagnosis of pancreatobiliary malignancy.120 In addition, secreted or membranous proteins expressed in pancreatic cancer, such as mesothelin or PSCA, shed into pancreatic secretions or blood, are under scrutiny as potential future markers for primary or recurrent disease.121 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
DNA ploidy analyses have yielded aneuploid patterns in approximately half of the tumors, the incidence being
545
higher with the poorly differentiated forms.122-125 Also, cytogenetic analyses of large series have revealed recurrent patterns of alterations in specific chromosomes,126-128 the most frequent whole chromosomal gains being chromosomes 20 and 7 and the most frequent whole chromosomal loss being chromosome 18.126,127 MicroRNAs (miRNAs), small noncoding RNAs that negatively regulate gene expression, are also altered in ductal adenocarcinoma.129 A number of studies have found that the STK11/ Lkb1 Peutz-Jeghers gene, a tumor suppressor gene, is inactivated in a minority (5%) of DA.130-135 This is clinically important, because patients with Peutz-Jeghers syndrome have a greater than 130-fold increased risk for pancreatic cancer.136 The STK11/Lkb1 PeutzJeghers gene is also commonly altered in IPMNs (see below). Among all human cancers, DA has the highest frequency of KRAS alterations, and the oncogene is constitutively activated in approximately 90% of DAs.79-82,101,132-135,137-141 However, it is important to point out that KRAS mutations are seen even in the earliest forms of neoplastic transformation (namely PanIN-1A), a very common incidental finding in the pancreas and in patients with chronic pancreatitis lacking invasive carcinoma,82,90 and thus by no means a specific marker for “cancer.” Other oncogenes found to be activated in pancreatic cancer include NCOA3 (formerly AIB1, amplified in 60%), BRAF (mutated in one third of the small minority of cancers with wild-type KRAS), MYC, and MYB.106,107,142-145 Microsatellite instability (MSI) is a very rare event in DA, and such cancers appear to have a distinct morphology referred to as medullary94,135,146,147 (see below) and an improved survival rate relative to conventional DAs.76,92,135,146 The MLH1 gene is often inactivated in MSI-H pancreatic carcinomas, characterized with loss of expression of its protein product, MLH1, at the IHC level, either by mutation or hypermethylation.135,148
KEY DIAGNOSTIC POINTS Ductal Adenocarcinoma • DAs are consistently positive for CK7 diffusely and strongly, whereas CK20 is often either very focal or absent. • Several mucin-related glycoproteins—MUC-1, MUC3, MUC4, and MUC5AC—and oncoproteins such as CA 19-9, CEA, B72.3, DUPAN-2, and CA 125 are also typically positive in DAs to variable degrees. None of these are entirely specific for this tumor type. • Scattered, possibly nonneoplastic neuroendocrine cells in DA can be demonstrated with immunostains for chromogranin A, synaptophysin, and NSE. • Loss of SMAD4 staining in the pancreatic ductal epithelium is suggestive of carcinoma, provided that this loss is confirmed by the presence of built-in controls. SMAD4 may also prove to be a helpful marker for differentiating pancreatic adenocarcinoma from other carcinomas in small fine-needle aspirate samples and in metastatic sites.
546
Immunohistology of Pancreas and Hepatobiliary Tract
KEY DIFFERENTIAL DIAGNOSIS Ductal Adenocarcinoma BENIGN, NONINVASIVE DUCTS VS. DUCTAL ADENOCARCINOMA • Strong cytoplasmic expression of MUC1 and CEA is often coupled with antigen leakage into stroma and is highly indicative of carcinoma (more common in high-grade areas). • Ki-67 and p53 are significantly more abundant in carcinoma than in normal epithelium, although overlaps are common and should be used cautiously. • Loss of SMAD4 is also a finding strongly in favor of adenocarcinoma, provided that built-in controls are working properly. • Mesothelin, fascin, S-100 protein, and PSCA are significantly more commonly expressed in carcinoma than in benign epithelium.58 NONDUCTAL TUMORS VS. DUCTAL ADENOCARCINOMA • Colon vs. DA: DA is consistently positive for CK7 diffusely and strongly. Approximately one third of DA expresses CK20 and CDX-2, but the expression is usually focal and weak. Colon carcinomas are negative for CK7, and they almost always express CK20 and CDX-2. Additionally, although DA is MUC1 and MUC5AC positive and MUC2 negative, colon carcinoma is more commonly MUC1 and MUC5AC negative and MUC2 positive. • Lung vs. DA: TTF-1 and surfactant apoprotein A (PE-10) are usually positive in lung adenocarcinomas and are negative in DAs. In contrast, antibodies that can be focally positive in DAs—such as CK20, CDX-2, and CA 125, are negative in nonmucinous lung carcinomas. • Müllerian (gynecologic tract) vs. DA: A panel composed of WT1, Pax-8, MUC5AC, and CK20 is advisable in distinguishing ovarian serous carcinoma from DA in omental or peritoneal biopsies, which often proves to be a challenging differential diagnosis. A WT1- and Pax-8– negative, MUC5AC-positive phenotype would point toward DA, whereas the WT1- and Pax-8–positive, MUC5ACnegative phenotype highly favors an ovarian primary. If present, CK20 and, less reliably, CDX-2 would also be more compatible with a diagnosis of DA.149 In one study, extensive CK17 reactivity has also been found to be supportive of a DA when the differential diagnosis includes ovarian serous and mucinous neoplasms.150
OTHER DUCTAL CARCINOMAS Undifferentiated Carcinoma
In some DAs, the hallmarks of ductal differentiation may be lacking. Such cases are classified as undifferentiated carcinoma.151 In some cases, the epithelial-tomesenchymal transition can be so complete that the tumor may resemble sarcoma (i.e., sarcomatoid carcinoma),151 and only after adjunct studies, such as IHC, can the ductal nature of the tumor be elucidated.151 Immunohistochemically, cytokeratins are expressed in the well-formed epithelial component; however, the sarcomatoid component might be negative or focal/ weak positive, hindering the differential diagnosis with sarcomas.25,152,153 It should be kept in mind that
sarcomas are exceedingly uncommon in the pancreas, and any sarcomatoid neoplasm ought to be regarded as suspicious for carcinoma rather than sarcoma. The keratins with the most diffuse and strongest staining has been reported to be the monoclonal so-called pancytokeratins (AE1/AE3);154 however, these are also more likely to be expressed in true sarcomas. CAM5.2 and CKs 7, 8, 18, and 19 are also positive in a variegated and less intense pattern.25,152,153 As is typical of most undifferentiated carcinomas, the neoplastic cells diffusely and strongly stain with vimentin.25,153,155,156 They may also express MUC1, CA 19-9, CEA, and DUPAN2,152,157 whereas EMA and B72.3 are negative in the majority of cases.153 Immunolabeling for chromogranin, synaptophysin, and NSE is also negative.4,158 In undifferentiated carcinomas with heterologous stromal elements, immunoreactivity may be consistent with the line of mesenchymal differentiation (e.g., myoglobin or myogenin in striated muscle, S-100 protein in chondroid elements).4 Recently, it has been demonstrated that noncohesive pancreatic cancers, including undifferentiated pancreatic carcinomas, are characterized by the loss of E-cadherin protein expression, which might explain the poor cohesion of many undifferentiated carcinomas.159 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications. Ductal adenocarcinomas develop through a multistep process initiated by an early activating KRAS mutation. Recent findings suggest that subsequent KRAS copy number changes and resulting KRAS mutant allele–specific imbalance in a subset of cases may lead to the progression to undifferentiated carcinomas. It seems that KRAS mutant allele–specific imbalance develops predominantly through KRAS amplification and/or chromosome 12 hyperploidy and monosomy and correlates with worse survival. Undifferentiated Carcinoma with Osteoclast-Like Giant Cells
Osteoclast-like giant cells are not uncommon in sarcomatoid carcinomas and mesotheliomas and even some sarcomatoid melanomas.160 In undifferentiated carcinoma with osteoclast-like giant cells of the pancreas, these cells are strikingly abundant and form a sea of giant cells that may, in some cases, obscure the epithelial component of the tumor.160 Recent studies confirmed that the osteoclast-like giant cells are in fact reactive in nature, and that the malignant cells are actually the smaller, atypical mononuclear cells in the background.160 Immunohistochemically, the atypical mononuclear cells in the background, which are the true malignant cells that represent the sarcomatoid carcinoma cells, almost always express vimentin, whereas only a minority express cytokeratins—CAM5.2, AE1/AE3 (Fig. 15-8, A), or CK7 and CEA.25,134,156,157,161-165 In some cases, all of the epithelial markers are negative. The osteoclast-like giant cells are positive for vimentin, leukocyte common antigen (LCA, also known as CD45), histiocytic markers (CD68, KP1; see Fig. 15-8, B), and α1-antichymotrypsin; they are nonreactive for CKs,
Pancreas
A
547
B
Figure 15-8 In undifferentiated carcinoma with osteoclast-like giant cells of the pancreas, such cells are negative for AE1/AE3 (A) and positive for CD68 (B).
EMA, and CEA.25,134,156,157,162-166 However, “tumor cannibalism,” presence of malignant cells in the benign giant cells, is fairly common in this entity and ought to be considered in evaluating these markers. Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications. These neoplasms often have TP53 gene mutations, and immunolabeling for the p53 protein has been shown to label the pleomorphic mononuclear cells but not the osteoclast-like giant cells.136 Additionally, genetic analyses have demonstrated that the atypical mononuclear cells harbor KRAS mutations in approximately 90% of these neoplasms.25,157,163,165,167,168 By contrast, the osteoclast-like giant cells do not harbor KRAS mutations.134,165 Medullary Carcinoma
As in other organs (such as breast and tubular GI tract), medullary carcinoma in the pancreas is defined as syncytial growth of poorly differentiated epithelioid tumor cells, often accompanied by a dense lymphoplasmacytic inflammatory infiltrate.135,146-148 Desmoplastic reaction is minimal.92,146,147,169 The tumors may arise sporadically or may occur in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome.147 Current experience is too limited to determine the biologic behavior of the disease or its prognosis; however, in one study, patients were found to have an improved survival rate relative to those with DA.135 Immunohistochemically, the epithelioid cells are labeled by antibodies to CK,146-148,169 whereas trypsin, chymotrypsin, lipase, chromogranin, and synaptophysin are usually negative. CD3 antibody highlights the presence of numerous intratumoral T lymphocytes.148 Rare examples also contain Epstein-Barr virus (EBV) RNA.148 Similar to their colorectal counterparts, medullary carcinomas of the pancreas often show MSI, which is usually caused by somatic hypermethylation of the MLH1 promoter in sporadic cases170 and by an inherited mutation in one of the mismatch repair (MMR) genes (MLH1, MSH2, etc.) in patients with HNPCC syndrome.147 Immunolabeling for MLH1 and MSH2 reveals
loss of expression of one of these DNA MMR proteins in many cases. The diagnosis of a medullary carcinoma has therapeutic implications; although not studied thoroughly in the pancreas, patients with microsatellite-unstable medullary colorectal adenocarcinomas do not benefit from fluorouracil-based chemotherapy.129 Adenosquamous Carcinoma
In the pancreas, squamous cells can be encountered in injured ductal epithelium as a result of a metaplastic process. The same metaplastic phenomenon also seems to take place focally in some examples of DA. When this finding is prominent (>25% of the tumor is the cutoff we use), the tumor is classified as adenosquamous carcinoma, and if it is exclusively squamous, then it is classified as squamous cell carcinoma (SCC).171 Most of the tumors express cytokeratins (CAM5.2, AE1/AE3, and CKs 5/6, 7, 8, 13, 18, 19, and 20), EMA, CA 19-9, CEA, and B72.3.171,172 Typically, CK5/6, CK13, and p63 (see Fig. 15-5) are limited to the areas of squamous differentiation, whereas CK7, CK20, CA 19-9, CEA, and B72.3 often label to the glandular elements.171,173,174 The majority of the cases show nuclear p53 staining and loss of SMAD4 protein, similar to the molecular signature found in DA,175 and KRAS mutations.171,176 Colloid Carcinoma
Colloid carcinomas are characterized by well-delineated pools of stromal extracellular mucin that contain scanty, floating carcinoma cells in clusters, strips, or as individual cells. By definition, the mucin/epithelium ratio is typically very high, and these lesions appear to have a distinctly better clinical course than other invasive carcinomas of ductal origin.177,178 Five-year survival of resected cases is 55% as opposed to 10% in DA.178,179 This indolent behavior has been attributed to a combination of two factors: 1) the inverse polarization of cells, which show secretory activity toward the “stroma-facing” surface of the cells, instead of the luminal surface; and 2) the mucin produced is a specific
548
Immunohistology of Pancreas and Hepatobiliary Tract
Figure 15-9 Strong and often diffuse intracytoplasmic MUC2 labeling is specific for colloid carcinoma.
gel-forming mucin (MUC2) that acts as a containing factor that prevents the spread of the cells.178,179 Immunohistochemically, in addition to the conventional epithelial and ductal markers such as CKs, CEA, CA 19-9, and B72.3, colloid carcinoma is unique among invasive carcinomas of the pancreas in its expression of intestinal differentiation markers MUC2 and CDX-2 (Figs. 15-9 and 15-10, respectively).178 Also, in contrast with DA, colloid carcinomas are negative for MUC1. Furthermore, the pattern of accentuated CEA labeling in the stroma-facing surface of the cells (Fig. 15-11) is specific to colloid carcinomas and is seldom seen in other invasive carcinomas of the pancreas. As opposed to DA, the expression of SMAD4 is intact in almost all cases,180 and only one third harbor KRAS oncogene mutations.178 PANCREATIC INTRAEPITHELIAL NEOPLASIA
A spectrum of intraductal proliferative lesions are presumed to be precursors of invasive carcinoma and are referred to as pancreatic intraepithelial neoplasia (PanIN).181 This spectrum is graded on a threetiered scale as PanIN-1A (the earliest step), progressing to 1B and 2, and finally to PanIN-3, which is considered
Figure 15-11 In contrast to MUC2, intracytoplasmic carcinoembryonic antigen (CEA) labeling is much weaker in colloid carcinoma; however, strong CEA labeling is seen in the stroma-facing surface of the cells.
carcinoma in situ (CIS). Lower grade PanINs are relatively frequent incidental findings.3 As a rule of thumb, it is considered that nearly 50% of adults older than 50 years have pancreatic foci of PanIN-1. However, highergrade PanINs, in particular PanIN-3 (CIS), is seldom encountered in isolation without a concomitant invasive adenocarcinoma.1 The IHC labeling pattern of PanINs parallels that of DA. None of the lesions express MUC2,182 but most express MUC1, MUC4, MUC5AC, and MUC6.51,182 In the multistep progression of DA, MUC1 expression within interlobular ducts appears to be decreased in the low-grade PanINs and is subsequently reexpressed in the advanced PanINs, increasing to 85% of PanIN-3.182 Unlike MUC1, MUC5AC is expressed relatively uniformly throughout all grades of PanIN.182 Increasing Ki-67 labeling indices has been shown with increasing grades of dysplasia in PanINs.183 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Figure 15-10 Colloid carcinomas also express CDX-2 diffusely.
Most of the molecular abnormalities identified within DA have also been detected within PanIN. Among these, KRAS, CDKN2A, GNAS, or BRAF mutations seems to be an “early” event.184 Their frequency increases with increasing grades of dysplasia, and it precedes both p53 mutation and SMAD4 inactivation.85,182 Based on nuclear cyclin D1 expression, seen in one third of PanIN-2 lesions, cyclin D1 abnormalities would best be classified as an “intermediate” event, also preceding p53
Pancreas
mutation and SMAD4 inactivation.182 In fact, p53 mutation, as assessed by nuclear overexpression of p53 protein (>25% nuclei), is a “late” event in the progression model of DA and occurs only in PanIN-3 (57% of the lesions). Similar to the case of the p53 gene, inactivation of the SMAD4 gene appears to be a “late” event, seen only in PanIN-3 (28% of the lesions).185 Comparative molecular/genetic analysis of microdissected normal and neoplastic ducts combined with IHC has disclosed the upregulation of a cluster of extrapancreatic foregut markers (pepsinogen C, MUC6, Kruppel-like factor 4 [KLF4], and trefoil factor 1 [TFF1]) and various gastric epithelial markers (Sox-2, gastrin, HoxA5, and others) in PanINs, whereas the intestinal markers CDX-1 and CDX-2 are rarely expressed, if at all, in both PanIN lesions or in invasive pancreatic cancer. These data suggest that PanIN development may involve Hedgehog gene–mediated conversion to a gastric epithelial differentiation program.186 INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS
IPMNs are characterized by intraductal proliferation of neoplastic mucinous cells, which usually form papillae and lead to cystic dilatation of the pancreatic ducts to form clinically and macroscopically detectable masses.4 Microscopically, papillae with four distinct morphologic patterns can be seen: 1) gastric/foveolar, reminiscent of gastric foveolar epithelium or resembling PanIN-1A with scattered goblet cells; 2) intestinal, which closely resembles colonic villous adenoma; 3) pancreatobiliary, characterized by more complex papillae lined by cuboidal cells187; and 4) oncocytic, characterized by exuberant, intricately branching papillae lined by oncocytic cells (because of an abundance of mitochondria) and intraepithelial lumina. The latter are round, punchedout spaces within the epithelium that often contain mucin. Scattered goblet cells may also be identified.188 Although the last pattern used to be regarded as a separate type of intraductal neoplasm, named intraductal oncocytic papillary neoplasm (IOPN),188 the 2010 World Health Organization (WHO) guidelines put this neoplasm under the general category of IPMN, as a variant thereof (Fig. 15-12).189 A spectrum of cytoarchitectural atypia exists (IPMN with low-, intermediate-, and high-grade dysplasia),4,158 and approximately 30% of resected IPMNs have an associated invasive carcinoma. Gastric/foveolar and intestinal types of IPMN are usually associated with colloid carcinoma, and the pancreatobiliary type is associated with tubular-type invasion with all the morphologic features of DA.187 Ki-67 and proliferating cell nuclear antigen (PCNA) labeling demonstrates a progressive increase in cell proliferation, from normal ductal epithelium to IPMN with low-grade dysplasia to IPMN with intermediate-dysplasia and then to IPMN with high-grade dysplasia.190,191 Also, immunostaining of p53 protein is seen only in IPMNs with intermediateand high-grade dysplasia and in DAs.192 The MUC expression profile of IPMNs has been instrumental in delineating the differentiation and
549
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% MUC1
MUC2
CDX2
Gastric-type papillae Intestinal-type papillae Pancreatobiliary-type papillae
Figure 15-12 Immunohistogram of intraductal papillary mucinous neoplasms with selected antibodies.
lineage of these neoplasias and in recognizing clinically significant subsets.10,51,57,193,194 First, the gastric/foveolar type papillae appear to be a full recapitulation of gastric mucosa, with more papillary areas expressing MUC5AC and only small glandular elements at the base labeling with MUC6; they are usually negative or only focally positive for MUC1 and CDX-2, and scattered goblet cells can be highlighted by MUC2 (Fig. 15-13).51,57,187,195,196 Secondly, intestinal type papillae do, in fact, show molecular characteristics of intestinal differentiation as evidenced by diffuse MUC2 and CDX-2 expression but not MUC1 (Fig. 15-14).51,57,187,195,196 Invasive carcinoma associated with the intestinal type, which is typically colloid carcinoma, also expresses MUC2 and CDX-2 but not MUC1. In addition, intestinal type papillae are positive for MUC5AC and negative for MUC6.51,57,178,187 Third, pancreatobiliary type papillae typically do not express MUC2 and CDX-2 but may instead express MUC1, a marker of an aggressive phenotype in the pancreas, as well as MUC5AC and, to a lesser degree, MUC6.12,57 The invasive component also expresses MUC1 but not MUC2.57 Fourth, oncocytic
Figure 15-13 Intraductal papillary mucinous neoplasms with gastric/foveolar-type papillae are usually negative for MUC1 (left). MUC2 expression is only focal, marking goblet cells (right).
550
Immunohistology of Pancreas and Hepatobiliary Tract
series.82,210,218,224 However, in contrast to DA, the protein product of the SMAD4 tumor suppressor gene is retained in virtually all cases, including in the invasive carcinomas, suggesting a substantial difference in the pathogenesis of these two neoplasms.57,82,180,206,210,218,225 Recently, human cripto-1 protein, which is thought to have a role in tumor progression, was reported to be expressed more abundantly in the pancreatobiliary type than in other types of neoplasms.226 MUCINOUS CYSTIC NEOPLASM
Figure 15-14 Most intestinal type intraductal papillary mucinous neoplasm papillae are negative for MUC1 (left). MUC2 expression is diffuse and strong (right).
type papillae usually label for MUC157,196,197 and MUC6,12 whereas MUC2 and MUC5AC are largely restricted to goblet cells.57,193,197 CDX-2 is rarely expressed.197 In general, IPMNs are reported to show weaker labeling for MUC4 (70%), MUC3 (60%), and MUC5B (35%)193 and are almost always negative for MUC7.4 As expected, virtually all IPMNs express cytokeratins. They are positive for CAM5.2, AE1/AE3, and cytokeratins 7, 8, 18, and 19 and variably for CK20.59,191,198 Some IPMNs, especially the intestinal type, also express CA 19-9 and CEA.59,199,200 In the intestinal type, the degree of CEA expression increases with the degree of dysplasia,201 and 20% of IPMNs label for DUPAN2.59,198,202 Scattered NE cells that are positive with chromogranin and synaptophysin are seen in most tumors but account for fewer than 5% of the tumor cells.203,204 Cyclooxygenase-2 (COX-2) is also expressed in 60% to 80% of IPMNs.205 Oncocytic type IPMNs also label strongly with antibodies against mitochondrial antigens such as 111.3.188,197 Interestingly, consistent immunolabeling is seen with hepatocyte paraffin 1 (HepPar1) antibodies; however, in situ hybridization (ISH) for albumin, a more specific test for hepatocellular differentiation, is consistently negative.4 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
KRAS oncogene mutations have been reported in 30% to 60% of IPMNs.82,192,198,203,206-216 However, oncocytic type IPMNs typically lack KRAS mutations,197,206 in stark contrast with any other tumor types in the pancreas characterized by ductal-mucinous differentiation, including more than 90% of invasive ductal carcinomas. Recently, it has also been shown that recurrent mutations at codon 201 of GNAS are also present in approximately two thirds of IPMNs and that either KRAS or GNAS mutations could be identified in approximately 95%,217 illustrating that molecular analyses are a highly sensitive assay for the identification of IPMNs. The frequency of TP5381,190,208,210-212,218-224 and CDKN2A tumor suppressor gene mutations varies greatly among reported
MCNs are typically seen in perimenopausal women (95% of patients are women; mean age, 50 years) and occur as a thick-walled cyst, often multilocular, in the tail of the pancreas. Microscopically the cysts are lined by mucin-producing epithelium. A distinctive ovarianlike stroma is invariably present in the septa of the cysts.227 Just as in IPMNs, MCNs also exhibit characteristics of an adenoma-carcinoma sequence (low-grade dysplasia, intermediate-grade dysplasia, and highgrade dysplasia or CIS).4,158,227 Invasive carcinoma can be seen in association with MCN in less than one fifth of MCNs and is almost exclusively of the tubular type with all the morphologic features of DA.228,229 On occasion, sarcomatoid neoplasms arise in MCNs, and it is debated whether these are sarcomatoid carcinomas that originate from the epithelial component or sarcomatous transformation of the ovarian-like stroma. The epithelial cells express immunoreactivity with cytokeratins 7, 8, 18, and 1938,48,198,230 and with EMA, CA 19-9, CEA, DUPAN-2, and CA 125.198,199,230-237 MUC1 is typically not expressed in noninvasive lesions of MCN but is a marker of invasion observed in more than 90% of cases with invasion, detected both in the invasive and in situ components.232,238 MUC2 and CDX-2 positivity can be seen, especially in the interspersed goblet cells; however, in contrast to intestinal– type IPMNs, the papillary dysplastic nodules are typically negative or are only focally positive for these markers.232,238 The lesions invariably express MUC5AC, which is also commonly expressed in DA.38,232 Although the papillary component of MCN is mostly MUC6 negative, the foveolar-like epithelium in nonpapillary areas is typically positive for MUC6.12 Chromograninor synaptophysin-positive scattered NE cells are frequently noted within the epithelium.231,234,239-241 The ovarian-like stroma is immunoreactive for vimentin, SMA, muscle-specific actin (MSA), desmin, h-caldesmon, Bcl-2, and CD99, usually in a patchy distribution.228,231,242-247 Progesterone receptors are also expressed fairly consistently (Fig. 15-15); in subtle cases, this may help establish the diagnosis by highlighting the presence of this pathognomonic finding. Estrogen receptors are often negative by the current antibodies available; however, it is suspected that this lack of detection is related to the receptor subunit specificities of these antibodies. If present, the luteinized cells label with antibodies to tyrosine hydroxylase, α-inhibin, and calretinin, which have been shown to recognize ovarian hilar cells and testicular Leydig cells.244,248
Pancreas
551
The lesions also express MUC1 (90%) and MUC6 (60%), whereas MUC2 is negative,196,255-258 placing them closer to pancreatobiliary-type IPMNs; however, MUC4 and MUC5AC are negative. The lesions almost always (>90%) show intact SMAD4 labeling (Fig. 15-16).13 Lack of acinar differentiation as demonstrated by negativity of trypsin and other acinar markers is essential in the differential diagnosis of intraductal tubular carcinomas from ACC, because the morphologic distinction of these two tumor types is often very challenging. SEROUS CYSTADENOMA
Figure 15-15 Progesterone receptors are expressed in the ovarianlike stroma cells and may be used to confirm the diagnosis in equivocal cases of mucinous cystic neoplasms.
By molecular/genetic analysis, KRAS mutations appear early and increase in frequency proportional to the degree of dysplasia.219,249-251 In addition, p53 overexpression appears to occur relatively late in invasive and in situ mucinous cystadenocarcinomas.219,249-251 Approximately half of the associated invasive carcinomas also show loss of SMAD4 expression, which is not surprising, because most of these carcinomas are conventional DAs.180,231,232,243 KEY DIAGNOSTIC POINTS
Serous cystadenoma (SCA) is the only nonmucinous example of ductal neoplasia in the pancreas, and unlike other ductal tumors, it has virtually no tendency for malignant transformation.259 It is also known to have a well-established association with von Hippel-Lindau (vHL) syndrome.4,158 The tumor cells in SCA have a clear cytoplasm, because of the abundant intracytoplasmic glycogen, and they label with CAM5.2, AE1/AE3, and cytokeratins 7, 8, 18, and 19 but usually do not label with CK20.259-265 A third of the cases are also positive for EMA.260,262,263,266 Even though CA 19-9 and B72.3 expression is reported,263,267 immunolabeling for mucin markers is generally lacking, with the exception of MUC6. CEA is uniformly negative, as are insulin, glucagon, somatostatin, vasoactive intestinal polypeptide (VIP), and vimentin.268 Chromogranin- or synaptophysinpositive scattered NE cells are commonly detected.230,235,260,261,263,267,269,270 Molecules implicated in clear
Mucinous Cystic Neoplasm • MCNs are mucinous ductal type neoplasia, thus the epithelial cells are positive for CKs and for EMA, CA 19-9, and CEA. • All MCNs express MUC5AC, but MUC1 is seen primarily in cases with an invasive component. • The ovarian-like stroma cells label with antibodies to SMA, MSA, desmin, and h-caldesmon and also stain variably for α-inhibin and calretinin. • Progesterone receptors are expressed in the ovarian-like stroma cells and may be used to confirm the diagnosis in equivocal cases of MCN.
INTRADUCTAL TUBULOPAPILLARY NEOPLASM
Intraductal tubulopapillary neoplasm (ITPN) is a recently described entity, the clinicopathologic characteristics of which has yet to be fully characterized.252-254 It resembles IPMN by its intraductal nature and mimics acinar cell carcinoma (ACC) by its often acinar pattern. Approximately one third of the lesions have foci of invasion that range from microscopic to somewhat larger.254 Immunohistochemically, ITPNs are positive for AE1/ AE3 (100%), CK7 (85%), and CK19 (85%) but not for CK20 and CDX-2.196,255-257 Most express CA 19.9 (95%),13 and focal linear immunoreactivity for CEA (40%) has been reported along the apical cytoplasm.
Figure 15-16 Intact SMAD4 expression in the nuclei of the intraductal tubulopapillary neoplasm cells.
552
Immunohistology of Pancreas and Hepatobiliary Tract
loss of heterozygosity (LOH) has also been reported on chromosome 10q.274 In contrast to DA, activating mutations in the KRAS oncogene and inactivation of the TP53 tumor suppressor gene have not been reported in SCA.219,251,267,273-275 KEY DIAGNOSTIC POINTS Serous Cystadenoma
Figure 15-17 Glucose uptake and transporter 1 expression in serous cystadenoma is detected predominantly in the cell membranes but is also found in the cytoplasm.
cell tumorigenesis—glucose uptake and transporter 1 (GLUT-1; Fig. 15-17), hypoxia-inducible factor 1α (HIF-1α), and carbonic anhydrase IX (CAIX)—are also consistently expressed.271 As in other vHL-related clear cell tumors, a prominent capillary network immediately adjacent to the epithelium confirms that the clear cell– angiogenesis association is also valid for this tumor type.271 This rich capillary network, which can be highlighted by CD31 stain (Fig. 15-18), may be helpful for surgical pathologists, especially now that needle biopsies are becoming the norm for initial workup.271 This finding may also be helpful in frozen sections, in which clear cell cytology is typically not evident because of the preservation of glycogen.271 By molecular/genetic analysis, VHL gene (chromosome 3p) allelic deletions are detected in SCAs from patients with vHL, providing further evidence of their neoplastic nature and integral association with vHL syndrome.272-274 Alterations of the VHL gene may also be detected in sporadic cases.251,272 Frequent (in 50%)
Figure 15-18 The remarkable intensity of the capillary network immediately adjacent to the epithelium is highlighted by CD31 in serous cystadenoma.
• SCA is virtually the only ductal tumor that does not show the pancreatic ductal differentiation markers (mucins, mucin-related oncoproteins, and KRAS mutation). • Instead, it commonly expresses markers of the centroacinar cell/intercalated duct system, such as MUC6, as well as markers of clear cell tumorigenesis, such as GLUT-1, HIF-1α, and carbonic anhydrase IX. • Epithelial differentiation markers, cytokeratins, and EMA are always positive. • The rich capillary network, which can be highlighted by CD31 stain, may be helpful, especially in needle biopsies and in frozen sections.
KEY DIFFERENTIAL DIAGNOSIS Serous Cystadenoma • Clear cell variant of pancreatic neuroendocrine tumor vs. SCA: Diffuse immunoreactivity for chromogranin and synaptophysin highly favors a neuroendocrine origin. • PEComa (“sugar tumor”) vs. SCA: HMB-45 and SMA are positive in PEComa, whereas the absence of such reactivity and immunolabeling for cytokeratin and EMA is typical for SCA.
ACINAR CELL CARCINOMA
Although prognostically not as dismal as DAs, acinar cell carcinomas (ACCs) are rare and fairly aggressive tumors3 that can be seen in any age group, but they are more common in elderly patients. They are typically solid, cellular, stroma-poor tumors characterized by sheets of relatively uniform cells. Variable amounts of NE elements in forms of scattered individual cells, large zones, and hybrid foci or even as separate, wellestablished nodules are quite common and are present in most cases if searched for carefully. If the NE component comprises more than 25% of the tumor, by convention the case is classified as “mixed.” ACC cells are almost always positive for AE1/AE3, CAM5.2, CK8, and CK18,276,277 whereas CK7 and CK19 are less common,278 and CK20 is generally negative. EMA is expressed in about half of the tumors.276 Glycoproteins characteristic of ductal differentiation— MUC1, MUC5AC, CEA, CA 19-9, DUPAN-2, B72.3 and CA-125—are either negative or only focally positive. However, nuclear immunoreactivity for the product of the pancreatic duodenal homeobox 1 (PDX1) gene is observed in a majority of the tumors.278 IHC identification of pancreatic enzyme production is helpful in confirming the diagnosis.67,68,279-282 Both trypsin (Fig. 15-19) and chymotrypsin are detectable in more than 95% of cases, although some studies have shown less
Pancreas
553
KEY DIFFERENTIAL DIAGNOSIS Acinar Cell Carcinoma • SPN vs. ACC: SPN typically expresses a nonspecific acinar marker, α1-antityripsin, similar to ACC; however, it also consistently expresses vimentin, CD56, β-catenin, CD10, and progesterone receptors. Furthermore, in contrast with ACC, more specific acinar markers (trypsin, chymotrypsin, and lipases) are not expressed in SPN, and epithelial markers are usually either focal or weak. • PanNET vs. ACC: Scattered neuroendocrine cells or a focal endocrine component are common in ACC; however, diffuse and strong reactivity for the neuroendocrine markers (chromogranin and synaptophysin) throughout the tumor is characteristic of PanNETs. Additionally, PanNETs do not show immunoreactivity for acinar markers. Figure 15-19 Immunohistochemical stains for acinar enzymes—in particular trypsin, shown here—are detectable in over 95% of acinar cell carcinomas.
sensitivity for chymotrypsin.279 Carboxyl ester lipase (CEL) and lipase are less commonly identified.278 Other enzymes reportedly positive in ACC are amylase, α1antitrypsin, α1-antichymotrypsin, phospholipase A2, and pancreatic secretory trypsin inhibitor. Recently, the antibody directed against the COOH-terminal portion of the Bcl-10 protein, which recognizes the COOHterminal portion of CEL, has been suggested as a useful tool for detecting ACC.278,283 Nuclear immunoreactivity for β-catenin can be seen in approximately 10% of cases.278 In daily practice, the most useful antibodies applied are trypsin and chymotrypsin. The NE component shows immunoreactivity for chromogranin or synaptophysin,67,262,279,284 and peptide hormones, such as glucagon or somatostatin, are expressed infrequently. In some cases of ACC, even the most typical acinar areas may show positivity with NE markers. Markers typically found in solid-pseudopapillary neoplasms—vimentin, CD56, and progesterone receptors—are negative. The literature on p53 expression and mutation is controversial,67,278,285-288 but SMAD4 is retained.81,289 In stark contrast with ductal cancers, ACCs very rarely if ever show KRAS mutations by molecular/genetic analysis.67,82,285,287,290,291 Recent work has identified a high frequency of allelic loss on chromosomes 4q, 11p, and 16q.289,292,293 In addition, 25% of ACCs have mutations in the APC/CTNNB1 (β-catenin) pathway, a pattern similar to that of pancreatoblastoma.67,287,289,292 KEY DIAGNOSTIC POINTS Acinar Cell Carcinoma • Immunohistochemical stains for acinar enzymes, in particular trypsin but also chymotrypsin, serve as highly specific markers of acinar differentiation. • Pancreatic ductal differentiation markers (mucins and mucin-related oncoproteins) are either negative or only focally positive. • The neuroendocrine component is very commonly present and shows immunoreactivity for chromogranin or synaptophysin as well as other neuroendocrine markers.
PANCREATOBLASTOMA
Pancreatoblastoma is a rare pancreatic tumor that shows differentiation toward all three lineages in the pancreas (acinar, ductal, and neuroendocrine) in variable amounts.3 It is the most common pancreatic neoplasm of childhood, although one third of reported cases occurr in adults. Microscopically, the tumors show large, solid, nesting, and acinar growth patterns and have characteristic squamoid corpuscles that occasionally have optically clear nuclei rich in biotin.254 Many cases show labeling for markers of acinar, ductal, and NE differentiation in the respective areas, although acinar differentiation is the most common and is the predominant pattern in the majority of the cases.254 The acinar component labels with antibodies to CAM5.2, AE1/AE3, and cytokeratins 7, 8, 18, and 19. Positivity for trypsin and chymotrypsin is found in nearly every case; lipase is less common.68,294-299 The ductal elements, present in 50% to 65% of cases, express glycoprotein markers such as CEA, B72.3, and DUPAN2.68,295,296,300,301 Finally, NE markers chromogranin and synaptophysin are positive in two thirds of cases in a highly variable proportion of the cells.68,300,301 Staining for islet peptides (insulin, glucagon, or somatostatin) is generally not found.296 IHC positivity for α-fetoprotein (AFP) has been detectable in cases with elevations in the serum levels of this marker.296,302 Morular formations known as squamoid corpuscles are characteristic and entity-defining features of pancreatoblastoma, present virtually in every case. IHC evaluation of the squamoid corpuscles has failed to define a reproducible line of differentiation for this component.296 In fact, both by morphology and immunophenotype, they show more striking similarities to morules seen in tumors such as endometrial carcinoma, pulmonary endodermal tumor, and the cribriform-morular variant of papillary thyroid carcinoma, all of which are associated with β-catenin alteration, just like squamoid corpuscles of pancreatoblastoma. In fact, the abnormal nuclear immunolabeling pattern for β-catenin is most prominent in squamoid corpuscles, more so than in other cell types that occur in pancreatoblastoma (Fig. 15-20). Squamoid corpuscles are also positive for EMA,
554
Immunohistology of Pancreas and Hepatobiliary Tract
SOLID-PSEUDOPAPILLARY NEOPLASM
10 na pt D1 op hy si n FL I-1
D
lin
yc
SE N
C
C
Sy
1 -A
nt PR itr yp si n
56
in
D
• Most pancreatoblastomas show at least some labeling for acinar enzymatic markers (trypsin, chymotrypsin, and lipases). • Neuroendocrine and ductal markers are also often positive but to a lesser degree, and positivity may be more focal. • An abnormal nuclear and cytoplasmic immunolabeling pattern for the product of the β-catenin gene CTNNB1 and overexpression of the product of its target gene, CCND1, is seen in most cases, usually in the squamoid corpuscles.
C
Pancreatoblastoma
n
KEY DIAGNOSTIC POINTS
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
en t
The most common genetic alteration identified to date is LOH of the highly imprinted region of chromosome 11p near the WT2 gene locus.305 Additionally, alterations in the APC/CTNNB1 (β-catenin) pathway have been reported in 50% to 80% of pancreatoblastomas.303 Most often, these involve the β-catenin gene CTNNB1.303,304 Unlike DAs, mutations of TP53 and KRAS have not been detected in pancreatoblastoma.303,305-307
at en i
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Vi m
The distribution pattern of the β-catenin immunolabeling is characteristic for pancreatoblastoma. The acinar/ ductular elements show mostly membranous (normal) expression of β-catenin, whereas the squamous corpuscles display diffuse nuclear/cytoplasmic (abnormal) expression (see Fig. 15-20) and overlapping cyclin D1 overexpression (>5% of tumor cells positive).303,304
-c
Genomic Applications of Immunohistochemistry
al
which accentuates their similarities with meningothelial whorls. CEA can be focally positive,296 and so can CK8, CK18, and CK19 but not CK7.297
Ab no rm
Figure 15-20 An abnormal cytoplasmic and nuclear immunolabeling pattern for the product of the β-catenin gene CTNNB1 is seen in most pancreatoblastomas, predominantly in the squamoid corpuscles. Other areas usually display normal membranous immunolabeling.
Solid-pseudopapillary neoplasm (SPN) is a peculiar tumor of indeterminate lineage. Although SPNs have been described in all age groups, the mean age at presentation is 30 years. They are seen almost exclusively in females, but they are also seen in males on occasion. Histomorphologically, SPNs typically show diffuse cellular proliferation of relatively bland cells admixed with a variable degree of stroma. The preferential dyscohesiveness of the cells away from the microvasculature, presumably related to alterations in cell-adhesion molecules (β-catenin and E-cadherin), leads to the highly distinctive arrangement of cells referred to as pseudopapillary, although it is not present in all cases (Fig. 15-21).187 Eosinophilic globules composed of α1antitrypsin might also be seen. SPNs are low-grade malignancies that are curable with complete removal in 85% of cases. No reliable criteria have been established to recognize the remaining 15% that will spread to the peritoneum or liver, but typically even these patients experience a protracted clinical course. Despite intensive study, the line of differentiation of these neoplasms remains uncertain.68,308-310 Both acinar and ductal markers discussed previously are consistently negative in SPN.68,311,312 The tumors are also consistently negative for chromogranin. In fewer than 5% of SPNs, chromogranin expression is very focal. Conflicting data on this issue have been published in the literature, but all investigators now agree68,311,313,314 that if a tumor shows substantial chromogranin expression, it is not an SPN. Peptide hormones are also usually negative or, at most, are focally positive.68,308-310,315,316 SPNs also fail to show any convincing neurosecretory granules by electron microscopy, which further collaborates that these are non-NE neoplasms. However, these tumors commonly react with some of the so-called NE markers such as synaptophysin, NSE, and CD56.68,306,308-311,314,316-325 Even the epithelial nature of SPN is dubious, although it has been referred as “carcinoma” in the past. Cytokeratins (CAM 5.2, AE1/AE3) and other epithelial
Figure 15-21 Immunohistogram of a solid-pseudopapillary neoplasm with selected antibodies. NSE, Neuron-specific enolase; PR, progesterone receptor.
Pancreas
markers are typically either negative or only very focal in rare cases. Ultrastructural evidence of epithelial differentiation is also lacking. However, the neoplastic cells express vimentin and α1-antitrypsin diffusely and stro ngly.68,289,308-311,314,316-325 Another marker consistently expressed in SPN is CD10; however, this marker should be used cautiously in the differential diagnosis, because DAs and PanNETs can also stain for CD10, also known as common acute lymphocytic leukemia antigen (CALLA).21,326 Progesterone receptors are also expressed in SPNs, both in women and in men.319,322,327,328 Recently, c-kit (CD117)329 and fli-1330 expression has been reported in a portion of SPNs. Usually, SPNs do not stain with S-100 protein,68 calretinin,331 or AFP.311 Genomic Applications of Immunohistochemistry
More than 90% of SPNs have mutations in exon 3 of the β-catenin gene, CTNNB1.2,67,68 In those cases not showing exon 3 mutations, which would account for the remaining 10% of cases, it is likely that mutations are present in other exons.21 Therefore, 100% of SPNs show an abnormal cytoplasmic/nuclear pattern of labeling with antibodies to the β-catenin protein (Fig. 15-22).306,319,330,332 This pattern suggests a CTNNB1 mutation activating the Wnt signaling pathway, which results in overexpression of cyclin D1 in the pathogenesis of these tumors.81,219,249,306,320,323,333 In fact, in more than two thirds of the cases, a concomitant cyclin D1 expression is also seen.306,323 In addition, through a mechanism that is not yet clear, the disruption of β-catenin interferes with E-cadherin and, as a result, use of an E-cadherin antibody in the extracellular domain of the molecule illustrates complete loss of membrane staining, whereas the antibody directed to the cytoplasmic fragment produces distinct nuclear staining of the tumor cells in virtually all cases.20,330 This loss of E-cadherin may be responsible for the distinctive dyscohesiveness of the cells that creates the characteristic (and entity name-defining) pseudopapillary appearance. Thus the most common genetic alterations in SPNs, CTNNB1 gene mutations, help explain the poor cohesion of the neoplastic cells and provide a useful diagnostic tool: immunolabeling for β-catenin protein.21,136
555
The expression of CD56, progesterone receptor, and fli-1 all located on chromosome 11q, has also been interpreted as evidence that chromosome 11q might be involved in a translocation or mutation that leads to the expression of some or all of these three proteins in SPN.330,334 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
In contrast to DAs, alterations in the KRAS, CDKN2A, TP53, and SMAD4/DPC4 genes have not been reported in SPNs. Besides, SPNs almost always exhibit CTNNB1 mutations.219,249,306,318,320,332 KEY DIAGNOSTIC POINTS Solid-Pseudopapillary Neoplasm • Abnormal nuclear/cytoplasmic expression of β-catenin, loss of membrane staining, and/or abnormal nuclear staining for E-cadherin combined with negative or very focal and/or weak cytokeratins (CAM5.2, AE1/AE3) can be used to confirm the diagnosis of SPN even in small biopsy specimens. • Most nonspecific neuroendocrine markers—synaptophysin, CD56, and NSE—are consistently positive in SPNs; however, the most specific neuroendocrine marker, chromogranin, is consistently negative in SPNs.
KEY DIFFERENTIAL DIAGNOSIS Solid-Pseudopapillary Neoplasm SOLID-PSEUDOPAPILLARY NEOPLASM VS. PANCREATIC NEUROENDOCRINE TUMOR • Nuclear expression of β-catenin is consistent in SPN but is very uncommon in PanNET. In contrast, most PanNETs show diffuse strong labeling for chromogranin and keratins, whereas virtually all SPNs are negative for these markers. • Strong positivity of vimentin and progesterone receptors is also more common in SPNs than in PanNETs. • Synaptophysin, NSE, and CD56 are expressed consistently in both tumors and thus cannot be used in this differential diagnosis. SOLID-PSEUDOPAPILLARY NEOPLASM VS. ACINAR CELL CARCINOMA VS. PANCREATOBLASTOMA • Expression of acinar enzymes, trypsin, and chymotrypsin coupled with diffuse keratin positivity are diagnostic of ACC and are also present in pancreatoblastomas. • Nuclear β-catenin expression and positivity of α1-antitrypsin and antichymotrypsin (not to be confused with trypsin and chymotrypsin) are common to all three tumors and cannot be used in this differential.
Neuroendocrine Neoplasms
Figure 15-22 Solid-pseudopapillary neoplasm with diffuse cytoplasmic and nuclear β-catenin labeling.
Focal NE differentiation, especially in the form of scattered cells, is quite common in pancreatic neoplasia of the ductal and acinar varieties and is of no known biologic significance. However, if a tumor is predominantly
Immunohistology of Pancreas and Hepatobiliary Tract
PANCREATIC NEUROENDOCRINE TUMORS
Pancreatic neuroendocrine tumors (PanNETs) represent the majority of the NE neoplasms in the pancreas.189 Most PanNETs are sporadic; however, these tumors also constitute one of the major components of the multiple endocrine neoplasia I (MEN 1) syndrome, and they may arise in patients with vHL syndrome. PanNETs associated with increased serum levels of hormones and lead to corresponding symptoms are referred as to functional and are named according to the hormone they secrete: insulinoma includes 42% of all functional variants; gastrinoma, 24%; glucagonoma, 14%; vasoactive intestinal polypeptide-secreting tumor (VIPoma), 10%; and somatostatinoma, 6%. Interestingly, the amount of hormone detected immunohistochemically in the tumor cells does not necessarily correlate with the functional status,254 thus routine use of a hormone panel in the diagnosis of PanNETs is still debated. Suffice it to say that serologic analysis or symptoms and signs of the tumor override the IHC findings in this regard. The tumor cells mimic the islet cells by forming nests, trabecules, and gyriform patterns, and they show the typical NE cytologic features that include round monotonous nuclei, “salt-and-pepper” chromatin, and a moderate amount of cytoplasm.4,158 Almost all PanNETs label for at least one of the NE differentiation markers: chromogranin, synaptophysin, NSE, CD56, and Leu-7 (CD57).335-351 Among these, chromogranin, the most specific NE marker, is detected in 85% to 95% of PanNETs. Synaptophysin is more consistently and diffusely expressed than chromogranin, but unfortunately it is less specific. For example, SPN, the main tumor in the differential diagnosis, is commonly positive for synaptophysin and for NSE and CD56. In addition to the conventional peptide hormones of pancreatic islets (insulin, glucagon, somatostatin, and pancreatic polypeptide352,353), studies have shown that these tumors can also secrete and express ectopic peptides—in particular, gastrin352-354 and VIP352-354 and, on occasion, adrenalcorticotropic hormone (ACTH)355; antidiuretic hormone; melanocyte-stimulating hor mone (MSH); calcitonin; neurotensin; secretoneurin,189,270,356,357 a parathyroid hormone–like peptide358; growth hormone and growth hormone releasing factor359-361; secretogranin II340; inhibin/activin362; prohormone convertases 2 and 3363; metallothionein364; and somatostatin receptors.365 The pattern of labeling for these hormones varies widely,352,366,367 and it is common to demonstrate the production of more than one hormone in a single PanNET. However, only occasionally does a PanNET produce detectable serotonin.4 Therefore for practical purposes, a serotonin-producing tumor should be regarded as a carcinoid, and if it is in the pancreas, the possibility of metastasis from the GI tract or elsewhere must be excluded. Most PanNETs also stain with CAM5.2, CK8, and CK18, and approximately half stain with AE1/AE3. In general, the degree of keratin expression is weaker than
in acinar and ductal tissue.368-370 CK7 and CK20 are usually either negative or stain only rare cells.44,347,371,372 Scattered cells that are positive for acinar differentiation markers, such as trypsin or chymotrypsin, are commonly seen in PanNETs.373-375 If more than 25% of the neoplastic cells in a predominantly NE tumor express markers of acinar differentiation, the neoplasm is classified as a mixed acinar-neuroendocrine carcinoma376 by convention. Limited experience suggests that these are more aggressive neoplasms that behave like acinar cell carcinomas. PanNETs may also show labeling for glycoprotein markers of ductal differentiation. This result may be encountered even in PanNETs with classic morphology and is not sufficient evidence for a diagnosis of mixed ductal-neuroendocrine carcinoma, unless a morphologically separate component of DA is recognized.4 Focal expression of DUPAN-2 or CA 19-9 is found in almost a quarter of conventional PanNETs.373,374,377 CEA is much less commonly expressed. Some oncocytic PanNETs also label with HepPar1, which may be important in the differential diagnosis, because these oncocytic PanNETs do resemble hepatocellular carcinomas (HCCs).378 Normal islet cells express progesterone receptors and CD99, but only some PanNETs retain expression of these markers (Fig. 15-23).373,379,380 Most PanNETs are low- to intermediate-grade malignancies, but it is somewhat difficult to predict which examples are more prone to recurrence and metastasis. By the 2010 WHO classification, NE neoplasms of the digestive system should be graded by mitotic rate and Ki-67 proliferation index. The proposed grading has three tiers, with the following definitions of mitotic count and Ki-67 index189: Grade 1 (G1): mitotic count, <2 per 10 hpf and/or ≤2% Ki-67 index Grade 2 (G2): mitotic count, 2 to 20 per 10 hpf and/ or 3% to 20% Ki-67 index Grade 3 (G3): mitotic count, >20 per 10 hpf and/or >20% Ki-67 index The grading requires mitotic count in at least 50 hpf and Ki-67 index by using the MIB antibody as a percentage of 500 to 2000 cells counted in areas of
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% na N C pto SE hr ph om y og sin ra C nin AM 5. 2 C D AE 56 1/ AE 3 C K1 9 C D 99 1 -A P nt itr R y D psi U PA n N C -2 A1 9m 9 C EA
composed of cells with NE lineage, it is classified as neuroendocrine.3
Sy
556
Figure 15-23 Immunohistogram of pancreatic neuroendocrine tumor with selected antibodies. CK, Cytokeratin; DUPAN, ductal of pancreas 2; mCEA, monoclonal carcinoembryonic antigen; NSE, neuron-specific enolase; PR, progesterone receptor.
Pancreas
strongest labeling (“hot spots”). If the grade differs for the mitotic count compared with Ki-67 index, it is suggested that the higher grade be assumed.189 Our recent data also support the notion that the mitotic rate and Ki-67 index–based grades of PanNETs can be discordant, and when the Ki-67 index is greater, the clinical outcome is significantly worse.381 We further demonstrated that morphologically well-differentiated PanNETs that are grade 3 by Ki-67 are still different from bona fide poorly differentiated neuroendocrine carcinomas (NECs, as defined in the lungs, with small and large cell variants), suggesting that the current grade 3 can be further separated into well-differentiated PanNET with increased proliferation index (G3) and poorly differentiated (high-grade) NECs (G4).381 We suspect that in the future, true poorly differentiated (high-grade) NECs, which are very uncommon in the pancreas, will nevertheless have to be regarded separately, either as G4 or a separate category. The best method to count Ki-67 is still debatable. In the North American Neuroendocrine Tumor Society (NANETS) guideline papers, it is stated that an “eyeballed estimate is adequate.”382 Although this may be true for experts who do this on a regular basis, clearly, as was illustrated by Tang and colleagues,383 the eyeballing approach is not an option for general practice. Our experience is very similar; the eyeballing method is highly subjective and inaccurate.384 In our experience, automated counting devices also have several challenges.384 They are highly operator dependent, and if used by inexperienced individuals, they are prone to miscounting, unless software modifications are implemented by experts to avoid overcounting of lymphocytes, hemosiderin, and other contaminating factors or to correct for cellular overlapping and other tissueprocessing variations. Moreover, unlike a microscope, no field area is defined in the automated systems, and the outlines of a “hot spot” can be highly operator dependent, impacting the numerator/denominator ratio. Availability of the instrument, which has an often prohibitively high cost, and the necessity of batching cases with the impact on turnaround time are also likely to limit widespread use of automated systems. The method we have found to be most effective and practical is to manually count the cells, not through the microscope, but either on a computer screen or on a printed version of the captured image, so the counted cells can be highlighted, crossed off, and circled for counting purposes.384 The latter can be performed with any digital camera set up with a microscope; the results are fairly reproducible, and it can be performed in a matter of minutes.384 Studies have shown that the Ki-67 approach labeling index in metastatic tumors has prognostic value as well.385 This is also advocated in the current guidelines. Considering the significant heterogeneity in the levels of Ki-67 expression within a given tumor,386 and occasional cases that seem to show a progression or dedifferentiation phenomenon, it is justified to also perform Ki-67 indexing in metastatic sites.387 Whether to perform Ki-67 labeling on a synchronous metastasis or only on the primary tumors (when removed at the
557
Figure 15-24 Cytokeratin 19 staining in pancreatic neuroendocrine tumors is considered by some authors to be an independent predictor of aggressive behavior.
same time) is an issue that requires further study and clarification. Recently, several studies from various institutions have also shown that CK19 staining (Fig. 15-24) may serve as another reliable independent predictor of aggressive behavior.368,369 This result has not yet been incorporated into the classification schemes, but many experts perform this stain routinely and report it in a comment. Functional status has also been traditionally used as a prognostic parameter. It is well documented that most insulinomas behave in a benign fashion, and most glucagonomas exhibit a malignant course. However, it is widely accepted that this association is through the stage of the tumor: most insulinomas manifest early with symptoms and signs—Whipple triad, symptoms of hypoglycemia, low serum glucose, and relief of symptoms with glucose administration—before they achieve a size of 2 cm, whereas many glucagonomas are large and metastatic at presentation. Other markers are under intense scrutiny as predictors of outcome in PanNETs. Chetty and Serra18,19 recently reported that aberrations of β-catenin (decrease in membranous staining compared with normal and/or abnormal cytoplasmic/nuclear staining) and E-cadherin expressions (decrease in membranous staining compared with normal and/or abnormal nuclear staining) occur in more than 50% of PanNETs. They also showed that PanNETs with aberrant β-catenin and E-cadherin expressions tend to be larger than those with normal staining patterns, and most of the cases with lymph node and liver involvement show concordant β-catenin and E-cadherin abnormal immunoexpression.18,19,369 Whether these markers may be of use in identification of PanNETs with a potential for spread, and hence poor outcome, has yet to be fully characterized. Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Cytogenetic and molecular genetic studies have identified many chromosomal alterations in PanNETs
558
Immunohistology of Pancreas and Hepatobiliary Tract
(chromosomal losses are more common than gains).388-391 Furthermore, a dominantly inherited defect in the MEN1 gene has been described in patients with the MEN 1 syndrome. Spontaneous MEN1 gene abnormalities also occur in approximately 20% of sporadic PanNETs,81,274,392-399 although a greater proportion show chromosomal losses in the same genetic region (11q13). It is believed that many of these PanNETs arise from the mutation in one locus of MEN1, followed by LOH. PanNETs that arise in patients with the vHL syndrome also usually show biallelic inactivation of the vHL gene.400 The vHL gene is normal in sporadic PanNETs.274 Genes involved in a chromatin remodeling complex (DAXX and ATRX) are somatically mutated, which includes numerous inactivating mutations, in approximately 45% of sporadic PanNETs and appear to be associated with better prognosis.129 Also, somatic mutations of genes in the cell-signaling pathway of mammalian target of rapamycin (mTOR)—including PIK3CA, PTEN, and TSC2—occur in approximately 15% of sporadic PanNETs.129 In contrast to DAs, KRAS, TP53, and SMAD4 are not mutated in most PanNETs, with the exception of CDKN2A abnormalities.274,285,394,401-403
KEY DIAGNOSTIC POINTS Pancreatic Neuroendocrine Tumors • Almost all PanNETs label for at least one of the neuroendocrine differentiation markers: chromogranin, synaptophysin, NSE, CD56, and Leu-7 (CD57). • PanNETs can express any of the pancreatic hormones and ectopic peptides. In some cases, more than one hormone is expressed. If an IHC “hormone panel” is to be used, the six hormones that cover the vast majority of the cases are insulin, glucagon, somatostatin, pancreatic peptide, gastrin and VIP; however, hormone ICH in the tumor does not necessarily correlate with the functional status in all cases. Discrepancies do occur, therefore serology and symptomatology are more reliable in classification of functional status. • For practical purposes, a serotonin-producing tumor should be regarded as a carcinoid, and if it is in the pancreas, the possibility of metastasis from the GI tract or elsewhere ought to be ruled out. • If more than 25% of the neoplastic cells in a predominantly neuroendocrine neoplasm express markers of acinar differentiation, such as trypsin or chymotrypsin, the neoplasm should be classified as a mixed acinarneuroendocrine carcinoma. • Ki-67 labeling index is essential for grading of PanNETs. • CK19 immunolabeling is also widely accepted to have prognostic significance. • It should be kept in mind that normal islet cells are prone to binding antibodies nonspecifically,16 and the same problem may conceivably exist for neoplastic neuroendocrine cells. Therefore caution should be exercised before classifying islets (and PanNETs) as positive for any marker not verified by Western blot or other confirmatory methods.
KEY DIFFERENTIAL DIAGNOSIS Pancreatic Neuroendocrine Tumors PRIMARY PANCREAS VS. PRIMARY GI TRACT VS. PRIMARY LUNG NEUROENDOCRINE NEOPLASMS Well-differentiated neuroendocrine (carcinoid) tumors of pulmonary and GI origin show some morphologic similarities to PanNETs, thus predicting the site of origin in a metastatic site may require aid from immunohistochemistry. It has been reported that404: • Nuclear expression of PDX-1 and NESP-55 (a new addition to the chromogranin family), especially if coupled with negative CDX-2 and TTF-1, would favor pancreatic origin.403 • Positivity of CDX-2 in the absence of PDX-1, NESP-55, and TTF-1 highly favors a carcinoid of GI primary. • TTF-1 positivity is compatible with pulmonary origin; however, it is not necessarily present in all lung neuroendocrine neoplasms. Distinguishing full-blown PanNETs from reactive megaislets (as much as several millimeters) can be accomplished by hormone stains that show a more clonal distribution in PanNETs with one or two peptides, whereas nonneoplastic islets often show more regional distribution of several hormones, similar to normal islets.
POORLY DIFFERENTIATED (HIGH-GRADE) NEUROENDOCRINE CARCINOMA
These highly aggressive and rapidly fatal tumors constitute less than 1% of all pancreatic NE neoplasms. They are so uncommon that some authors believe a poorly differentiated NEC that occurs in the pancreas is most likely a metastasis from an occult primary in the lung. However, cases of primary tumor in this region have been proved.4,405-407 Many are in fact of ampullary origin, and few are pancreatic.4,405-407 Some are akin to small cell carcinomas as defined in the lung, but more commonly they resemble large cell NECs such as those common in the lung or elsewhere in the GI tract.158 Although unfortunately these are now classified as grade 3 PanNETs by the 2010 WHO criteria, as if they are in a continuum with the ordinary PanNETs; in fact, overwhelming evidence now suggests that they actually belong in a category of their own.381 We suspect that in the future, true poorly differentiated (high-grade) NECs will nevertheless have to be regarded separately, either as grade 4 tumors or in a separate category. IHC labeling commonly reveals positivity for chromogranin and synaptophysin even in the most poorly differentiated NECs of the pancreas; however, it may be very focal, especially for chromogranin.4 CD56 and CD57 are frequently strongly positive in a membranous pattern.408,409 In some studies, in contrast to DAs and well-differentiated PanNETs, poorly differentiated NECs were found to express the cell-adhesion molecule L1 (CD171).410,411 Parallel to the degree of mitotic activity and necrosis, the Ki-67 labeling index also tends to be very high in these tumors. Primitive neuroectodermal tumor (PNET), which shares some cytologic features with small cell carcinoma but occurs in younger
Extrahepatic Biliary Tract (Gallbladder and Extrahepatic Bile Ducts)
559
patients, stains immunohistochemically for CD99412 and fli-1.413
KEY DIFFERENTIAL DIAGNOSIS Poorly Differentiated (High-Grade) Neuroendocrine Carcinoma POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMA VS. ACINAR CELL CARCINOMA • Acinar cell carcinoma often gets misdiagnosed as poorly differentiated NEC.413 Therefore, thorough immunohistochemical evaluation—especially with trypsin, chymotrypsin, and Bcl-10—should be performed before a poorly differentiated NEC diagnosis is rendered. Figure 15-25 MUC6 is consistently expressed in pyloric type intracholecystic papillary tubular neoplasms of the gallbladder.
Extrahepatic Biliary Tract (Gallbladder and Extrahepatic Bile Ducts) Epithelial Neoplasms INTRADUCTAL AND INTRACHOLECYSTIC NEOPLASMS (TUMORAL INTRAEPITHELIAL NEOPLASMS)
Mass-forming preinvasive neoplasms (tumoral intraepithelial neoplasms) of the extrahepatic biliary tract (EHBT) are known by a plethora of designations that include adenomas; papillary, tubular, and tubulopapillary neoplasms; and papillomatosis based on the pattern of growth, degree of dysplasia, or extent of the lesion. In the extrahepatic bile ducts, the analogy of these neoplasms to their pancreatic counterparts (see IPMN and ITPN in the sections above) is now well established and, in the 2010 WHO guidelines, the papillary variety are referred to as intraductal papillary neoplasms (IPNs).189 In contrast, these tumors in the gallbladder are classified under two separate categories as adenoma and intracystic papillary neoplasm, presumably based on the degree of papilla formation and/or dysplastic transformation. However, no specific criteria are provided for their distinction from each other, which seems to be problematic even for experts.414 We believe these two categories can be regarded under a generic category, for which we prefer the term intracholecystic papillarytubular neoplasm (ICPN),415 because they share common features and also exhibit many similarities to pancreatic IPMNs and ITPNs: they demonstrate a spectrum of dysplastic change (adenoma-carcinoma sequence), variable configuration, and different cell lineages that recapitulate the cell types in the GI tract. Transitional forms, mixed areas, and unclassifiable patterns are more common and are seen to some degree in 90% of the cases. This renders the cell lineage– based classification more difficult to apply. However, when cases are classified on the basis of the predominant (>75%) pattern, four distinct cell lineages can be
identified.415 The most common phenotype is classifiable as a biliary type and commonly expresses MUC1; it resembles gallbladder epithelium or pancreatobiliarytype IPMNs. The gastric phenotype has two distinct subtypes: the foveolar type, with uniform MUC5AC expression, is closely related to the biliary type and is also commonly accompanied by invasive carcinoma (in 60% of the cases); the pyloric type is characterized by diffuse/uniform MUC6 expression (Fig. 15-25) and is often large and homogenous with morule formations. Similar to other morule-forming tumors—also called biotin-rich optically clear nuclei (BROCNs),416 including pancreatoblastoma—this subtype is also commonly associated with aberrant nuclear localization of β-catenin, gene mutation, and common expression of estrogen receptors. More importantly, it has a significantly lower frequency of associated invasive carcinoma. The third intestinal phenotype, which may or may not express CDX-2 and MUC2, is relatively uncommon; when it does occur, it is less complete than it is in pancreatic IPMNs. As in other areas of the pancreatobiliary tract, the fourth phenotype, oncocytic, is fairly uncommon in ICPNs. More importantly, it does not show the classic immunophenotypic features of oncocytic IPMNs or IPNs417-420 and often lacks HepPar1 and MUC6 expression; instead, it consistently shows MUC1 expression. In addition to the aid it provides in verifying the cell lineages discussed above, immunophenotyping also discloses some general characteristics of ICPNs. True to their biliary origin, ICPNs are typically positive for CK7, even in the cases that show intestinal differentiation. It is also noteworthy that MUC1, in addition to being a fairly good indicator of biliary phenotype, is also expressed in the high-grade areas of any type and thus may serve as a marker of high-grade dysplasia. It may be important to reiterate here that a significant proportion of the ICPNs have hybrid patterns and heterogeneous areas that coexpress different IHC markers. In fact, this pronounced proclivity to form hybrid
560
Immunohistology of Pancreas and Hepatobiliary Tract
phenotypes further necessitates the classification of these lesions under one umbrella. Of note, expression of p53 is noted in approximately one third of cases, mostly in biliary examples with high-grade dysplasia. Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Mutation in KRAS is uncommon (<25%), as is the case for other gallbladder neoplasms.421 GNAS codon 201 mutation, seen in approximately two thirds of pancreatic IPMNs, is also rarely seen in these tumors.422 MUCINOUS CYSTIC NEOPLASMS
Mucinous cystic neoplasms (MCNs) represent the biliary counterpart of pancreatic mucinous cystic neoplasm (see the “Pancreas” section above for details).423 DYSPLASIA
Microscopic incidental forms of preinvasive neoplasia in the EHBT are considered under the category of nontumoral (“flat”) lesions to distinguish them from the massforming preinvasive neoplasms (tumoral intraepithelial neoplasms) discussed above. These so-called flat lesions also often have papillary configuration but by definition do not form a grossly or radiographically recognizable compact, distinct mass. In the past, these were referred to as epithelial atypia, but currently the preferred terminology is dysplasia or biliary intraepithelial neoplasia (BilIN). Dysplasia is reported in 40% to 60% of patients with invasive adenocarcinoma in EHBT; however, the incidence of dysplasia outside the setting of invasive adenocarcinoma is difficult to determine. It is also clear that the frequency varies significantly among different populations and risk groups and parallels that of adenocarcinoma.424,425 Microscopically, dysplasia is characterized by the disorderly intraepithelial proliferation of atypical cells and is generally graded as low or high grade. However, biliary epithelium has a tremendous capacity to develop marked cytologic atypia secondary to injury that may, at times, be impossible to distinguish from a true neoplastic process.2 If used cautiously, IHC can be used as an adjunct in the differential diagnosis of reactive versus dysplastic lesions. Nuclear p53 expression is present in more than 30% of all dysplasia in the EHBT (Fig. 15-26), and the incidence and degree of expression is significantly higher in high-grade lesions,426 whereas p53 is relatively uncommon in nonneoplastic epithelium. On the other hand, it can be present in areas of marked regenerative changes as well, which limits its value as a sole diagnostic marker. Similarly, although Ki-67 labeling index is often substantially greater in dysplastic lesions, and it increases in quantity with increasing degrees of dysplasia, it can also be marked in areas of regenerative change.1 Of note, it is being recognized that gallbladder dysplasia may also manifest along different cell lineages, including biliary, gastric, intestinal, and oncocytic lines. Preliminary evidence suggests that the
Figure 15-26 Immunohistochemical overexpression of p53 protein by dysplastic cells in gallbladder.
immunophenotype of these may correspond to the cell lineage markers discussed above, but more studies are needed to confirm this.427 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Analysis of normal and dysplastic gallbladder epithelium has shown much. First, allelic losses at chromosome 8p occur in normal mucosa, which was interpreted as the earliest genetic steps in the carcinogenesis. Second, allelic loss at chromosome 3p appears to be common in low-grade dysplastic lesions, which was regarded as “intermediate change.” This loss also corresponds to progressive loss of fragile histidine triad (FHIT) protein in dysplastic lesions with increasing grade, as demonstrated immunohistochemically. Third, LOH for 5q has been found in various grades of dysplasia.427 Telomere shortening is also very common in dysplastic lesions, as it is in invasive carcinoma, but it is typically absent in normal mucosa.428 However, metaplastic changes also commonly show telomere shortening. Interestingly, mutation of the KRAS oncogene is highly uncommon in upper biliary dysplasias, including those of the proximal extrahepatic bile ducts (EHBDs) and gallbladder. Even in distal common bile duct, the incidence is lower than it is in pancreatic intraepithelial neoplasia.427 INVASIVE ADENOCARCINOMA
Carcinomas of the EHBDs and gallbladder are rare.2,429 They occur in elderly patients, seen predominantly in the seventh and eighth decades of life,430 although those associated with primary sclerosing cholangitis (PSC) tend to occur in younger patients. Most occur in the gallbladder, followed by the upper third of the EHBDs (above the cystic duct junction). Certain risk factors are well established, such as gallstones for gallbladder cancer431 and parasitic infections for EHBD carcinoma.432 The most common growth pattern is a scirrhous, gray-white, firm mass because of the prominence of desmoplasia.430 Calculi are present in more than 80% of gallbladder cancers.1,2
Extrahepatic Biliary Tract (Gallbladder and Extrahepatic Bile Ducts)
561
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Figure 15-27 Carcinoembryonic antigen, which is generally limited to the apical membrane of the benign cells, often shows intracytoplasmic staining in invasive carcinoma cells.
Extrahepatic biliary tract (EHBT) carcinomas are very similar, both morphologically and immunophenotypically, to other foregut carcinomas, namely pancreatic and gastroesophageal cancers. Their similarity to pancreatic ductal carcinoma is such that they are often classified together as “pancreatobiliary type” adenocarcinoma.1 Mucin, in particular sialomucin433 (nonsulfated or neutral types), is demonstrable by histochemical or IHC stains in almost all cases and may be abundant.1 Immunohistochemically, CK7 is nearly always positive. CK20 may also be positive in some cases, which contrasts with intrahepatic cholangiocarcinomas that tend to be negative for CK20. Many surface glycoproteins such as MUC1 and CEA (Fig. 15-27), normally limited to the apical membrane of the benign cells, are commonly detected in the cytoplasm of the adenocarcinoma cells.1 For these markers, a progressive increase is apparent in the level of expression from preinvasive to invasive and poorly differentiated carcinomas, with dense intracytoplasmic expression detected mostly in advanced carcinomas. The tumors also usually express MUC5AC, CA 19-9, B72.3, EGFR, and pepsinogen I and II.2,430,434 Estrogen receptors are only detectable in a small percentage of cases.435 Scattered NE cells, positive for chromogranin and synaptophysin stains, may also be found.430 Genomic Applications of Immunohistochemistry
In approximately 65% of cases, a positive immunoreactivity for the protein product of the TP53 gene is noted.436,437 Some studies have shown this to be fairly specific for carcinoma, as opposed to the nonneoplastic changes associated with PSC.426 However, it ought to be used cautiously because of significant overlap. Loss of SMAD4, present in approximately half of pancreatic DA, is almost as common in distal common bile duct carcinomas. However, SMAD4 is retained in most proximal EHBD carcinomas.119
Deletion of the CDKN2A gene is observed in half of all gallbladder cancers426,438,439 and is reported to be associated with a poor prognosis in gallbladder cancers.440,441 Although the frequency of KRAS mutations has differed widely among studies, most investigators have found these mutations to be significantly higher in EHBD carcinomas than in gallbladder carcinomas.437,442-446 Amplification of the ERBB2 gene is detected in half of the tumors as well.447 Overexpression of ERBB family receptors, including EGFR, has also been reported.448 Loss of retinoblastoma protein (pRb) expression is rare in non–small cell gallbladder carcinoma but is common in small cell carcinoma of the gallbladder.438 High throughput microarrays have shown aberrant expression of several epithelial antigens such as mesothelin, prostate stem cell antigen, fascin, 14-33σ, topoisomerase II, and many peritumoral stromal proteins.58 These have not yet been fully tested as diagnostic or prognostic markers. KEY DIFFERENTIAL DIAGNOSIS Invasive Adenocarcinoma BENIGN, NONINVASIVE EPITHELIUM VS. INVASIVE CARCINOMA • Gallbladder adenocarcinomas may be deceptively bland appearing and may therefore be difficult to distinguish from Aschoff-Rokitansky sinuses or Luschka ducts. Although p53 and Ki-67 are significantly more common in neoplastic epithelium, unfortunately, overlaps are too common for them to have a conclusive role in this distinction. Similarly, dense cytoplasmic expression of MUC1 and CEA are more characteristic of invasive adenocarcinomas. Unfortunately, this feature is not as evident in the more problematic well-differentiated carcinomas, in which the expression is more typically luminal membranous.
OTHER INVASIVE CARCINOMAS OF THE EXTRAHEPATIC BILIARY TRACT
Intestinal type adenocarcinoma with all the features of conventional colonic adenocarcinoma is very uncommon in the gallbladder; however, on occasion, ordinary gallbladder adenocarcinoma may exhibit foci of columnar cells and pseudostratification and thus may resemble intestinal carcinoma. Further complicating the issue, intestinal markers such as MUC2 and CDX-2 may show some positivity in these tumors1 and may be taken as further evidence of intestinal differentiation, although it should be kept in mind that many classic foregut carcinomas also express these markers. It is advisable not to classify such cases as intestinal-type adenocarcinoma, unless they exhibit all the characteristic morphologic features of colonic adenocarcinomas.2 Most mucinous carcinomas in the gallbladder are of the mixed type. Pure mucinous (colloid) carcinomas (>50 of the tumor showing stromal mucin deposition)
562
Immunohistology of Pancreas and Hepatobiliary Tract
as seen in the breast, skin, or pancreas178 are exceedingly rare in the gallbladder and constitute 2.5% of all gallbladder carcinomas.449 Immunophenotypically, these mucinous carcinomas differ from conventional gallbladder adenocarcinomas by MUC2 positivity, from intestinal carcinomas by an often inverse CK7/20 profile, from breast colloid carcinomas by a lack of MUC6, and from pancreatic colloid carcinomas by CDX-2 negativity. Unlike GI mucinous (colloid) carcinomas, they appear to be microsatellite stable.449 Hepatoid carcinomas show many characteristics of hepatoid differentiation, including positivity for HepPar1 and AFP,450 and a canalicular pattern with polyclonal CEA (pCEA) or CD10 may be seen, although this is very uncommon. However, unlike true HCCs, they also express CK19 and CK20, which are more characteristic of biliary differentiation.451,452 Squamous differentiation is not uncommon in EHBT carcinomas, especially in the gallbladder. Most are focal and are not reported. If the squamous areas constitute more than 25% of the tumor, the term adenosquamous is applied. By convention, even a small glandular component qualifies the tumor as adenosquamous, not squamous. The term squamous cell carcinoma is reserved for those rare “pure” examples in which glandular elements cannot be documented by extensive sampling. The areas of squamous differentiation are typically positive for CK5/6, CK13, and nuclear p63, whereas glandular areas show CEA and B72.3 positivity.2 In some studies, squamous/adenosquamous carcinomas of the gallbladder are reported to be associated with a better prognosis,453,454 unlike those of the pancreas. However, in our experience, the overall prognosis of adenosquamous carcinoma/SCC appears to be even worse than that of ordinary adenocarcinoma.455 Similarly, Chan and colleagues456 reported a tendency for squamous carcinoma of the gallbladder to be of a higher stage at the time of diagnosis. Although the majority of EHBT carcinomas show overt glandular differentiation, in a substantial minority, gland formation may not be as evident; these tumors have a sheetlike growth pattern and lack gland formation,457 and some may have more monotonous, rhabdoidappearing cells. IHC is very helpful in elucidating the epithelial nature of these malignancies by demonstrating keratins, EMA, and other markers and thus differentiating these neoplasms from metastatic melanoma and lymphoma. Expression of mucin-related glycoproteins such as MUC1, CA 19-9, CEA, and others may also help further establish the identity of these tumors and sometimes may also highlight the abortive glandular elements that confirm the diagnosis of adenocarcinoma. Certain variants of poorly differentiated/undifferentiated carcinoma ought to be recognized separately. For example, although rare, medullary-type carcinomas akin to those that occur in the other parts of the GI tract may be seen in EHBT, this pattern is typically associated with lymphoplasmacytic infiltrates, nodular growth, and distinctive cytology that includes round to ovoid large cells with vesicular chromatin and single, prominent nucleoli. In our experience, some of these are associated
with loss of one of the more common MSI markers, such as MLH1 or MSH2. Spindle cell (sarcomatoid) carcinomas also occur in the gallbladder.457,458 Similar to their counterparts in other organs, mesenchymal differentiation in these neoplasms at the morphologic level is also accompanied by immunophenotypic transformation into mesenchymaltype cells, manifested by increased expression of vimentin and acquisition of aberrant expression of actin and other markers. Often, keratins are either weak or focal. A distinctive variant of sarcomatoid carcinoma is the osteoclastic giant cell carcinoma, in which abundant benign osteoclastic giant cells are positive for CD68 and other histiocytic lineage markers.
Neuroendocrine Neoplasms Scattered NE cells may be identified in most gallbladder tumors, including adenomas and carcinomas, and a spectrum of NE differentiation may be encountered in other gallbladder neoplasias.1,2 However, if a tumor is predominantly composed of cells with NE lineage, it is classified as neuroendocrine. Neuroendocrine tumors (NETs) are relatively uncommon in the EHBT,459-461 and they have the typical characteristics of NETs elsewhere in the digestive system (G1, mitotic count <2 per 10 hpf and/or ≤2% Ki-67 index; G2, mitotic count 2 to 20 per 10 hpf and/ or 3% to 20% Ki-67 index and minimal or no necrosis). NETs are commonly but not always positive for chromogranin (Fig. 15-28), synaptophysin, and CD56.1,2 Hormones commonly detected at the IHC level are serotonin and somatostatin, although these do not necessarily correlate with serologic findings.2 True poorly differentiated (high-grade) NECs must be distinguished from NETs, and most are of the small cell type, characterized by diffuse or nested growth patterns, cells with a high nuclear/cytoplasmic ratio, high mitotic activity (typically more than 30 per 10 hpf), and necrosis. They are defined by their morphologic
Figure 15-28 Well-differentiated neuroendocrine tumor, so-called carcinoid, in gallbladder. The tumor cells are diffusely and strongly positive for chromogranin. Dysplastic glandular cells on the surface are negative.
Ampulla
features, and IHC evidence of NE differentiation is not required; chromogranin may be focal or weak and may appear only as fine granules within the cytoplasm. Some tumors would also fit the description of large cell NEC as defined in the lung.438,462,463 For the diagnosis of these so-called large cell NECs, IHC support is desirable to differentiate them from their non-NE counterparts.
Nonepithelial Neoplasms Granular cell tumors (GCTs) are not uncommon in the EHBT, especially in the common bile duct, and they may be multicentric or may coexist with GCTs in other sites, such as skin. These have the characteristic morphologic features of GCTs that arise elsewhere and are immunoreactive to S-100 protein, NSE, Leu-7 (CD57), and vimentin.1,430 Embryonal rhabdomyosarcoma (E-RMS) is the most common malignant tumor of the biliary tract in children; however, it represents less than 1% of all rhabdomyosarcoma (RMS). It may arise from any segment of the EHBDs and in the gallbladder, but the most common site is the common bile duct.1 Macroscopically, the tumor usually consists of a conglomerate of soft, mucosacovered polyps. Microscopically, the polypoid fragments are usually covered by a layer of flattened biliary epithelium, which may be focally denuded. Beneath the surface is a dense zone of primitive spindle cells that form a cambium layer. Muscular differentiation is demonstrable by IHC labeling with MSA, desmin, and muscle transcription factors myogenin and MyoD1.1 Genetic analyses of E-RMS have shown expression of the MYOD1 gene and LOH at loci on chromosome 11.430
Ampulla Premalignant Lesions In the past decade, major developments have occurred in the classification of and terminology for preinvasive neoplasms of the pancreatic ductal system and biliary tract. It is now well established that mass-forming preinvasive neoplasms (tumoral intraepithelial neoplasms) in these regions constitute a distinct group that is different from both conventional adenocarcinoma and from “flat” (ordinary) dysplasia. In the pancreas, intraductal papillary mucinous neoplasm (IPMN) has been the term widely accepted as a unifying category.3,181,189 Meanwhile, the intraampullary counterpart of these lesions remains poorly characterized. Although conventional intestinal-type adenomas, which can also involve the papilla of Vater, have been fairly well documented, as have virtually all intestinal adenomas,464-466 the data on those that arise specifically within the ampulla have been very limited. Such cases have thus far been analyzed either as a part of studies on duodenal adenomas or those on conventional cancers of the ampulla.467,468 In the 2010 WHO blue book, cases that resemble intestinal adenomas were still classified as intestinal-type
563
adenomas, along with adenomas of duodenal surface, whereas those with the pancreatobiliary phenotype are now recognized under a separate category, noninvasive pancreatobiliary papillary neoplasms.189 Because of their analogy to pancreatic or biliary intraductal papillary and tubulopapillary neoplasms, as evidenced by their papillary and/or tubular growth, variable cell lineage, and spectrum of dysplastic change (adenoma-carcinoma sequence), we prefer to classify these as intraampullary papillary-tubular neoplasms (IAPNs).469 In terms of cell-lineage morphology, almost half of IAPNs display a mixture of patterns. However, when evaluated with a forced-binary approach, as intestinal versus gastric/pancreatobiliary based on the predominant pattern, almost two thirds can be classified as intestinal; the remainder can be classified as gastric/ pancreatobiliary. Most intestinal-type papillae reveal CDX-2 (94%) and MUC2 (85%) expression (Fig. 15-29); however, the specificity of these markers for this phenotype is fairly low. In contrast, most gastric/ pancreatobiliary–type papillae are at least focally positive for MUC5AC (95%) and MUC1 (89%). CK7 and CK20 are coexpressed in approximately half of all IAPNs regardless of the cell lineage,469 therefore they are not reliable in this distinction. Much more importantly, in a majority of patients, an associated invasive carcinoma is found. Cell lineage of the invasive component is usually the same as that of the preinvasive component. As expected, the overall survival of patients with IAPN and associated invasive carcinoma is significantly worse than that of patients with IAPN only. However, when compared with conventional invasive carcinomas of the ampullary region, IAPNs with an associated invasive carcinoma have significantly better prognosis.469
Adenocarcinoma More than 80% of ampullary epithelial neoplasms are adenocarcinomas. They are seen predominantly in the seventh decade of life and may be associated with polyposis syndromes and neurofibromatosis. Because ampullary carcinomas result in early symptoms and detection, they are often relatively small at the time of diagnosis. Most have an exophytic component.1 The majority of lesions are intestinal-type adenocarcinomas that exhibit features typical of intestinal adenocarcinomas, including large, elongated tubular units lined by columnar to cuboidal cells. The second most common type is pancreatobiliary-type adenocarcinoma, characterized by smaller glandular units lined by cuboidal cells and surrounded by desmoplastic stroma. Because patients with intestinal-type ampullary adenocarcinoma are reported to have a significantly better prognosis than patients with the pancreatobiliary type,470 the differentiation between the two tumor types is important.1,2 Although IHC has had only limited value in unequivocally differentiating these types, several authors have suggested that a CDX-2 and mucin (MUC) expression profile may be useful for this purpose.471-475 Recently, Sessa and colleagues476 showed that all intestinal-type ampullary adenocarcinomas are
564
Immunohistology of Pancreas and Hepatobiliary Tract
Intestinal type
CDX2
MUC2 Gastric/pancreatobiliary type
MUC1
MUC5AC
Figure 15-29 Top: Most intestinal-type intraampullary papillary-tubular neoplasm (IAPN) papillae reveal CDX-2 and membrane-associated mucin (MUC) 2 expression. Bottom: In contrast, most of the gastric/pancreatobiliary-type IAPN papillae are at least focally positive for MUC5AC and MUC1.
diffusely immunoreactive for CDX-2 (Fig. 15-30, A) compared with rare (30%) pancreatobiliary-type ampullary adenocarcinomas that exhibit only focal CDX-2 (see Fig. 15-30, B). In addition, a significantly higher frequency of MUC1 and MUC5AC expression is detected in the pancreatobiliary type than in the intestinal type, whereas a significantly higher percentage of positivity for MUC2 was found among the intestinal type compared with the pancreatobiliary type.471,476 Chu and colleagues472 reported that CDX-2 and MUC2 can be used as positive markers for intestinal-type ampullary adenocarcinoma with a positive predictive value of 82%. According to the same study, CK17 and MUC1 can be used as positive markers for pancreatobiliary-type ampullary adenocarcinoma and for pancreatic DA and cholangiocarcinoma, with positive predictive values of 83%, 76%, and 58% respectively.472 Moreover, CK7 is also often strongly positive, and CK20 is more focal, in the pancreatobiliary type, whereas the intestinal type tends to have more CK20 and less CK7.476 It is very important to note that all
of these markers must be used in the context of morphology, because none is entirely specific, and the overlaps are too common to allow their usage as a sole marker. Additionally, in 75% of invasive carcinomas, a diffuse, strong cytoplasmic reaction for monoclonal CEA (mCEA) is seen that occurs only in the luminal membrane of benign epithelium. Intestinal-type ampullary adenocarcinomas are more likely to stain than pancreatobiliary types. Similarly, 60% of invasive carcinomas express DUPAN-2; however, pancreatobiliary types are more frequently positive for DUPAN-2 than the intestinal type. Most ampullary adenocarcinomas also stain for CA 19-9, and CA 19-9–positive cases are reported to be more aggressive.477 NE cells are commonly found in ampullary adenocarcinomas.1,2 Although they may not be evident by routine microscopy, IHC staining for chromogranin reveals scattered NE cells in one third of the cases.430 The distribution of these cells is random; in some cases they are scattered evenly throughout the tumor, whereas
Ampulla
A
565
B
Figure 15-30 A, Intestinal-type ampullary adenocarcinomas are diffusely immunoreactive for CDX-2. B, In contrast, pancreatobiliary-type ampullary adenocarcinomas are either negative for CDX-2 or exhibit it only focally.
in others they may be clustered. In general, they are more common in intestinal-type or mucinous adenocarcinomas than in the pancreatobiliary type.430 Other forms of carcinoma that occur in the ampullary region include mucinous adenocarcinoma, signetring cell adenocarcinoma, and so-called invasive papillary carcinoma,430 which may mimic in situ neoplasia because of its exophytic growth, papilla formation, branching architecture, and relatively smooth contours. Adenosquamous and squamous carcinomas of this region are very rare. Similarly, sarcomatoid carcinomas are exceedingly uncommon.1,430 The immunoprofiles of these tumors are very similar to their counterparts in the EHBT and pancreas, as discussed in detail above. GENOMIC APPLICATIONS OF IMMUNOHISTOCHEMISTRY
Mutations of p53 have been detected in the majority of ampullary carcinomas with corresponding accumulation of the abnormal product as detected immunohistochemically.468,478 EGFR is overexpressed in 50% to 65% of invasive ampullary carcinomas. Pancreatobiliary types of adenocarcinoma are more likely to overexpress EGFR than are intestinal-type tumors. Related growth factors c-erbB-2 and c-erbB-3 are also overexpressed in ampullary carcinoma.430 BEYOND IMMUNOHISTOCHEMISTRY: ANATOMIC MOLECULAR DIAGNOSTIC APPLICATIONS
Ampullary adenocarcinomas are less likely to show loss of SMAD4 gene expression479 and KRAS gene mutations than are pancreatic DAs,480 probably corresponding to the incidences of these mutations in intestinal versus pancreatobiliary types of adenocarcinoma. Poorly differentiated ampullary carcinomas with morphologic features that resemble medullary carcinomas of the large bowel are rare but have been reported to demonstrate MSI.476
KEY DIAGNOSTIC POINTS Ampullary Adenocarcinoma • Most pancreatobiliary type ampullary adenocarcinomas are CDX-2, MUC1, and MUC5AC positive and MUC2 negative, and conversely, many intestinal-type ampullary adenocarcinomas are CDX2 and MUC2 positive and MUC1 and MUC5AC negative. • CK7 is often strongly positive, and CK20 is more focal, in pancreatobiliary type ampullary adenocarcinomas, whereas intestinal-type ampullary adenocarcinomas tend to have more CK20 and less CK7.
Neuroendocrine Neoplasms A spectrum of NE differentiation may be seen in the ampullary region that occurs in 3% of ampullary tumors.430 Most are ordinary NETs, that is, grade 1 (carcinoid) and grade 2 in the 2010 WHO classification. Although the general characteristics of these are not too different from NETs elsewhere in the digestive system, some peculiarities are worth mentioning. The majority of “functioning” somatostatinomas occur in the ampulla. Moreover, in addition to the classic features of low-grade NE neoplasms, somatostatin-positive tumors of this region, whether functional or not, also display tubule formation, focal intraluminal mucin, and psammomatous calcifications, and they may be associated with neurofibromatosis. This phenomenon is fairly specific to the ampulla.481 All express diffuse and strong chromogranin (Fig. 15-31), synaptophysin, and somatostatin; however, they are not associated with the stigma of somatostatin secretion, and therefore the term glandular psammomatous carcinoid of the ampulla is preferable to somatostatinoma. Other peptides may also be found. Although lymph node metastasis is seen at presentation in fewer than 50% of the cases, surrogate signs of aggressiveness, high Ki-67 index and CK19 positivity, are not seen, and Ki-67 index is typically low (<5%). Common
566
Immunohistology of Pancreas and Hepatobiliary Tract
retinoblastoma protein is reported in 60% of poorly differentiated (high-grade) NECs but not in non-NE carcinomas. In contrast, loss of p27 is common in non-NE carcinomas but not in poorly differentiated (high-grade) NECs.482 KEY DIFFERENTIAL DIAGNOSIS Ampullary Neuroendocrine Neoplasms POORLY DIFFERENTIATED (HIGH-GRADE) NEUROENDOCRINE CARCINOMA VS. NEUROENDOCRINE TUMOR
Figure 15-31 Diffuse and strong chromogranin positivity in ampullary somatostatinoma (glandular psammomatous carcinoid).
S-100 protein expression is intriguing, especially considering the association with neurofibromatosis. Gland formation, intraluminal but not intracytoplasmic mucin, and an infiltrative appearance may be mistaken as adenocarcinoma in small biopsies. Moreover, many cases show focal CA 19-9 and CEA positivity.481 The ampulla is also included in the “gastrinoma triangle,” and NETs associated with Zollinger-Ellison syndrome and MEN 1 are often localized in this region430 and may be microscopic; thus they may be difficult to identify preoperatively and grossly. Poorly differentiated (high-grade) NECs may also be seen in the ampulla.1,2 Although these account for an exceedingly small percentage of malignancies in the GI tract, they appear to occur at a relatively higher proportion in the ampulla. Whether this is related to the abundance of NE cells seen also in the adenomas of this region is not known. Both small and large cell variants are recognized.1 Although these are now regarded as grade 3 NETs in the 2010 WHO classification, it is clear that they actually make up a category of their own. We suspect that in the future, true poorly differentiated (high-grade) NECs will have to be regarded separately, either as grade 4 or a separate category. Most poorly differentiated NECs are of the small cell type, similar to pulmonary small cell carcinoma, and tend to have a diffuse growth pattern. The tumor cells have minimal cytoplasm, indistinct cell borders, and polygonal nuclei with finely stippled chromatin and indistinct nucleoli. Some cases have more of a large cell phenotype with a more nested pattern and a moderate amount of cytoplasm. The nuclei are round and vesicular with often prominent nucleoli. Poorly differentiated (high-grade) NECs are identified by brisk mitosis and easily identifiable necrosis. Cytokeratin and NE markers such as chromogranin, synaptophysin, and NSE are often positive, but these may be focal and weak. As with their counterparts in other organs, small cell types are mostly defined by morphologic characteristics rather than detectability of NE markers. CEA may be positive, and Ki-67 labeling index is typically very high, displaying positivity in the majority of the cells.430 Loss of
• In general, positivity is more diffuse for general neuroendocrine markers in NETs. By definition,483 proliferation markers such as Ki-67 are expressed significantly more often in poorly differentiated (high-grade) carcinomas versus NETs. POORLY DIFFERENTIATED (HIGH-GRADE) NEUROENDOCRINE CARCINOMA VS. LYMPHOMA • IHC positivity for keratin and no staining with lymphoid markers help exclude lymphoma. POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMA VS. POORLY DIFFERENTIATED NONNEUROENDOCRINE CARCINOMA • By IHC, poorly differentiated NECs are positive for CKs AE1/AE3, CAM5.2, CK7, and in a lesser percentage CK20, similar to the pattern found in poorly differentiated non-NECs. However, if present, IHC expression of neuroendocrine markers is helpful in this regard. • Loss of retinoblastoma protein expression, a characteristic finding in pulmonary small cell carcinomas, is present in almost half of ampullary poorly differentiated (high-grade) NECs. In contrast, p27 expression is lost in poorly differentiated non-NECs and is retained in most poorly differentiated (high-grade) NECs.482
Duodenal Gangliocytic Paraganglioma Duodenal gangliocytic paraganglioma is a lesion that is fairly specific to this area. It is a peculiar tumor of unknown origin that exhibits a mixture of 1) epithelioid elements (paraganglioma-like or carcinoid-like), which are positive for keratins and for NE markers, such as chromogranin, synaptophysin, and NSE; 2) ganglionlike cells, and 3) spindle cells of the nerve sheath type,430 which are negative for keratins and express S-100 protein instead. Specific peptides may also be found, especially pancreatic polypeptide and somatostanin.430 The latter may be important in the differential diagnosis, to distinguish these lesions from somatostatinomas; because short of the ganglion-like and nerve sheath components, duodenal gangliocytic paragangliomas may be very difficult to distinguish from glandular carcinoids (somatostatinomas) of this region.
Autoimmune Diseases of the Pancreas and Ampulla Autoimmune pancreatitis (AIP) is a pancreatobiliarycentric inflammatory disease that is frequently mass
Ampulla
forming and constitutes an appreciable proportion of pancreas resections.484,485 The first descriptions of what has become known as AIP appeared in the 1950s, most notably among patients with ulcerative colitis and pancreatitis,486 and in patients with sclerosing pancreatitis and hypergammaglobulinemia.484,485,487,488 The term autoimmune pancreatitis gained popularity in the 1990s.489 IMMUNOGLOBULIN G4–ASSOCIATED AUTOIMMUNE PANCREATITIS
Immunoglobulin (Ig) G4–associated AIP has been a topic of interest recently, because it probably constitutes the majority of AIP cases. Other terms for this disorder used in the literature include lymphoplasmacytic sclerosing pancreatitis with cholangitis, chronic sclerosing pancreatitis, and nonalcoholic ductdestructive chronic pancreatitis.490 Although it will not be discussed in detail here, IgG4 is an unusual IgG, and it is somewhat peculiar in that it is associated with this disorder.491,492 IgG4-associated AIP is often a multicentric disease, as evidenced by the presence of extrapancreatic manifestations in approximately one third of cases, among which lesions of the biliary system and liver are quite noteworthy.484 The disease can involve the bile ducts (sclerosing cholangitis),493 gallbladder (lymphoplasmacytic sclerosing cholecystitis),494 kidney (interstitial nephritis and pseudotumors),491 and the salivary glands in a disorder previously referred to as a Kuttner tumor,495 and it can involve the lungs by forming inflammatory masses.484,485,496 Such multiorgan involvement may be part of a systemic IgG4-related disease, which has been termed IgG4-associated immune complex multiorgan autoimmune disease (IMAD)491 and, more recently, IgG4-related disease, as in a recent consensus document.497 AIP patients can be seen with vague abdominal pain; obstructive jaundice, when the disorder affects the bile ducts (in ~75% to 80%); and with other autoimmune diseases (in ~20%; includes disorders such as idiopathic retroperitoneal fibrosis, Sjögren syndrome, ulcerative colitis, and lymphocytic thyroiditis). The elderly and patients in later middle age are affected disproportionately.485 Elevated serum IgG4 levels can often be useful in recognizing the disease, and suspecting IgG4associated disease is important, because it is often responsive to steroid therapy.498 The diagnosis can often be made before pancreatectomy by using a combination of clinical findings, the presence of elevated IgG4 levels, radiologic features, response to steroids, and endoscopic ultrasound-guided FNA biopsy findings to potentially avoid surgery in selected patients.485 Autoantibodies may be detected in the clinical laboratory, including antinuclear antibodies (ANAs), anti–smooth muscle antibodies, anti–carbonic anhydrase II antibodies, and antilactoferrin antibodies.499-501 Radiologic studies such as magnetic resonance imagine (MRI), computed tomography (CT), and ultrasonography often show a diffuse focal or segmental enlargement (mass formation) or hypoechoic enlargement of the pancreas, referred to as the sausage sign, and may be useful in
567
demonstrating stenosis of pancreatic and bile ducts.485,490 Endoscopic retrograde cholangiopancreatography (ERCP) can be used to recognize the disorder and often shows a diffusely irregular main pancreatic duct and strictures of the common bile duct.485 Grossly, the pancreas is most often most noticeably abnormal in the pancreatic head, where it is gray to yellowish white, indurated, variably enlarged, and shows a loss of the normal lobular architecture. Bile duct/main pancreatic duct obstruction or stenosis may be observed, particularly in the distal portions, even extending to the papilla. Involvement in the tail and body or diffuse involvement throughout the pancreas is seen in a minority of cases. Pseudocysts are not usually seen, and calculi are uncommonly seen, typically only in advanced cases.485,490,502 Histologic findings in AIP are characterized by dense lymphoplasmacytic pancreatic parenchymal infiltrates with secondary fibrosis and acinar atrophy. Inflammation and injury may be spotty and may show prominently involved areas that alternate with relatively uninvolved areas; ducts are typically involved first, followed by acinar involvement and sclerosis. The fibroinflammatory process involves the pancreatic head in about 80% of cases; however, it can extend up the bile duct, leading to bile duct and gallbladder wall thickening and inflammation of the liver hilum. Microscopic features of AIP include obliterative phlebitis and periductal inflammation that forms a collar around small, medium-sized, and large interlobular ducts; the smaller ducts are affected mostly in advanced cases. Perineural inflammation is also a useful feature in suggesting the diagnosis of AIP; other clues include periductal granulomas and pseudotumors, and storiform fibrosis may be admixed with myofibroblast-type cells, leading to changes reminiscent of inflammatory pseudotumors.484,485,490 Some studies have categorized cases of AIP as ductocentric (AIP-D), characterized by fibroinflammatory processes primarily in the periductal region with or without lobular involvement, and lobulocentric (AIP-L), distinguished by an almost exclusively lobular lymphoplasmacytic infiltrate with a paucity of periductal inflammation.502 Frank pseudotumor formation may occur, as evidenced in notable reports; and cases may be associated with idiopathic retroperitoneal fibrosis.503-506 The pseudotumor formation may extend up the bile duct to involve the liver hilus, and there may be an overlap with cases previously referred to as PSC.493,507 Lymph nodes that surround the pancreas and bile duct are commonly enlarged and show follicular hyperplasia. Of note, the typical stigmata of alcoholic pancreatitis are absent.484,485,490 The lymphoplasmacytic inflammation is characteristically composed of IgG4-positive plasma cells, but admixed macrophages, neutrophils, and eosinophils may also be present. Vasculitis may involve small veins and, less commonly, arteries, sometimes leading to an obliterative arteritis. Lymphocytes are predominately CD4- and CD8-positive T cells with fewer B cells, and sometimes small follicles of B cells can be appreciated.484,485,490
568
Immunohistology of Pancreas and Hepatobiliary Tract
GRANULOCYTIC EPITHELIAL LESION–FORMING PANCREATITIS
A category of AIP in which the inflammatory infiltrate contains a pronounced component of neutrophils, which has been termed the granulocytic epithelial lesion (GEL)– forming (idiopathic duct centric) form of AIP. Although also associated with prominent populations of IgG4positive plasma cells in most cases, GEL-forming AIP appears to occur in a younger subset of patients in their mid-40s and seems to affect equal numbers of men and women, unlike the other form of IgG4-associated AIP, which affects men more than women by a ratio of approximately 3 : 1. Cases with neutrophilic infiltrates also tend to be seen in younger individuals with inflammatory bowel disease (IBD) as opposed to cases associated with Sjögren syndrome, which affects older male patients preferentially and is not associated with pancreatic neutrophilic infiltrates.304,490,508 Histologically, GEL-forming lesions are characterized by ductal epithelial detachment and injury secondary to invasion of the epithelium by neutrophils and sometimes eosinophils, which may cluster underneath the epithelium, sometimes extending into small intralobular acini and ducts. Although the injury tends to be severe, it has been suggested that ductal scarring and complete obliteration are not common end points.490,508 In what is known as “The Honolulu Consensus Document,” AIP has been broken into two types. The type 1 designation is used for cases typically associated with IgG4, including most of the cases of lymphoplasmacytic sclerosing pancreatitis (LPSP); type 2 is used for idiopathic duct centric pancreatitis (IDCP) or GEL-positive pancreatitis. In the preparation of the Honolulu document, many experts had concerns about the use of the term autoimmune when discussing these forms of pancreatitis, particularly the type 2 form of disease, which is not typically associated with increased serum or tissue IgG4 positivity.509 DIAGNOSIS OF AUTOIMMUNE PANCREATITIS
Since shortly after AIP gained attention as an entity, interest has been mounting in the diagnosis of AIP on the basis of biopsy material. The features typically associated with AIP can be appreciated on biopsies, notably obliterative venulitis, granulocytic epithelial lesions, and periductal lymphocytic inflammation. Studies have investigated the use of IgG4 IHC as an ancillary test to make the diagnosis of IgG4-related AIP. Recommendations vary on the density of IgG4-positive plasma cells needed to diagnose AIP.484,490,508,510-513 Other studies have recommended that a ratio of IgG4-positive plasma cells to IgG-positive plasma cells, obtained by counting the number of cells on both IgG4- and IgG-stained tissue sections, is more useful diagnostically, because in a sense this measures the percent of IgG produced by plasma cells that is composed of IgG4. For the diagnosis of AIP, it has been proposed that this IgG4/IgG ratio can be useful as a surrogate marker of disease in the
pancreas, in ampullary biopsies,514 and in cholecystectomy specimens.494 Multiple criteria sets have been developed to assist in the diagnosis of AIP, including those of the Japan Pancreas Society. Recently, some clinicians have used the Mayo Clinic “HISORT Criteria,” which rely on histology, imaging, serology, organs of involvement, and respose to corticosteroid therapy.512 In a recent consensus document published by a number of IgG4-related disease (IgG4-RD) experts, diagnostic criteria for IgG4-RD were set forth for a number of organs.497 For all organs, characteristic histologic features include a dense lymphoplasmacytic infiltrate, fibrosis that typically has a storiform character, and obliterative phlebitis. In the document, the number of IgG4-positive plasma cells per hpf is specified for a number of organs. In the pancreas, cases with two or more histopathologic features are required to have more than 50 and more than 10 IgG4-positive plasma cells per hpf in surgical specimens and biopsies, respectively, to be deemed histologically highly suggestive of IgG4-RD. Cases with only one histopathologic feature are required to have the same number of IgG4positive plasma cells, but the diagnosis in this case is only deemed to have “probable” histologic features of IgG4-RD. In addition, an IgG4/IgG-positive cell ratio greater than 40% is mandatory for a histological diagnosis of IgG4-RD.497 Although some authors have found IgG4 immunostaining of duodenal/ampullary biopsies to be helpful in the diagnosis of AIP, in our experience, the specificity and sensitivity of these mucosal biopsies for this purpose is extremely low and often misleading.
Liver A wide variety of nonneoplastic, also referred to as medical, and neoplastic diseases may affect the liver. Proper diagnosis of these entities is important, because effective therapeutic options are increasingly available, including medical therapy for nonneoplastic diseases and resection for neoplastic diseases. In addition, liver transplantation has become an important modality for many chronic neoplastic and nonneoplastic conditions. IHC has an important role in the diagnosis of liver diseases, and as will be discussed, it can be very useful in identifying hepatic infections, evaluating transplant biopsies, and classifying hepatic tumors. IHC has also been instrumental in elucidating the pathogenesis of many disorders of the liver.
Normal Hepatic Parenchyma HEPATOCYTES
Cytokeratins are the principal intermediate filaments of epithelial cells. Embryonal hepatocytes contain cytokeratins 8, 18, and 19; however, mature cells contain only CK8 and CK18, and CK19 is negative by the tenth week of gestation. CAM5.2 stains hepatocytes in the
Liver
periportal zones and adjacent venules. Cytokeratins 7, 19, and 20 are negative, as are EMA and vimentin. Although often not detectable in normal hepatocytes, AFP can be positive in cirrhotic nodules.42,515-524 HepPar1 stains hepatocytes in a diffuse granular cytoplasmic pattern without canalicular accentuation.525-527 Thyroid transcription factor 1 (TTF-1) also stains the cytoplasm of hepatocytes in a coarsely granular pattern but does not stain the nuclei.527 Bile canaliculi stain with antibodies to pCEA,520,528 CD10,520,528 and CD25.529 Hepatocytes can display a number of artifacts that can lead to difficulty in IHC staining and stain interpretation. They contain relatively abundant biotin, which may lead to hepatocytes staining positively with standard IHC techniques, if the endogenous biotin activity is not blocked to prevent the major potential problem of false positivity.522-524 Hepatocyte intracellular contents can sometimes lead to confusion with regard to IHC interpretation. Although not typically present in a quantity or distribution that is problematic, lipofuscin can be mistaken as positive staining in a dotlike pattern. Intracellular pigments, such as bile and iron, can sometimes also lead to confusion. BILE DUCTS
Intrahepatic bile ducts and peribiliary glands stain for cytokeratins 7, 8, 18, 19, 34βH11, 34βH12, and AE1/ AE3.515-517,526,530 CK20, CA 19-9, and CEA are generally negative.42,531 Portal interstitial cells of Cajal can be identified with C-KIT (CD 117) immunohistochemistry, particularly around extrahepatic portal vasculature.532 This likely parallels the nerve supply of the liver, which is thought to play a role in liver regeneration and osmoreception.533 VASCULATURE
The expression of many endothelial markers—CD34, factor VIII (FVIII), CD31, Ulex europaeus lectin—is fairly weak in normal vasculature, whereas it can become quite prominent in pathologic conditions such as chronic liver disease and HCC.520,534-537
569
with fascin.553 In some studies, they have been shown to have features of neural/neuroectodermal differentiation and stain with antibodies to synaptophysin, glial fibrillary acidic protein (GFAP), and NCAM.520,554-556 Activated stellate cells often acquire myofibroblastic features and show expression of vimentin, desmin, and SMA.520,538,557 The use of other ancillary IHC stains for assessing fibrosis, including peptidylarginine deiminase, have been advocated by some.558
Medical Liver Diseases STEATOHEPATITIS AND MALLORY BODIES
Accumulation of fat in the liver eventually leads to inflammation and ultimately to fibrosis in some cases. The process is likely the result of actions brought about by inflammation mediated by both the innate and adaptive immune systems.559,560 First described in alcoholic patients by Frank B. Mallory in 1911, Mallory hyaline bodies also appear in other chronic liver diseases. Steatohepatitis, both alcoholic and non–alcohol-related, is one disorder in which the presence of Mallory bodies is frequently encountered and is used for the diagnosis.561 Although standard histology is the modality most frequently applied in the diagnosis of steatohepatitis, sometimes this diagnosis can be difficult, and some have suggested that IHC is of use in steatohepatitis in certain circumstances.562 Mallory hyalines can be difficult to distinguish on routine biopsies, and ancillary IHC with keratins CK18, 34βE12, and CAM5.2, as well as antibodies to ubiquitin (Fig. 15-32), may help highlight them. They are also occasionally positive for CK7 and CK19.520,563,564 In an attempt to distinguish nonalcoholic steatohepatitis (NASH) from alcoholic steatohepatitis (ASH), IHC was performed for protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the insulin receptor (IR). This staining showed a trend toward decreased IR in obesity-related NASH and increased
INTERSTITIUM
The interstitial matrix of the liver is composed of collagens, glycoproteins, proteoglycans, and glycosaminoglycans. Alterations in the extracellular matrix (ECM) of the liver play an important role in fibrosis and in the stromal milieu of both neoplastic and nonneoplastic processes. Fibrosis is typically evaluated with trichrome or Sirius red staining. IHC staining for collagens I, III, and IV can all be used to evaluate hepatic fibrosis.538 Type I collagen is the main collagen of portal tracts and in fibrotic liver and appears as thick, deep blue fibers on trichrome stains. Newly formed collagen is often composed of collagen type III and appears as fine, light blue fibers on trichrome staining.538-552 Hepatic stellate cells have an important role in mediation of hepatic interstitial fibrosis and remodeling538-552 and can be highlighted
Figure 15-32 Ubiquitin Mallory hyalines.
immunohistochemistry
demonstrates
570
Immunohistology of Pancreas and Hepatobiliary Tract
PTP1B expression, with more normal IR expression and lower levels of PTP1B expression in ASH. In nonalcoholic fatty liver disease (NAFLD), hepatic progenitor cells (HPCs) can express a variety of adipokines such as adiponectin, resistin, and glucagon-like peptide 1 (GLP-1), and increased expression of these adipokines is strongly associated with the severity of NAFLD and the progression toward NASH and resultant fibrosis.565 PRIMARY BILIARY CIRRHOSIS AND SCLEROSING CHOLANGITIS
Cytokeratin IHC directed toward biliary epithelium can be useful in highlighting bile ductular proliferations that occur in a variety of conditions, including primary biliary cirrhosis (PBC) and PSC, regardless of the mechanism, whether it is a result of “ductular metaplasia” of limiting plate hepatocytes or primary proliferation of native ducts.566,567 The utility of CK7 in this context has been demonstrated,568,569 because it can be particularly useful in highlighting the bile ductular reaction in cases in which chronic biliary disease is being considered.570 According to one study, IHC characterization of bile ductular proliferation shows that a type P (primitive) reaction is CD56 positive and EMA and CD10 negative; a type D (differentiating) reaction is CD56, EMA, and CD10 positive; and a type O (obstuctive) reaction is EMA positive and CD56 and CD10 negative. Most PBC and focal nodular hyperplasia cases fall into the type P category, type D is seen in cirrhotic samples and in regenerating liver following fulminant hepatic failure, and type O is seen in obstructive reactions.571 IHC for endothelial markers such as CD31, CD34, FVIII-related antigen, U. europaeus, and other lectins may also reveal differences in the microvasculature of the portal tracts in PSC and PBC with the overall commonality to both disorders being a loss of capillary microvasculature. In PBC, vessels are often obliterated by granulomatous inflammation; whereas in PSC, the vessels are displaced by collagen deposits.572-574 Immunophenotyping of inflammatory cells may be of use in autoimmune hepatitis (AIH), PBC, and overlap syndromes.575-577 IHC studies suggest that CD8- and CD57-positive T cells accumulate around the portal areas in PBC.578 Hepatic progenitor cells are present in inflammatory disorders such as PBC, showing activation of pathways such as the Notch signaling pathway, and this is demonstrable by using progenitor cell markers.579 Investigators have demonstrated that immunostaining of plasma cells with antibodies for IgM and IgG may be useful in the distinction between PBC and AIH; most of the plasma cells stain for IgM in PBC, and most of the plasma cells stain for IgG in AIH. This parallels an increase in IgG that can be seen in the serum in AIH and increase in serum IgM that can be seen in PBC.580 An IgG predominance has also been observed in the AIH PBC overlap syndrome.577 However, this staining is not entirely specific. For example, IgG is not entirely specific for AIH, and it must be noted that IgG plasma cells can be seen in the liver in a number of disorders.577,580
IMMUNOGLOBULIN G4–RELATED CHOLANGITIS
Recently, lesions that contain large numbers of IgG4bearing plasma cells have been identified in a number of organs. Much interest has focused on these lesions in the pancreas;485 however, lesions of the intrahepatic and extrahepatic bile ducts may also contain large numbers of IgG4-bearing plasma cells. This lesion, referred to by a number of designations, most notably IgG4-associated cholangitis or IgG4-related sclerosing cholangitis, may be confused with other lesions, particularly PSC and AIH. The lesions usually also contain prominent fibrous tissue in a stellate pattern with admixed inflammatory cells that include lymphocytes and eosinophils, and obliterative phlebitis may be observed. The lesions may be quite florid with pseudotumor formation. IHC for IgG4 may be useful in properly categorizing these lesions.485,581-587 A review of liver transplants for presumed PSC indicates that nearly one quarter of explanted livers that carry a diagnosis of PSC contain increased IgG4-positive plasma cell infiltrates and positive IgG4 serum levels, and PSC cases with IgG4 positivity can have a more aggressive clinical course with a shorter time to transplant and a higher likelihood of recurrence than IgG4-negative PSC.587 The numerical threshold of IgG4positive plasma cells required to place a lesion into this category has yet to be established. In the pancreas, this number has been advocated as a minimum of 10 per hpf, and some authors require 20 or even 30.493,510,512,513 Other studies advocate an IgG4/IgG ratio in establishing a suspicion of IgG4-related sclerosing cholangitis.588 Steroids are typically used in the therapy of IgG4-related disease; however, rituximab has also been used.589 CIRRHOSIS
When fibrosis progresses because of chronic injury in the liver, collagen and other ECM molecules are deposited, and fibrous bands often eventually impart a nodular architecture on the liver that culminates in cirrhosis. Hepatic stellate cells (HSCs) are thought to be the major source of collagen-producing myofibroblasts in cirrhotic livers. Fibroblast secretory protein 1 (FSP1), also referred to as S-100A4 protein, is a marker of fibroblasts in a variety of organs, and increased numbers of FSP1-positive cells can be identified by IHC in a number of liver diseases, including hepatitis C virus (HCV) infection, alcoholic liver disease, NAFLD, hereditary hemochromatosis, and others; and studies indicate that these FSP1-positive cells are a subset of inflammatory macrophages.590 Studies have suggested that small and medium-sized portal tract bile ducts and ductular reaction cells undergo epithelial to mesenchymal transition (EMT), leading to portal tract fibrogenesis in chronic liver disease. This has been illustrated in tissue sections using in situ hybridization (ISH) staining for transforming growth factor-β (TGF-β) and S-100A4 mRNA and IHC for phosphorylated SMAD2 and 3 (p-SMAD2 and p-SMAD3),591 and data suggest that EMT can contribute to fibrosis and chronic T-cell–mediated allograft rejection.592
Liver
Cirrhotic nodules may acquire some staining for AFP; however, macroregenerative nodules are typi cally negative for this marker.521,593 In cirrhotic regen erative nodules, peripheral hepatocytes may show ductular transformation and express both CK7 and CK19.563,566,567,594 It is suggested that this is evidence of a stem-cell phenotype,595-598 and the focal expression of AFP is also interpreted to provide further evidence for this.521,599 Endothelial cells at the periphery of cirrhotic nodules adjacent to fibrous septa also have an altered phenotype and express CD31 and CD34, a feature that may be seen in other conditions, including nodular regenerative hyperplasia.520,534-536 VIRAL INFECTIONS
Viral infections may lead to hepatitis, which may become chronic and may eventually lead to cirrhosis. Interface hepatitis and lobular inflammation is usually present in these infections, as is some degree of inflammatory mononuclear infiltrate in portal tracts; however, because the inflammation is variable and does not always parallel the infectious activity, IHC may need to be used to highlight infected hepatocytes. In chronic hepatitis B virus (HBV) infection, “ground-glass” hepatocytes can be appreciated and can be confirmed by cytoplasmic staining for the hepatitis B surface antigen (HBsAg) by both IHC and immunofluorescence (IF).600-602 Membranous staining for HBsAg is often indicative of active viral replication (Fig. 15-33, A). Detection of HBsAg usually denotes chronic hepatitis, because it is usually not detected in acute hepatitis. Hepatitis B core antigen (HBcAg) reactivity, on the other hand—which is usually appreciated in the nucleus (see Fig. 15-33, B), rather than in the cytoplasm—is used to measure the degree of viral replication. Patients who are immunocompromised and who have received HBV through vertical transmission have both membranous HBsAg and nuclear HBcAg staining without a great deal of inflammation.603-607 HBV IHC may be particularly useful in identification of recurrent HBV
A
571
after transplant for chronic HBV infection.600,601 Delta virus, or hepatitis D, can also be detected immunohistochemically with both cytoplasmic and nuclear staining.604,608,609 It has been difficult to develop IHC antibodies that accurately detect HCV that can be applied on routine clinical specimens.604,610-615 Research studies have used IHC directed against the hepatitis core virus protein (HCVc), notably in a study that connected HCVc protein to cholangiocarcinoma invasion and metastasis.616 IHC has been used to demonstrate patterns of inflammation in HCV, demonstrating the presence of intrahepatic CD4- and FoxP3-positive T cells accompanied by scarce numbers of CD8- and FoxP3-positive T cells. The CD4- and FoxP3-positive T cells were located in necroinflammatory areas in contact with CD8positive T cells.617 ISH techniques to detect hepatitis B, C, and D virus have been developed and have shown some utility.618,619 Epstein-Barr virus (EBV) infection can be detected both immunohistochemically and through ISH. This is of particular use in EBV-driven posttransplant lymphoproliferative disorders (PTLDs) and in lymphoepithelioma-like HCCs and cholangiocarcinomas, which are associated with EBV.604,620-623 Cytomegalovirus (CMV) is another important infection that can involve the liver, and it can be detected with CMV IHC stains604,624,625 and ISH.618 Other infections of the liver that can be detected immunohistochemically include herpes simplex virus, herpes zoster virus, adenovirus, mycobacteria, and amoebiasis.604,626,627 FOCAL NODULAR HYPERPLASIA
Focal nodular hyperplasia (FNH) is considered to be a hyperplastic reactive proliferation of hepatocytes as a result of localized abnormalities in blood flow. Classically, FNH has a central scar with radiating septa and hyperplastic hepatocyte nodules but is devoid of bile ducts.628,629 Because of the localized nodularity, FNH is sometimes thought of as a localized cirrhosis.630
B
Figure 15-33 A, Hepatitis B surface antigen immunohistochemistry shows both cytoplasmic and membranous positivity. B, Hepatitis B core antigen immunohistochemistry shows nuclear positivity.
572
Immunohistology of Pancreas and Hepatobiliary Tract
The diagnosis of FNH can be aided by IHC. CD34 IHC highlights sinusoidal endothelial cells in the vicinity of fibrous septa in a linear pattern.628,629 Hepatocytes at the periphery of cirrhotic-like nodules of FNH may stain with cytokeratins 7 and 19, and the CKpositive cells may be continuous with fibrous septa.515,567 Glumatine synthetase shows a maplike (broad and anastomosing) pattern of staining adjacent to hepatic veins in FNH.631,632 METABOLIC DISORDERS
Alpha-1–antitrypsin (α1-AT) deficiency disease is associated with periodic acid–Schiff (PAS)-positive, diastaseresistant globules in the hepatocytes (Fig. 15-34, A). IHC staining for α1-AT may be useful in verifying the nature of these granules (see Fig. 15-34, B), which can also occur in cirrhosis, chronic hepatitis, and neonatal hepatitis; these are distinct from α1-AT deficiency. In addition, early in α1-AT deficiency, these globules may not be as visible, and IHC may be useful in highlighting α1-AT (see Fig. 15-34, B). The α1-AT stain may be positive as early as 19 weeks’ gestation.633-635 Afibrinogenemia and hypofibrinogenemia (type I fibrinogen deficiencies) are rare congenital disorders in which plasma fibrinogen levels are low or unmeasurable. Some cases of hypofibrinogenemia may actually be present as a “fibrinogen storage disease,” in which fibrinogen is present in large quantities in hepatocytes and is appreciable on routine stains as eosinophilic globules in hepatocytes, some of which have a dark core and some of which are vacuolated. Fibrinogen antibodies may be useful in determining that the intrahepatocytic material is fibrinogen.635,636 LIVER TRANSPLANTATION
IHC has two important applications in the evaluation of transplant biopsies: 1) identification of infectious agents and 2) determination of the mechanism, and thus the etiology, of the immune injury. IHC for various
A
organisms, particularly viral organisms, may be crucial in the interpretation of liver transplant biopsies. This may be particularly important for the identification of recurrent hepatitis B or C, as previously discussed.637 Diagnosing liver allograft rejection, particularly humoral rejection, can unfortunately be problematic. IHC for C4d, a marker frequently used in the interpretation of renal allograft biopsies, has been investigated in liver allografts with mixed results.638-651 Antibody-mediated rejection in the liver does appear to occur, as evidenced by antibody-mediated rejection of ABO-incompatible livers. Deposition of C4d, the marker typically used in renal and other allografts, has been demonstrated in liver allografts and is associated with decreased survival.638 However, studies have pointed out different patterns of deposition of C4d in the liver, including stromal,638 portal,646,652 sinusoidal,643-645,652,653 and hepatocytic patterns.644,645 It has also been noted that portal vessels, predominantly capillaries but also veins and arteries, can be positive in acute rejection;640,643,648,654 however, others have noted that this pattern of staining in small arteries and capillaries is indeterminate.638 The sinusoidal and portal capillary endothelium patterns seem to be most promising,643,645,655 although it is unclear which of these patterns actually has prognostic significance.638 Studies have suggested that IF is the most sensitive technique for detecting C4d deposition; however, IF is typically performed on frozen tissue, which may require additional biopsy material.653,655 Therefore if optimal techniques can be established on IHC, then it will likely be more widely applicable clinically. This field, though yet imperfect, is in a rapid evolution, and it is likely that IHC will have an increasing role in the diagnosis of immune injury in the transplant setting in the future. Of note, C4d staining can also be used in the identification of antibody-mediated rejection in pancreatic allografts. In this scenario, C4d deposits in the interacinar capillaries.656 Differentiation of recurrent HCV from acute cellular rejection can be difficult. MxA protein is a marker of interferon type 1 production, and it stained a higher
B
Figure 15-34 A, Periodic acid–Schiff diastase stain shows prominent intracytoplasmic diastase-resistant globules. B, Immunohistochemistry with α1-antitrypsin confirms that globules contain this marker.
Liver
proportion of recurrent HCV patients than acute cellular rejection patients in one study.657 OTHER NONNEOPLASTIC LIVER DISEASES
Sinusoidal dilatation and congestion can be seen when there is impairment in venous outflow from conditions such as heart failure. Aberrant expression of CK7 can be seen in perivenular hepatocytes in patients with heart failure, which correlates with a cholestatic chemistry profile (increased bilirubin) and fibrosis.658 IHC and immunocytochemistry can be useful in metabolic diseases of the liver. For example, in the investigation of peroxisomal disorders, immunogold labeling of material for electron microscopy can be useful in demonstrating abnormalities in peroxisome morphology. These techniques can be coupled with catalase staining used in the investigation of peroxisome function.631,659-661 In hypofibrinogenemia, hepatocytes have intracytoplasmic globular inclusions that react strongly with human antifibrinogen antibodies on IHC.662
Neoplastic Liver Diseases Neoplasia displays a wide diversity in the liver, both from primary liver tumors and from metastatic disease, as discussed below. Most primary liver cancer fits under the rubric of HCC, and adenomas are thought to be a precursor lesion. Some adenomas show dysplasia, and it is thought by many that adenomas go through a transition to carcinoma in an adenoma-carcinoma sequence. Some of the lesions that affect the liver are discussed below, and a number of useful reviews are available on the topic.663-666 HEPATIC ADENOMAS
Hepatic adenomas are benign tumors composed of plates of cells that resemble normal hepatocytes separated by sinusoids. Grossly, they are usually
TABLE 15-1
573
well-demarcated yellow or tan masses that sometimes contain fibrosis, hemorrhage, or necrosis.667 Adenomas can be identified through radiology methods, notably MRI.668 Hepatic adenomas express β-catenin;664,669,670 however, this has also been observed in HCC associated with HCV infection.667,671 HCC expresses more glypican-3 than cirrhotic nodules, adenomas, FNH-like lesions, or low- or high-grade dysplastic nodules.664,672-674 CD34 usually shows incomplete staining of the sinusoidal vessels, in contrast with HCCs, which typically show a “complete” pattern. Arterialized sinusoids that are CD34 positive are more typical of HCCs but can be seen in hepatocellular adenomas (Table 15-1).189 Low- and high-grade dysplastic nodules are thought to be on the continuum from adenomas to HCC. Highgrade dysplastic nodules can be thought of “borderline” lesions that some consider well-differentiated or early HCC. IHC has been conducted in an attempt to distinguish dysplastic nodules from other hepatic nodules, including CK7/19 to evaluate stromal invasion, CD31 or SMA to evaluate the vascular pattern, or tumor markers (e.g., heat shock protein 70 [HSP70], glutamine synthetase, and glypican-3).675-679 Approximately 10% to 15% of hepatocellular adenomas have β-catenin–activating mutations. These can be termed β-catenin–activated adenomas, and they typically have diffuse glutamine synthetase positivity as a result of upregulation of GLUL, which encodes glutamine synthetase, a target of β-catenin. These adenomas are associated with specific conditions such as glycogenosis, administration of male hormones, and male sex; and except in the condition of glycogenosis, these lesions are typically solitary. Malignant transformation occurs more frequently in this hepatocellular adenoma subtype than in other subtypes. Pseudogland formation and nuclear atypia are frequent in this subtype, often making it difficult to distinguish these adenomas from welldifferentiated HCC. Steatosis and inflammation are typically absent, and both cytoplasmic and nuclear β-catenin positivity can be seen. Glutamine synthetase
Immunohistochemical Features of Hepatocellular Adenoma Subtypes and Focal Nodular Hyperplasia L-FABP
Glutamine Synthetase*
β-Catenin
SAA/CRP
HNF-1α–inactivated HCA
−
−
−
β-Catenin–activated HCA
+
+ (diffuse)
Nuclear staining
−†
Inflammatory HCA
+
−‡
‡
+ to +++†
Unclassified HCA
+
−
FNH
+
+ (maplike)
− Membranous staining
From Bosman FT, Carneiro F, Hruban RH, et al (eds): WHO Classification of Tumors of the Digestive System. International Agency for Research on Cancer: Lyon, 2010; and Shafizadeh N, Kakar S: Diagnosis of well-differentiated hepatocellular lesions: role of immunohistochemistry and other ancillary techniques. Adv Anat Pathol 2011;18(6):438-445. *Glutamine synthetase is occasionally expressed around veins and at the periphery of HCA without β-catenin mutations. † β-catenin–activated HCA is usually negative for SAA/CRP but maybe positive in 10% of inflammatory HCA with β-catenin activation. ‡ Inflammatory HCA is typically negative for these proteins, except when CTNNB1 is mutated. FNH, Focal nodular hyperplasia; HCA, hepatocellular adenoma; HNF-1α, hepatocyte nuclear factor 1α; L-FABP, liver fatty acid–binding protein (normally expressed in nontumoral liver); SAA/CRP, serum amyloid adenocarcinoma/C-reactive protein.
574
Immunohistology of Pancreas and Hepatobiliary Tract
positivity is diffuse and strong, as opposed to that of FNH, which shows maplike positivity.189,631 A variant of hepatocellular adenoma, termed inflammatory hepatocellular adenoma, shows either patchy or diffuse cytoplasmic staining with serum amyloid– associated (SAA) protein, and reverse transcription polymerase chain reaction (RT-PCR) shows an upregulation of SAA in these inflammatory hepatocellular adenomas. These adenomas also are referred to as telangiectatic adenomas and are thought to account for more than half of hepatocellular adenoma cases. These adenomas are more common in obese women with fatty liver disease, and multiple adenomas may be present that are characterized by focal or diffuse inflammation, sinusoidal dilatation, congestion, peliotic areas, and thickwalled arteries. C-reactive protein (CRP) is expressed in the cytoplasm of these adenomas as well as SAA, and it is thought that both of these proteins are increased in hepatocellular adenoma, because of the fact that they are inflammation-associated proteins. CRP and erythrocyte sedimentation rate may also be increased in the serum in approximately 50% of cases. Approximately 60% of these adenomas have mutations in gp130, which coexist with β-catenin gene mutations in approximately 10% of inflammatory hepatocellular adenomas. Malignant transformation can occur in these adenomas, particularly when β-catenin is mutant.189,631,632,680 Hepatocellular adenomas can have inactivation of HNF-1α. The HNF1A gene encodes HNF-1α, a transcription factor involved in hepatocyte differentiation. Biallelic inactivating mutations of the HNF1A gene are present in 35% to 40% of hepatocellular adenomas. Heterozygous germline mutations in HNF1A are also responsible for an autosomal dominant form of diabetes, maturity-onset diabetes of the young type 3 (MODY3). HNF1A-inactivated hepatocellular adenomas can show characteristic features that include marked and diffuse steatosis, absence of inflammation, and lack of nuclear atypia. These adenomas occur almost exclusively in women and can be solitary or multiple. The FABP1 gene, which encodes liver fatty acid–binding protein (L-FABP), is positively regulated by HNF-1α expressed in normal liver tissue and is downregulated in HNF1α–inactivated hepatocellular adenoma. Immunohistochemically, complete absence of L-FABP staining can be shown in this adenoma subtype.189,681-683 To characterize liver nodules, one study used L-FABP, SAA, CRP, glutamine synthase (GS), and β-catenin. In most cases (~90%), the characteristic staining pattern was seen. FNH stained for GS in a maplike pattern, and inflammatory hepatocellular adenoma stained for SAA.684
and pleomorphic. In addition, fatty change may be present.667 Cytokeratins
HCC reacts with antibodies directed against a variety of cytokeratins, particularly low-molecular-weight (LMW) cytokeratins—CK8, CK18, and CAM5.2—in addition to HepPar1, AFP, α1-AT, α1-antichymotripsin, CD10, and villin. Antibodies directed against CK7, CK20, and AE1 are usually negative,42,49,685,686 which may be important to delineate these tumors from biliary epithelial lesions. A proportion of HCCs can be CK7 positive, shown to be approximately 33% of conventional HCCs in one study. In contrast, 72% of fibrolamellar HCCs were CK7 positive.687 CK19 positivity is less common in fibrolamellar carcinoma than in conventional HCCs, which show positivity in approximately 20% of cases, as opposed to only 5% of fibrolamellar HCCs. CK19 positivity may be indicative of aggressive features, possibly as a result of increased epidermal growth factor–induced disease progression.688,689 Carcinoembryonic Antigen
A canalicular staining pattern for polyclonal carcinoembryonic antigen (pCEA) is seen in approximately 90% of HCCs (Fig. 15-35). It is sometimes useful in discriminating hepatocellular tumors from other malignancies,594,664 although it should be remembered that abortive lumina formation in poorly differentiated adenocarcinomas can mimic a canalicular pattern. Polyclonal CEA can also be useful in FNA specimens.690 The canalicular pattern of pCEA can also be seen with antibodies to CD10 and ABC1/MDR1.189 HepPar1
HepPar1 is an antigen that reflects hepatocytic differentiation (Fig. 15-36). It stains both normal fetal and adult liver and also stains neoplastic hepatocytic tissue, including 80% to 100% of HCC cases. The staining is usually of a granular, cytoplasmic pattern. Some investigators have reported decreased staining with more
HEPATOCELLULAR CARCINOMA
Hepatocellular carcinoma (HCC) is a malignant tumor generally considered to be derived from hepatocytes, and the cells of HCC resemble variably pleomorphic hepatocytes. Architecturally, HCC may have a number of different patterns, the most common of which is a trabecular or platelike pattern. Other patterns include acinar, pseudoglandular, scirrhous, clear cell, spindle cell,
Figure 15-35 Polyclonal carcinoembryonic antigen positivity in hepatocellular carcinoma. Note the canalicular staining pattern.
Liver
575
nodules may also stain with glypican-3.665,672-674,678,693-695 Measurement of glypican-3 expression with IHC staining has been used as a marker of prognosis, with higher levels of glypican-3 seen in cases with shorter survival times,699 higher histologic grade, and intrahepatic metastases.700 Arginase-1
Figure 15-36 Hepatocellular carcinoma with positive HepPar1 immunohistochemistry.
poorly differentiated HCC; however, this distinction is not completely clear. HepPar1 stains conventional adult HCC, as well as fibrolamellar and clear cell variants, but not sclerosing HCC. Furthermore, HepPar1 may be positive in a number of tumors not limited to HCC that contain tumor cells with eosinophilic, granular cytoplasm including intraductal or cystic oncocytic neoplasia.519,526,594,664-666,691 Glypican-3
Glypican-3 is a placental and fetal hepatic heparan sulfate proteoglycan that is not normally expressed in normal adult liver. Recently, much interest has been focused on the utility of glypican-3 as an oncofetal antigen in the diagnosis of HCC (Fig. 15-37).665,672-674,678,692-698 Several studies have shown that it is more sensitive than HepPar1 for the diagnosis of HCC, particularly poorly differentiated HCC. It is used in the differential diagnosis of HCC versus cholangiocarcinoma (positive in 50% to 90% of HCCs and usually negative in cholangiocarcinomas). However, cirrhotic
Figure 15-37 Hepatocellular carcinoma with focal glypican-3 staining.
Arginase-1 (Arg-1) has recently shown utility in the identification of hepatocellular differentiation. It is a binuclear manganese metalloenzyme that is key in the urea cycle, in which it catalyzes the hydrolysis of arginine to ornithine and urea. Arginase-1 is both a sensitive and specific marker of benign and malignant hepatocytes. In one study, arginase-1 stained 100%, 96.2%, and 85.7% of well, moderately, and poorly differentiated HCCs.700 In a study of FNA material, arginase-1 showed a higher sensitivity than HepPar1 or glypican-3.701 CD34
CD34 in conjunction with glypican-3 may be of some use in distinguishing HCC from its benign mimics. Virtually all sinusoidal spaces in HCC tend to have staining for CD34, classified as a “complete” staining pattern, which is quite uncommon in benign lesions, with the exception of a few hepatic adenomas or FNH.674 Albumin
Albumin is a major serum transport protein synthesized by hepatocytes.666 ISH for the messenger RNA (mRNA) that encodes albumin shows positivity in nonneoplastic hepatocytes and in HCC, hepatic adenomas, and hepatoblastomas. Unfortunately, IHC is not amendable to the detection of albumin, because albumin is abundant in the serum, which leads to nonspecific tissue staining. Up to 96% of HCC can show positivity with ISH, although staining can be diffuse, patchy, or focal.666,702-707 One caveat is that other tumors, including clear cell carcinoma of the ovary708 and hepatoid carcinomas—for example, hepatoid gastric and bladder carcinomas702,709-712—are also positive. α-Fetoprotein
An oncofetal glycoprotein, α-fetoprotein (AFP) can show positivity in HCC with 17% to 70% of HCC cases showing positivity. This positivity parallels an increase in AFP seen in the serum of patients with HCC; however, serum increases and IHC staining of the tumor can be seen in cases of primary and metastatic hepatoid adenocarcinomas and yolk sac tumors. It should be kept in mind that although very high levels of serum AFP levels are fairly specific for HCC, some increase can also be seen in hepatitis and cirrhosis.593,666,707,713 However, AFP has limitations in its sensitivity and specificity for HCC; it has been demonstrated that a high proportion of clear cell carcinomas of the liver are negative for AFP.666,714 Unfortunately, both primary and metastatic hepatoid carcinomas of the liver can be positive for AFP. For example, hepatoid carcinomas such as those that can be seen in the ovary, stomach, lung, renal pelvis, pancreas, and bladder can show AFP positivity, therefore AFP is of limited utility when trying to differentiate HCC from
576
Immunohistology of Pancreas and Hepatobiliary Tract
many other neoplasms that may metastasize to the liver.715-717 Miscellaneous Markers Used in Hepatocellular Carcinoma
Thyroid transcription factor 1 (TTF-1) is normally expressed in the nuclei of certain cells and commonly shows cytoplasmic positivity in HCC (Fig. 15-38). Some studies have reported no staining, however. It appears that this result is highly dependent on the manufacturer and antibody clone used.527,718,719 Although the significance of this cytoplasmic labeling—and in fact, whether it is “real” staining or not—has yet to be determined, it can be of some value in identifying HCC. EMA can be positive in HCC, and studies report from 20% to 40% of HCC cases with positivity. There is some indication that higher-grade neoplasms have increased staining.26,720-723 HCC can also show reactivity for CD10 (common acute lymphoblastic leukemia antigen, CALLA) with approximately half of the tumors showing staining. It is often positive in the canalicular pattern similar to that of pCEA.593,724 MOC-31 is a cell surface glycoprotein typically used as a marker of adenocarcinoma. Only a minority of HCC cases show staining for this marker, and studies show positivity in 0% to 22% of tumors.666,725-727 It has been shown that α1-AT can be positive in HCC, and some studies indicate a high proportion (up to 86%) of HCC cases that show positivity. Other studies have shown positivity in much lower proportions of tumors (as low as 22%). Cholangiocarcinoma is typically negative, and reports indicate that 0% to 10% of tumors stain.728 However, this marker often shows some nonspecific labeling, which limits its usability. Vascular markers can be useful in HCC, because some show characteristic staining patterns in HCC, whereas others can be useful in identifying lymphovascular invasion. FVIII-related antigen (von Willebrand factor [vWF]) positivity can be seen, at least focally, in nearly all HCC cases. Only periportal sinusoids typically
Figure 15-38 Hepatocellular carcinoma with granular cytoplasmic thyroid transcription factor 1 staining.
stain in normal liver. In HCC, the staining is in sinusoidal endothelium, although staining can be very focal and may even be interpreted as being absent. Some authors advocate the use of FVIII in conjunction with CD34.536,729-733 Other studies have investigated similar patterns of the vascular endothelium in HCC with such markers as CD31, U. europaeus lectin, and ABH blood group antigens; unfortunately, these markers have not been found to be as reliable as CD34 in this regard.535,732,733 Assessment of microvessels in HCC reveals at least two different types: sinusoid-like and capillary-like microvessels. Investigations show that disease-free survival and overall survival of the capillary-like group are better than those of the sinusoid-like group. Capillarylike vessels are more common in small HCC and typically occur with a higher microvessel density than with the sinusoid-like vessels.734,735 Downregulation of the tight junction protein claudin-5 leads to leaky vasculature and a worse prognosis, likely attributable to an increased risk for lymphovascular invasion.736 Claudin10 expression is highly linked to angiogenesis and shorter overall survival.737 D2-40 (podoplanin) can be used to identify lymphatic vessels, which are present primarily in the portal tracts that accompany portal veins, arterites, and bile ductules.738 Factor XIIIa (FXIIIa), a blood coagulation proenzyme, can be positive in HCC. FXIIIa also shows positivity in histiocytes/macrophages and benign hepatocytes.739 Some have suggested that FXIIIa can be useful in differentiating HCC from cholangiocarcinoma, but other studies have indicated that it is noncontributory in this differential diagnosis.526,593,594,728 Of note, HCC may also react with antibodies directed against alkaline phosphatase, α1-antichymotripsin, CRP, inhibin, thioredoxin (RX), and villin.664,666 Staining for the transferrin receptor has been demonstrated in HCC using the monoclonal antibody BK 19.9.740 Staining has also been demonstrated in HCC for acidic isoferritin and erythropoietin (Fig. 15-39).664,666 Molecular and Genomic Applications of Immunohistochemistry
Molecular markers of cell-cycle status and nuclear regulation have been linked with prognosis in HCC.741 Numerous studies have suggested the utility of commonly investigated molecules such as p53742-744 and Ki-67742 in HCC. The product of CDKN2A, p16 is an important regulator of the cell cycle, controlling entry into the mitotic S phase; and p16 positivity can be seen in HCC by IHC. However, p16 positivity can also be seen in normal liver.745 Histone deacetylases (HDACs) control chromatin remodeling and tumor suppressor genes, and some HDACs—specifically HDACs 1, 2, and 3—can show increased staining in HCC.746 Ribosomal proteins (e.g., ribosomal protein L36 [RPL36]) are possibly involved in hepatocarcinogenesis and are expressed in HCC at a level linked to overall survival.747 Nuclear dUTP pyrophosphatase (dUTPase) expression correlates with a poor prognosis in HCC.748 Growth arrest homeobox (MEOX2, formerly GAX) downregulation is associated with poor survival in HCC patients.749
Liver
577
Zinc finger proteins have been investigated in HCC and can be evaluated with IHC.750-752 Some of these, such as retinoblastoma-interacting zinc finger gene PRDM2 (formerly RIZ1), are involved in epigenetic modification through DNA methylation and histone modification.752 The zinc finger ZBTB20 is expressed in HCC and is associated with a worse prognosis.753 Zinc finger and homeobox 2 (ZHX2) represses transcription of genes associated with HCC, leading to inhibition of HCC growth.751 Kinases responsible for signaling and other enzymes responsible for DNA repair may be important targets for chemotherapy agents. The S-phase kinase-associated protein 2 regulates numerous genes involved in cellcycle progression in HCC.754 HCC also stains for protein kinase C, a protein important in cell regulation, tumor formation, and signal transduction.755 The cyclindependent kinase 10 (Cdk10) is a Cdc2-related kinase involved in progression from the G2 phase to the M phase of the cell cycle. Cdk10 overexpression enhances the chemosensitivity of HCC cells to cisplatin and epidoxorubicin, two chemotherapeutic agents used in HCC.756 Similarly, excision repair cross-complementation group 1 (ERCC1), a key enzyme in DNA repair, is important in chemosensitivity to cisplatin. In a study of HCC specimens and cell lines, increased ERCC1 expression is associated with CDDP resistance.757 Control of apoptosis is also important in HCC. Bcl-2, caspase-8, and survivin, key molecular markers of apoptosis, have been investigated in HCC758,759 and have shown correlation with a high proliferative index and poor prognosis.758 Survivin, an inhibitor of apoptosis, shows cytoplasmic staining in cirrhosis and nuclear staining in HCC. In liver cirrhosis, survivin expression correlates with expression of signal transducer and activator of transcription 3 (p-STAT3) and cyclin D1 and downregulation of E-cadherin.760 A poorer prognosis is predicted by IHC with higher levels of osteopontin and lower levels of caspase-3.761 In one study, COX-2 expression correlated with survivin, and both correlated with survival.759 The activation of signal transducer and activator of p-STAT3 is inhibited by β-escin; furthermore, β-escin potentiates the apoptotic effects of
Ar r-1 gi na nt se itr yp si n pC C G E yt l A op yp ic la sm an ic -3 TT F1 C D 10 AF P C D 34 EM A C K7 C K2 0
III
1 -A
Pa
FV
ep H
IS H
in
AM
m
C
bu Al
Figure 15-39 Immunohistogram of hepatocellular carcinoma with selected antibodies. AFP, α-Fetoprotein; CK, cytokeratin; EMA, epithelial membrane antigen; FVIII, factor VIII; ISH, in situ hybridization; pCEA, polyclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor 1.
5. 2
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
paclitaxel and doxorubicin in HCC cells and may help suppress the proliferation of HCC.762 Stem cells have been investigated in HCC.763 Studies indicate that HCC can arise from stem cells, including, for example, in cirrhotic livers from viral hepatitis;764 however, other studies indicate that the positivity for stem cell markers in HCC simply represents a transdifferentiation of HCC cells rather than a malignant transformation of stem/progenitor cells.765 Hepatoblasts are considered to be fetal liver stem/progenitor cells, capable of differentiating into hepatocytes and cholangiocytes; and facultative hepatic stem/progenitor cells in adults are considered to be oval cells, which have been difficult to isolate.766 Gene expression and pathway analyses of HCC reveal that epithelial cell adhesion molecule (EpCAM)- and AFP-positive cells have stem/progenitor cell features, and some of the EpCAM-positive tumors have increased invasiveness and growth, at least partially because of Wnt/β-catenin signaling activation.767 Stem cell markers such as CD44, CD133, and Thy1/CD90 have been used.768,769 In particular, Thy1/CD90 has been associated with a worse prognosis in HCC.770 The stem cell transcription factors Oct-4 and Nanog are increased in HCC with aggressive tumor behavior.771 Tumor-infiltrating lymphocytes (TILs) have been investigated in a number of neoplasms, and investigating TILs may be important in HCC. T-lymphocyte–related antigens can be simultaneously detected by using fluorescence-based IHC of paraffin-embedded tissue. For example, investigators have developed a method to simultaneously detect CD4, CD8, CD57, and T-cell receptor-β (TCR-β).772 Programmed death 1 (PD-1) and its ligand (PD-L1) control apoptosis in CD8-positive T lymphocytes, and CD8-positive T cells induced PD-L1 expression on hepatoma cells in vitro, suggesting that this could be a therapeutic target in HCC.773 IHC staining with various markers has been useful in elucidating potential explanations for the molecular pathogenesis of HCC. Nuclear accumulation of β-catenin, a component of the wingless/Wnt pathway, has been observed by IHC in HCC.774 This correlates with mutations in the β-catenin gene, which were detected in 2% to 41% of HCCs,667,671 particularly those
578
Immunohistology of Pancreas and Hepatobiliary Tract
associated with HCV infection.671 Investigators have also demonstrated the presence of MSI in HCC by both molecular studies and with IHC for the microsatellite markers (MLH1, MSH2, MSH6, and PMS2),775-777 although MIS does not appear to play a big role in HCC carcinogenesis and is less common among older individuals.775,777 In addition, the phosphoinositol-3 kinase/mammalian target of rapamycin (mTOR) pathway has been investigated in HCC by using a number of methods, including immunostaining, suggesting that HCC can be treated with blockade of mTOR with drugs such as everolimus.629,630 The mTOR pathway—including such downstream effectors as PTEN, pAkt, p27, and pS6—is altered in high-grade tumors and tumors with poor prognostic factors.778 Other metabolic and molecular derangements may be detected in HCC. For example, upregulation of phosphoglycerate mutase 1 (PGAM1) can be found in HCC.779 As mentioned previously, arginase is expressed at a higher level in HCC.701,780 Leptin expression significantly correlates with HCC proliferation as evaluated by Ki-67 staining, whereas adiponectin expression correlates significantly with increased disease-free survival and inversely with tumor size and local recurrence.781 Based on measurements in cell lines, hepatocyte growth factor (HGF) upregulation promotes carcinogenesis and EMT in HCC via COX-2 and Akt pathways.782 Overexpression of the homeoprotein Six1 in HCC patients is associated with venous infiltration, metastasis, and poor survival; however, Six1 expression can be suppressed in metastatic HCC by using short hairpin RNA (shRNA) interference against Six1.783 Heat shock protein (HSP) molecules may be useful as markers in HCC. Staining for HSP70 correlates with vascular invasion, large tumor size, higher Edmonson-Steiner grade, and shorter diseasefree survival.784 HCC patients with Forkhead box M1 (FOXM1)–positive tumors had a poorer recurrence-free and overall survival after hepatectomy than those with FOXM1-negative tumors.785 Diacylglycerol kinase alpha (DASGK) enhances HCC progression by activation of the Ras-Raf-MEK-ERK pathway as measured by IHC.786 Insulin-like growth factor–binding protein 7 (IGFBP7) downregulation is associated with tumor progression and clinical outcome in HCC.787 In the pertumoral tissues of HCC, high placental growth factor (PGF) expression is associated with hypoxia-inducible factor 1-alpha (HIF-1α) expression and tumor recurrence.788 Phosphatidylethanolamine binding protein 1 (PEBP1, also RKIP) downregulation is associated with aggressive tumor behavior in HCC.789 Reduced expression of N-cadherin is associated with metastatic potential of HCC and poorer surgical prognosis.790 LZAP, the binding protein of the Cdk5 activator p35, typically acts as a tumor suppressor; and LZAP overexpression suppresses HCC tumorigenicity, suggesting that this could be a therapeutic target.791 Studies have also shown how markers of vascularity in HCC can be important.792 This has been illustrated by a number of studies that have utilized markers of microvessels. For example, one study used double staining for Ki-67/CD34 and caspase-3/CD34 in HCC to
illustrate that the microvessels are mostly mature and have a similar density in HCC in both cirrhotic and noncirrhotic livers.793 Studies have shown how high vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) are linked to a poor prognosis in HCC.737,756,794 Data suggest that tumors that stain for both EpCAM and AFP have a higher microvessel density, higher level of VEGF expression, later tumor-node-metastasis (TNM) stage, increased incidence of venous invasion, and may be therapeutic candidates for antiangiogenesis therapy.795 C1QTNF6 is overexpressed and possibly contributes to tumor angiogenesis by activating the Akt pathway in many HCCs.796 The endogenous angiogenesis inhibitor vasohibin-1 shows increased expression in HCC and increased vasohibin-1 expression is linked with higher microvessel density, VEGF expression, microvessel invasion, and worse survival.797 Currently, advanced HCC is treated with sorafenib, a tyrosine kinase inhibitor with a broad inhibitory profile (e.g., inhibition of VEGFR, PDGFR, and BRAF); the use of this inhibitor is largely thought to be useful because of its inhibition of the neovascularization in HCC.798 HCC undergoes changes in hypoxia that allow tumoral cell survival. HIF-1α is a molecule that is involved in this process and that is a predictive factor for recurrence in HCC.799,800 High HIF-1α is associated with poorer overall survival.800 Hypoxia also stimulates expression of the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) gene via the HIF-1 pathway, and PLOD2 expression, measured by RT-PCR and IHC, is associated with such features as increased tumor size, intrahepatic metastasis, and poor prognosis.799 Human carbonyl reductase 1 (CBR1) upregulated by hypoxia renders resistance to apoptosis in HCC cells.801 Interaction of HCC with the ECM has yielded important insights. Matrix metalloproteinases (MMPs) are important in cell migration, and increased staining for MMPs by IHC (e.g., MMP-9) has been observed at a higher frequency in HCC with large vessel invasion. In addition, increased MMP-9 staining is associated with HCC with a worse prognosis.802 A similar molecule, a disintegrin and metalloproteinase-9 (ADAM9), is overexpressed in HCC.803 Evidence suggests that the process of epithelial to mesenchymal transition (EMT) occurs in HCC. CD151 amplifies signaling by integrin α-6–β-1, promoting EMT and invasiveness of HCC cells.804 Snail protein, thought to be a key regulator in EMT through its suppression of E-cadherin, shows increased expression in poorly differentiated HCC, decreased E-cadherin expression, and an association with postoperative recurrence.805 Periostin, a molecule putatively involved in EMT, is expressed in some HCCs and bile duct carcinomas.806 Molecular dysregulation may be specifically brought about by HCV. For example, some data suggest that the dominant genotype found in patients with HCC is HCV-1b.807 In the HCV inflammation-fibrosiscarcinoma sequence, no data are available to suggest that Toll-like receptors (TLRs) are upregulated along with proinflammatory mediators (e.g., TNF-α and COX-2)808; these findings are supported by other data
Liver
to suggest that TLR cell-surface expression promotes cell-surface survival in human HCC.809 Markers that stain in HCC by IHC may also be useful as serum markers for the noninvasive detection of disease. This knowledge has been applied for a number of markers, including human hepatocyte growth factor, insulinlike growth factor, VEGF, and AFP.803,810-813 However, a perfect noninvasive detection method for HCC is still elusive and will need validation before it can be implemented diagnostically.810,814-816 Molecular insights into the pathogenesis of HCC have also helped guide therapy and will likely be a big player in guiding future therapies. The use of everolimus, a rapamycin analog, to block mTOR signaling decelerated HCC tumor growth in vitro and in a xenograft model and increased survival.817,818 In one study, platelet-derived growth factor receptor alpha (PDGFRA) was found to be overexpressed in the endothelium of HCC with a high metastatic potential.819 In this study, STI-571, also known as imatinib mesylate (Gleevec, Novartis), inhibited tumor growth, apparently through antiangiogenesis via inactivation of PDGFRA.819 Another study showed that high expression of PDGFRA may be linked to decreased overall survival.794 These studies help illustrate how PDGFRA can potentially serve as a biomarker for predicting metastasis and as a therapeutic target. HEPATOCELLULAR CARCINOMA VARIANTS Fibrolamellar Hepatocellular Carcinoma
Fibrolamellar HCC is a distinct variant of HCC that occurs most often in noncirrhotic individuals from adolescence to young adulthood. It is characterized by sheets or trabeculae of neoplastic cells separated by collagen bundles in a lamellar configuration.667 Tumor cells have granular eosinophilic (oncocytoid) cytoplasm and are large and polygonal.820,821 Fibrolamellar carcinoma is associated with a better prognosis than conventional HCC and cholangiocarcinoma.822 Fibrolamellar HCC has immunophenotypic similarities to the usual type of HCC820,821; however, unlike usual HCC, it may show strong CK7 expression.823 Thus, because of its positivity for CK7 and EMA, it has been suggested that fibrolamellar carcinoma shows both hepatocellular and bile duct differentiation.824 One study suggested that fibrolamellar HCC shows “stemness” based on positive staining for stem cell markers such as CD133 and CD44.825 Fibrolamellar HCC may also stain for synaptophysin, but whether there is true positivity with chromogranin665,826,827 is a debated issue. Fibrinogen has been found in fibrolamellar HCC,821 and studies indicate that these correlate with the pale bodies of the fibrolamellar HCC cells.820,821 Fibrolamellar HCC also has a characteristic staining pattern for the claudin tight junction proteins, and tricellulin is decreased in fibrolamellar carcinoma and in other liver tumors compared with normal liver.828 Increased expression of EGFR may also be seen in fibrolamellar carcinoma, suggesting that treatment with EGFR antagonists may be considered in the future.817,829,830
579
Spindle Cell Hepatocellular Carcinoma
High-grade/poorly differentiated HCC may have a spindle cell morphology.667 Poorly differentiated HCC may be frankly sarcomatous, and some cases have heterologous differentiation, such as chondrosarcomatous.831 The sarcomatous component often loses its epithelial differentiation and becomes negative for the conventional epithelial markers. In addition, they show the appropriate lineage markers that reflect their differentiation at the histomorphologic level. For example, areas with chondrosarcomatous differentiation stain with S-100 and vimentin. However, some cells of the sarcomatous component also retain keratin in some cases.831 Spindle cell carcinomas may also have osteoclast like giant cells, in which case the osteoclast like giant cells express histiocytic differentiation markers.832 Clear Cell Hepotocellular Carcinoma
Clear cell HCC may be difficult to distinguish from other clear cell tumors, such as adrenal cortical carcinoma and renal cell carcinoma (RCC), by using routine histology.664 RCC usually shows reactivity with vimentin; whereas only high-grade HCC or HCC with spindled morphology shows reactivity with vimentin.664 Adrenal cortical carcinoma stains with melan-A and inhibin, and HCC is usually negative with antibodies to these molecules. Furthermore, adrenal cortical carcinoma is negative for CAM5.2.664,833 NEC can sometimes have clear cell morphology, and NECs are usually positive for synaptophysin and chromogranin, whereas HCC is usually negative for these markers.664 Medullary (Lymphoepithelioma-Like) Carcinoma
Carcinoma with a medullary (lymphoepithelioma like) morphology has been reported in the liver. These cases consist of carcinoma admixed with abundant lymphoid stroma. The carcinoma component in these cases is reported to be HCC with positivity for HepPar1. The presence of EBV has been demonstrated in these lesions with positivity for EBV-encoded RNA (EBER) by ISH.621,623,834 Biliary-Type Differentiation in Hepatocellular Carcinoma
Some HCCs may express markers that are otherwise considered specific for biliary-duct differentiation and do not stain HCCs. The expression of these markers alone is not considered enough by most authors to qualify the tumor as “combined HCC and cholangiocarcinoma.” This is often referred to as biliary-type differentiation. HCC with this sort of differentiation stains with mCEA, CK7, CK19, and AE1/AE3.835-847 Combined Hepatocellular and Cholangiocarcinoma
Tumors that have both HCC and cholangiocarcinoma components have the distinctive morphologies of both of these carcinoma types, respectively, in which there are clear-cut areas of both HCC and cholangiocarcinoma; however, it is controversial whether the cholangiocarcinoma component of these tumors is actually
580
Immunohistology of Pancreas and Hepatobiliary Tract
C AM 5.
2 EM A pC EA C K7 C K1 M O 9 C Be -31 r-E P4 C D 1 C 5 A1 9C 9 A1 25 B7 2. 3 C K2 0
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Figure 15-40 Immunohistogram of cholangiocarcinoma with selected antibodies. CK, Cytokeratin; EMA, epithelial membrane antigen; pCEA, polyclonal carcinoembryonic antigen.
cholangiocarcinoma that arises in HCC or simply a form of malignant metaplasia involving HCC. In contrast, some draw the distinction with the entity referred to as cholangiocellular carcinoma, a term that includes cases that morphologically consist of biliary adenocarcinomas that have the IHC staining pattern of HCC.838-841 In general, studies have shown that their molecular signature is closer to cholangiocarcinoma, suggesting they may arise from cholangiocarcinoma. Overall, the cholangiocarcinoma component is usually more aggressive and dictates prognosis. The staining patterns of the HCC and cholangiocarcinoma components are similar to the carcinomas of each type respectively. However, some antibodies can produce seemingly discrepant results. For example, HepPar1 may stain some cholangiocarcinomas (Fig. 15-40).526,664,842-846 KEY DIAGNOSTIC POINTS Hepatocellular Carcinoma • HCC is positive for certain CKs, particularly low-molecularweight cytokeratins CK8, CK18, and CAM5.2. • The markers HepPar1, glypican-3, and arginase are particularly useful for the diagnosis of HCC. • pCEA usually shows canalicular staining in HCC. • Additional miscellaneous markers (α-fetoprotein, CD10, thyroid transcription factor, etc.) may also be positive in HCC. • In situ hybridization for albumin may also be useful in HCC.
Differential Diagnoses
Hepatocellular Carcinoma Versus Benign Hepatic Tissue. In the past, routine histology and histochemical stains were the mainstay in the distinction of benign hepatic tissue from HCC. Assessment of routine morphology provides the first insight into the true nature of a lesion. Reticulin outlines the normal sinusoidal architecture in benign hepatic tissue and shows thickened hepatocyte plates and abnormal nodules in HCC.666 Dealing with this differential diagnosis is also aided by IHC. Glypican-3 has gained attention for its utility
in this regard, because antibodies directed against it stain mainly neoplastic liver and only rarely stain cirrhotic and normal liver.530,666,673,674 CD34 stains portal and periportal sinusoidal areas in benign liver, whereas sinusoids throughout the tissue stain for CD34 in HCC.666,674 Nuclear staining for β-catenin and/or diffuse staining with glutamine synthetase favor a diagnosis of HCC or an adenoma with increased risk for HCC development.631 Agrin, a multifunctional heparan sulfate proteoglycan that accumulates in HCC and cholangiocarcinoma, stains malignant lesions and is negative in the majority of benign lesions.847 In favor of HCC over a high-grade dysplastic nodule in the setting of cirrhosis, stromal invasion can be favored by the absence of a CK7-positive ductular reaction.631 Studies indicate that positivity of two or more of HSP70, glutamine synthetase, and glypican-3 show a high specificity for HCC.631 HCC can show gains in chromosomes 1 and 8 on cytogenetic studies.631 Molecular studies that also may be useful include telomerase activity, comparative genomic hybridization, and measurement of proliferation status through counting of argyrophilic nucleolar organizer regions (AgNORs) with special silver staining.666 Hepatocellular Carcinoma Versus Cholangiocarcinomas. Cholangiocarcinoma is positive for both CK7 and CK19, whereas HCC is usually negative for these antibodies. HepPar1, a marker commonly used to identify HCC, is not typically expressed in cholangiocarcinoma.526,665 Hepatocellular Carcinoma Versus Metastatic Lesions. Because metastatic lesions are quite common in the liver, HCC must be distinguished from such entities. This distinction can be aided by IHC guided by careful examination of routine histology and determination of a likely differential diagnosis.664 Markers differentially expressed in HCC as opposed to other malignant neoplasms can help in this distinction.666 Canalicular staining with pCEA, CD10, and villin is rather specific for HCC.848 MOC-31, an antibody directed against a cell-surface glycoprotein, stains metastatic adenocarcinoma and cholangiocarcinoma but not HCC, which helps distinguish HCC from other tumors.725,727 Agrin IHC stains HCC microvasculature but not the microvasculature of cholangiocarcinoma or metastatic carcinoma.849 Furthermore, analysis for antigens expressed in neoplasms of particular origins can help to essentially rule out HCC in the identification of a liver tumor.665 BILE DUCT ADENOMAS
Bile duct adenomas are benign collections of bile ducts. In addition to secreting acid mucin, peribiliary glands and bile duct adenomas typically express foregut antigens such as D10, F6, MUC5AC, and also MUC6, which is seen in a higher proportion of inflamed peribiliary glands. Staining with these antigens suggests that these lesions are localized healing responses, akin to pyloric gland metaplasia in the foregut.850
Liver
581
CHOLANGIOCARCINOMA
Cholangiocarcinoma is a malignant tumor composed of cells resembling those of the bile duct, described as intrahepatic (or peripheral) when arising in the liver or hilar when arising from the right or left hepatic ducts near their junction. Histologically, cholangiocarcinoma often consists of malignant glands in a tubular configuration embedded in a fibrous stroma. Variants include clear cell, clear cell papillary, mucinous, pleomorphic, and spindle cell types. Areas of adenosquamous, mucinous, and signet-ring cell carcinoma may be evident.851,852 Cholangiocarcinoma usually reacts with CK7 and CK19, CAM5.2, AE1/AE3 (Fig. 15-41), and MOC-31. Cholangiocarcinoma is more likely to react with CK7 than are other pancreatobiliary carcinomas, and it is less likely to react with CK17 and CK20.664 CK19 can be particularly useful in the diagnosis of cholangiocarcinoma and shows positivity in a high proportion of cholangiocarcinomas.853 BerEP4 stains cholangiocarcinoma in a similar pattern to MOC-31.727 Both pCEA and mCEA stain in a noncanalicular pattern.664 Although not always included in diagnostic panels of cholangiocarcinoma, CA 19-9 can be positive in up to 90% to 100% of cholangiocarcinomas (Fig. 15-42),721,854-856 and around one third stain with mesothelin.857 Cholangiocarcinoma is typically negative for p53,664 but it may stain with parathyroid hormone–related peptide.580,666 One study that used IHC staining for the HBV core and surface antigens indicated that HBV may be associated with intrahepatic cholangiocarcinoma but not extrahepatic cholangiocarcinoma.858 A number of other markers may be positive in cholangiocarcinoma and may be useful in predicting prognosis. Mucins such as MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 may be useful in classifying cholangiocarcinomas and predicting prognosis. A gastric mucin phenotype has been associated with a worse prognosis.474,664,842,859,860 CD151, a hydrophobic protein that interacts with the MET and c-Met proteins, is overexpressed in intrahepatic cholangiocarcinoma
Figure 15-42 CA 19-9 positivity in cholangiocarcinoma.
with more aggressive features, such as larger tumor size, poor differentiation, multinodularity, microvascular/ bile duct invasion, and lymphatic metastasis.861 The protein p38 mitogen-activated protein (MAP) kinase, p38δ, also known as MAPK13 (formerly SAPK4), is upregulated in cholangiocarcinoma relative to normal biliary tract tissues and HCC.862 The expression of phosphorylated AKT1 and phosphorylated mTOR is associated with a favorable prognosis in cholangiocarcinoma, and this effect is independent of phosphatase and tensin homolog deleted on chromosome 10 (PTEN).863 Phosphatase of regenerating liver 3 (PRL-3) expression correlates with progression and metastasis of intrahepatic cholangiocarcinoma.864 EGFR expression is present in 80% of biliary tract and gallbladder adenocarcinoma cases by IHC, and fluorescence in situ hybridization (FISH) shows EGFR overexpression in 46% of tumors. ERBB2 (HER2/neu) is positive in only 4% of cases.865 Molecules that mediate stromal invasion appear to be particularly important in cholangiocarcinoma. The cell-adhesion molecules P-cadherin (CDH3) and CD24 are important in the control of cell motility, invasive growth of tumor cells, and morphogenic processes; both are expressed at high frequency in the early stages of biliary tract carcinogenesis.806 Expression of MMP-2 and ECM metalloproteinase inducer are unfavorable prognostic factors in intrahepatic cholangiocarcinoma.752 Studies have shown that α-V–β-6 integrin is a highly specific IHC marker for cholangiocarcinoma and shows staining in cholangiocarcinoma but not in HCC.866 Tumor-associated angiogenesis (microvascular density) and lymphangiogenesis (lymphatic microvessel density) correlate with the progression of intrahepatic cholangiocarcinoma.867 CHOLANGIOCARCINOMA VARIANTS Spindle Cell (Sarcomatoid) Cholangiocarcinoma
Figure 15-41 AE1/AE3 staining in cholangiocarcinoma. Note the interface of the tumor with normal liver.
Spindle cell (sarcomatoid) cholangiocarcinoma usually occurs as a component of a more conventional or poorly differentiated cholangiocarcinoma. The spindled cell areas may stain only focally for cytokeratins, obscuring
582
Immunohistology of Pancreas and Hepatobiliary Tract
the cholangiocarcinoma component; therefore, more conventional epithelioid areas must be selected for cytokeratin IHC.868-871 Cases with a variety of sarcomatous differentiation have been reported that included chondrosarcomatous elements872 and rhabdoid elements.873-876 Cells of the sarcomatous elements stain in a typical pattern for those sarcomas, and the spindled areas may also be positive, albeit focally, for LMW cytokeratins such as CAM5.2, AE1/AE3, 34βH11, and EMA.868,870,874 For example, in the case with chondrosarcomatous differentiation, the chondrosarcomatous elements showed S-100 protein positivity but were negative for keratins; and transitional areas had positivity for both S-100 protein and keratins.872 Rhabdoid elements are typically positive for vimentin,873,874 which is typically true of other spindled/sarcomatous elements.870 CEA positivity may be seen,869,870 and AFP is usually negative.868,869
KEY DIAGNOSTIC POINTS Cholangiocarcinoma • Cholangiocarcinoma is positive for particular cytokeratins, notably CK7 and CK19. • Cholangiocarcinoma is positive for both monoclonal and polyclonal CEA in a noncanalicular pattern. • Cholangiocarcinoma is also positive for various markers of adenocarcinoma (MOC-31, Ber-EP4, etc.).
Differential Diagnoses
Cholangiocarcinoma Versus Hepatocellular Carcinoma. The distinction of cholangiocarcinoma from HCC can be aided by cytokeratin staining, because cholangiocarcinoma shows staining for cytokeratins such as CK7 and CK19, whereas HCC is usually negative. Cholangiocarcinoma is usually negative for TTF-1, whereas HCC is often positive. Claudins, which are positive in cholangiocarcinoma and typically negative in HCC, have also been proposed as useful in the distinction of cholangiocarcinoma from HCC.664,719,877,878 Staining for PDZ binding kinase/lymphokine-activated killer T-cell–originated protein kinase (PBK/topk) is positive in cholangiocarcinoma and normal bile duct epithelial cells and is negative in HCC, and low expression of PBK/topk is predictive of poor survival in cholangiocarcinoma patients.879 Cholangiocarcinoma Versus Metastatic Lesions. Cholangiocarcinoma can be difficult to differentiate from adenocarcinoma metastatic from other sites, particularly GI primaries—such as gallbladder, stomach, extrahepatic biliary tree, or pancreas—by using IHC, because cholangiocarcinoma shows an overlapping staining pattern with carcinomas of many sites.665,666 Cholangiocarcinoma may react with CA 125, making distinction from müllerian carcinomas difficult. However, cholangiocarcinoma does not usually react with estrogen receptor.664,880 Cholangiocarcinoma Prognosis and Treatment. The prognosis of cholangiocarcinoma has been linked to several molecular markers. Overexpression of the
hydrophobic protein CD151 has been demonstrated in metastasis/invasion of intrahepatic cholangiocarcinoma. CD151 forms a functional complex with the protooncogene that encodes MET protein, c-Met, which is also involved in the invasion/metastasis of several tumors. CD151 and c-Met overexpression is a potential therapeutic target in ICC.881 Worse prognosis in cholangiocarcinoma has also been linked with strong CD133 expression.882 Studies have indicated that HDAC expression measured by IHC correlates significantly with higher stage carcinoma, vascular invasion, and lymph node metastasis.883 IHC measurement of expression of the transcription factor HIF-1α serves as an independent prognostic factor for both overall and disease-free survival and correlates significantly with higher stage and tends to correlate with vascular infiltration, tumor diameter (>4 cm), and intrahepatic metastasis.883 POORLY DIFFERENTIATED AND UNDIFFERENTIATED CARCINOMA
Carcinosarcoma of the liver can occur, although it is rare.872,884,885 In a series from China in which all of the patients were HBV surface antigen positive, conventional HCC merged with areas of rhabomyosarcoma, “malignant fibrous histocytoma,” and fibrosarcoma; and IHC supported the diagnosis of carcinosarcoma.885 BILIARY MUCINOUS CYSTIC NEOPLASMS: CYSTADENOMAS AND CYSTADENOCARCINOMAS
Biliary cystadenomas and cystadenocarcinomas have an IHC profile similar to pancreatobiliary cystadenomas and cystadenocarcinomas.423,704,886-889 The epithelium typically shows staining for CK7, CK20, CA 125, AE1/ AE3, CA 19-9, and CEA.423 Ovarian-type stroma is required for this diagnosis and typically shows staining for estrogen and progesterone receptors.890,891 Biliary cystic tumors without ovarian stroma are either other types of cysts or intraductal papillary neoplasms as discussed above. HEPATOBLASTOMA
Hepatoblastoma is a tumor in the liver that typically occurs in children. It is a malignant tumor with embryonal features and divergent differentiation, with fetal epithelial hepatocytes and other embryonal and differentiated tissue that includes striated muscle, fibrous tissue, and material that resembles osteoid. Approximately one third of cases have a pure fetal epithelial differentiation that resembles developing hepatocytes. Most tumor cells are positive for AFP.892 U. europaeus agglutinin type 1 (UEA-1) and anti-CD34 stain in a more diffuse pattern than Kupffer and endothelial cells that line sinusoids in normal liver.703,892,893 Other diagnostic considerations for malignant liver tumors in the pediatric age group include malignant rhabdoid tumor, which shows loss of staining for INI1b.894-896 Activation of β-catenin is important in hepatoblastoma, and IHC for total and phosphorylated β-catenin shows cytoplasmic and nuclear staining, as opposed to the membranous staining seen in normal liver. In this
Summary
583
circumstance, β-catenin is thought to be a good surrogate marker of HGF/c-Met activation.897 In addition, data indicate that cyclin D1 and Ki-67 can be useful in predicting prognosis in hepatoblastoma. Cyclin D1 stains the nuclei of the mixed epithelialmesenchymal type of hepatoblastoma more than the pure fetal type, and higher cyclin D1 correlates with a longer event-free survival. High Ki-67 correlates with recurrence and death.898 SERPINB3 (SB3) is found overexpressed in human HCC and hepatoblastoma. And in hepatoblastoma, SB3 correlates with Myc expression and high tumor stage.899 NEUROENDOCRINE NEOPLASMS
By definition, any NET in the liver should be regarded as a metastasis, either from the GI tract, pancreas, lung, thyroid (medullary carcinoma), or elsewhere. NETs stain with antibodies for synaptophysin, chromogranin, and CD56. NETs may also stain for MOC-31 but do not stain for HepPar1. HCC may show focal NE differentiation and may stain for CD56,900,901 synaptophysin, and chromogranin.665,902 OTHER TUMORS THAT OCCUR IN THE LIVER AND EXTRAHEPATIC BILE DUCTS
Angiomyolipoma is a benign tumor that consists of variable combinations of blood vessels, adipose tissue, and smooth muscle. Blood vessels may have thick walls and may be hyalinized. Angiomyolipomas are thought to arise from perivascular epithelioid cells, also known as PEC cells,903-905 and some cases may have a prominent epithelioid component.906 Extramedullary hematopoiesis may also be present. Smooth muscle cells of angiolipomas express the melanoma markers human melanoma black 45 and melan-A in addition to SMA.903 Epithelioid hemangioendothelioma (EHE) is a tumor of the liver that may display benign or malignant behavior.907 Histologically, tumors grow along vessels or form new vessels and are composed of epithelioid or spindle cells, often with intracellular vascular lumina. Tumor cells express FVIII-related antigens (vWF) and endothelial cell markers, such as CD31 (Fig. 15-43) and CD34.713,903 Infantile hemangioendothelioma is another variant of hemangioendothelioma that has a similar immunophenotype.908 Fewer than 50% of these lesions react with SMA, AE1/AE3, or CAM5.2.664 Expression of VEGF has been demonstrated by IF in EHEs, suggesting that anti-VEGF therapy may be useful in treating hepatic EHE.909 Mesenchymal hamartoma is a hepatic neoplasm that primarily occurs in children. Grossly, mesenchymal hamartoma can be cystic. Cysts are surrounded by a mesenchymal component with mature connective tissue with variable desmin positivity in arteries, veins, and nerves and in CK7- and CK19-positive ductal structures. The stroma of these neoplasms can be edematous and rich in mucopolysaccharides.910 Undifferentiated or embryonal sarcoma of the liver (UESL) may also rarely occur, primarily among children and young adults. It consists of a malignant mesenchymal neoplasm. Some data suggest that it is
Figure 15-43 Endothelial markers are commonly expressed in epithelioid hemangioendothelioma and are extremely helpful in the differential diagnosis with adenocarcinoma and hepatocellular carcinoma; CD31 is shown here.
linked to another hepatic neoplasm, mesenchymal hamartoma.911,912 UESL often grows rapidly to form an appreciable abdominal mass. These tumors stain for CD56 in a membranous pattern and also stain for various cytokeratins—such as AE1/AE3, CAM5.2, and OSCAR—in a paranuclear, dotlike pattern.913 Lymphomas and pseudolymphomas can occur in the liver, and ancillary IHC is often crucial in the evaluation of these lesions, as covered in other chapters.914,915 Gamma/delta T-cell hepatosplenic lymphoma preferentially affects the liver.916 A variety of other tumors can occur in the liver and in EHBDs. Mesotheliomas can involve the surface of the liver and can be identified with IHC, by using markers such as podoplanin (D2-40).917 Lymphangioma may occur in the liver and stains with such markers as D2-40 and LYVE-1.918 Primary leiomyosarcoma is a rare tumor in the liver with a typical immunophenotype for that neoplasm and characteristic positivity for markers such as SMA.919,920 Some vimentin-positive tumors have also been described.921 Follicular dendritic cell tumors have been reported in the liver that stain for markers such as CD21 and CD35, and data suggest that these follicular dendritic cell tumors are related to EBV, based on positive EBV-encoded RNA (EBER) ISH staining.922 Anaplastic lymphoma kinase (ALK)–positive inflammatory pseudotumors have also been reported in the liver.923,924
Summary IHC can be a powerful tool in hepatopancreatobiliary pathology. In the future, multiplex quantum dot staining, whole-slide digital imaging, and image analysis may be helpful in providing an even greater level of information than that provided by standard IHC.925 REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Adsay NV: Gallbladder, Extrahepatic Biliary Tree And Ampulla. In Mills SE, editor: Sternberg’s Diagnostic Surgical Pathology, ed 5, Vol. 2, Philadelphia, 2009, Lippincott Williams & Wilkins, pp 1600–1652. 2. Adsay NV, Klimstra DS: Benign and Malignant Tumors of the Gallbladder and Extrahepatic Biliary Tract. In Odze RD, Goldblum JR, editors: Surgical Pathology of the GI tract, Liver, Biliary Tract, and Pancreas, ed 2, Philadelphia, 2009, Elsevier, pp 845–875. 3. Klimstra DS, Adsay NV: Tumors of the Pancreas and Ampulla of Vater. In Odze RD, Goldblum JR, editors: Surgical Pathology of the GI tract, Liver, Biliary Tract, and Pancreas, Philadelphia, 2009, Elsevier, pp 909–960. 4. Hruban RH, Pitman MB, Klimstra DS: Tumors of the Pancreas, ed 4, Washington, DC, 2007, ARP Press. 5. Moniaux N, Escande F, Porchet N, et al: Structural organization and classification of the human mucin genes. Front Biosci. 6:D1192–D1206, 2001. 6. Nagata K, Horinouchi M, Saitou M, et al: Mucin expression profile in pancreatic cancer and the precursor lesions. J Hepatobiliary Pancreat Surg. 14(3):243–254, 2007. 7. Levi E, Klimstra DS, Andea A, et al: MUC1 and MUC2 in pancreatic neoplasia. J Clin Pathol. 57(5):456–462, 2004. 8. Yonezawa S, Goto M, Yamada N, et al: Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior. Proteomics. 8(16):3329–3341, 2008. 9. Yonezawa S, Nakamura A, Horinouchi M, et al: The expression of several types of mucin is related to the biological behavior of pancreatic neoplasms. J Hepatobiliary Pancreat Surg. 9(3):328– 341, 2002. 10. Nakamura A, Horinouchi M, Goto M, et al: New classification of pancreatic intraductal papillary-mucinous tumour by mucin expression: its relationship with potential for malignancy. J Pathol. 197(2):201–210, 2002. 11. Saitou M, Goto M, Horinouchi M, et al: MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas. J Clin Pathol. 58(8):845–852, 2005. 12. Khayyata S, Basturk O, Klimstra DS, et al: MUC6 Expression Distinguishes Oncocytic and Pancreatobiliary Type from Intestinal Type Papillae in Pancreatic Neoplasia: Delineation of a Pyloropancreatic Pathway. Mod Pathol. 19(1):275A, 2006. 13. Klimstra DS, Adsay NV, Dhall D, et al: Intraductal tubular carcinoma of the pancreas: Clinicopathologic and immunohistochemical analysis of 18 cases. Mod Pathol. 20(2):285A, 2007. 14. Koshikawa N, Hasegawa S, Nagashima Y, et al: Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. Am J Pathol. 153(3):937–944, 1998. 15. Portela-Gomes GM, Hacker GW, Weitgasser R: Neuroendocrine cell markers for pancreatic islets and tumors. Appl Immunohistochem Mol Morphol. 12(3):183–192, 2004. 16. Nalbantoglu I, Basturk O, Thirabanjasak D, et al: Islets, the king of antigens? Non-specific reactivity of (especially the) islets is a common pitfall in immunohistochemical evaluation of the pancreas. Mod Pathol. 20(2):288A, 2007. 17. Pignatelli M, Ansari TW, Gunter P, et al: Loss of membranous E-cadherin expression in pancreatic cancer: correlation with lymph node metastasis, high grade, and advanced stage. J Pathol. 174(4):243–248, 1994. 18. Chetty R, Serra S, Asa SL: Loss of membrane localization and aberrant nuclear E-cadherin expression correlates with invasion in pancreatic endocrine tumors. Am J Surg Pathol. 32(3):413– 419, 2008. 19. Chetty R, Serra S: Nuclear E-cadherin immunoexpression: from biology to potential applications in diagnostic pathology. Adv Anat Pathol. 15(4):234–240, 2008. 20. Chetty R, Serra S: Membrane loss and aberrant nuclear localization of E-cadherin are consistent features of solid pseudopapillary tumour of the pancreas. An immunohistochemical study using two antibodies recognizing different domains of the E-cadherin molecule. Histopathology. 52(3):325–330, 2008.
583.e1
21. Serra S, Chetty R: Revision 2: an immunohistochemical approach and evaluation of solid pseudopapillary tumour of the pancreas. J Clin Pathol. 61(11):1153–1159, 2008. 22. Evans DB, Abruzzese JL, Rich TA: Cancer of the pancreas. In Devita VT, Hellman S, Rosenberg SA, editors: Cancer: Principles and practice of oncology, ed 6, MA, 2001, Lippincott-Raven, pp 1126–1161. 23. Moll R: Cytokeratins as markers of differentiation in the diagnosis of epithelial tumors. Subcell Biochem. 31:205–262, 1998. 24. Luttges J, Vogel I, Menke M, et al: Clear cell carcinoma of the pancreas: an adenocarcinoma with ductal phenotype. Histopathology. 32(5):444–448, 1998. 25. Hoorens A, Prenzel K, Lemoine NR, et al: Undifferentiated carcinoma of the pancreas: analysis of intermediate filament profile and Ki-ras mutations provides evidence of a ductal origin. J Pathol. 185(1):53–60, 1998. 26. Thomas P, Battifora H: Keratins versus epithelial membrane antigen in tumor diagnosis: an immunohistochemical comparison of five monoclonal antibodies. Hum Pathol. 18(7):728–734, 1987. 27. Shimonishi T, Miyazaki K, Nakanuma Y: Cytokeratin profile relates to histological subtypes and intrahepatic location of intrahepatic cholangiocarcinoma and primary sites of metastatic adenocarcinoma of liver. Histopathology. 37(1):55–63, 2000. 28. Alexander J, Krishnamurthy S, Kovacs D, et al: Cytokeratin profile of extrahepatic pancreatobiliary epithelia and their carcinomas. Appl Immunohistochem Mol Morphol. 5:216–222, 1997. 29. Real FX, Vila MR, Skoudy A, et al: Intermediate filaments as differentiation markers of exocrine pancreas. II. Expression of cytokeratins of complex and stratified epithelia in normal pancreas and in pancreas cancer. Int J Cancer. 54(5):720–727, 1993. 30. Vlasoff DM, Baschinsky DY, Frankel WL: Cytokeratin 5/6 immunostaining in hepatobiliary and pancreatic neoplasms. Appl Immunohistochem Mol Morphol. 10(2):147–151, 2002. 31. Duval JV, Savas L, Banner BF: Expression of cytokeratins 7 and 20 in carcinomas of the extrahepatic biliary tract, pancreas, and gallbladder. Arch Pathol Lab Med. 124(8):1196–1200, 2000. 32. Goldstein NS, Bassi D: Cytokeratins 7, 17, and 20 reactivity in pancreatic and ampulla of vater adenocarcinomas. Percentage of positivity and distribution is affected by the cut-point threshold. Am J Clin Pathol. 115(5):695–702, 2001. 33. Lee MJ, Lee HS, Kim WH, et al: Expression of mucins and cytokeratins in primary carcinomas of the digestive system. Mod Pathol. 16(5):403–410, 2003. 34. Ramaekers F, van Niekerk C, Poels L, et al: Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocarcinomas. Am J Pathol. 136(3):641–655, 1990. 35. Tot T: Adenocarcinomas metastatic to the liver: the value of cytokeratins 20 and 7 in the search for unknown primary tumors. Cancer. 85(1):171–177, 1999. 36. Baars JH, De Ruijter JL, Smedts F, et al: The applicability of a keratin 7 monoclonal antibody in routinely Papanicolaou-stained cytologic specimens for the differential diagnosis of carcinomas. Am J Clin Pathol. 101(3):257–261, 1994. 37. Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 13(9):962–972, 2000. 38. Ji H, Isacson C, Seidman JD, et al: Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 21(4):391–400, 2002. 39. Tot T: Cytokeratins 20 and 7 as biomarkers: usefulness in discriminating primary from metastatic adenocarcinoma. Eur J Cancer. 38(6):758–763, 2002. 40. Rullier A, Le Bail B, Fawaz R, et al: Cytokeratin 7 and 20 expression in cholangiocarcinomas varies along the biliary tract but still differs from that in colorectal carcinoma metastasis. Am J Surg Pathol. 24(6):870–876, 2000. 41. Tot T: The value of cytokeratins 20 and 7 in discriminating metastatic adenocarcinomas from pleural mesotheliomas. Cancer. 92(10):2727–2732, 2001. 42. Moll R, Lowe A, Laufer J, et al: Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol. 140(2):427–447, 1992.
583.e2
Immunohistology of Pancreas and Hepatobiliary Tract
43. Kaufmann O, Deidesheimer T, Muehlenberg M, et al: Immunohistochemical differentiation of metastatic breast carcinomas from metastatic adenocarcinomas of other common primary sites. Histopathology. 29(3):233–240, 1996. 44. Miettinen M: Keratin 20: immunohistochemical marker for gastrointestinal, urothelial, and Merkel cell carcinomas. Mod Pathol. 8(4):384–388, 1995. 45. Wauters CC, Smedts F, Gerrits LG, et al: Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary. Hum Pathol. 26(8):852–855, 1995. 46. Schussler MH, Skoudy A, Ramaekers F, et al: Intermediate filaments as differentiation markers of normal pancreas and pancreas cancer. Am J Pathol. 140(3):559–568, 1992. 47. Chen J, Baithun SI: Morphological study of 391 cases of exocrine pancreatic tumours with special reference to the classification of exocrine pancreatic carcinoma. J Pathol. 146(1):17–29, 1985. 48. Cathro HP, Stoler MH: Expression of cytokeratins 7 and 20 in ovarian neoplasia. Am J Clin Pathol. 117(6):944–951, 2002. 49. Chu PG, Weiss LM: Keratin expression in human tissues and neoplasms. Histopathology. 40(5):403–439, 2002. 50. Di Como CJ, Urist MJ, Babayan I, et al: p63 expression profiles in human normal and tumor tissues. Clin Cancer Res. 8(2):494– 501, 2002. 51. Adsay NV, Merati K, Andea A, et al: The dichotomy in the preinvasive neoplasia to invasive carcinoma sequence in the pancreas: differential expression of MUC1 and MUC2 supports the existence of two separate pathways of carcinogenesis. Mod Pathol. 15(10):1087–1095, 2002. 52. Monges GM, Mathoulin-Portier MP, Acres RB, et al: Differential MUC 1 expression in normal and neoplastic human pancreatic tissue. An immunohistochemical study of 60 samples. Am J Clin Pathol. 112(5):635–640, 1999. 53. Swartz MJ, Batra SK, Varshney GC, et al: MUC4 expression increases progressively in pancreatic intraepithelial neoplasia. Am J Clin Pathol. 117(5):791–796, 2002. 54. Masaki Y, Oka M, Ogura Y, et al: Sialylated MUC1 mucin expression in normal pancreas, benign pancreatic lesions, and pancreatic ductal adenocarcinoma. Hepatogastroenterology. 46(28):2240–2245, 1999. 55. Andrianifahanana M, Moniaux N, Schmied BM, et al: Mucin (MUC) gene expression in human pancreatic adenocarcinoma and chronic pancreatitis: a potential role of MUC4 as a tumor marker of diagnostic significance. Clin Cancer Res. 7(12):4033– 4040, 2001. 56. Kim GE, Bae HI, Park HU, et al: Aberrant expression of MUC5AC and MUC6 gastric mucins and sialyl Tn antigen in intraepithelial neoplasms of the pancreas. Gastroenterology. 123(4):1052–1060, 2002. 57. Luttges J, Zamboni G, Longnecker D, et al: The immunohistochemical mucin expression pattern distinguishes different types of intraductal papillary mucinous neoplasms of the pancreas and determines their relationship to mucinous noncystic carcinoma and ductal adenocarcinoma. Am J Surg Pathol. 25(7):942–948, 2001. 58. Swierczynski SL, Maitra A, Abraham SC, et al: Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays. Hum Pathol. 35(3):357–366, 2004. 59. Terada T, Ohta T, Nakanuma Y: Expression of oncogene products, anti-oncogene products and oncofetal antigens in intraductal papillary-mucinous neoplasm of the pancreas. Histopathology. 29(4):355–361, 1996. 60. Yonezawa S, Sato E: Expression of mucin antigens in human cancers and its relationship with malignancy potential. Pathol Int. 47(12):813–830, 1997. 61. Zhang H, Maitra A, Tabaczka P, et al: Differential MUC1, MUC2 and MUC5AC expression in colorectal, ampullary and pancreatobiliary carcinomas: Potential biologic and diagnostic implications (abstract). Mod Pathol. 16(1):180A, 2003. 62. Werling RW, Yaziji H, Bacchi CE, et al: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 27(3):303–310, 2003. 63. Moskaluk CA, Zhang H, Powell SM, et al: Cdx2 protein expression in normal and malignant human tissues: an
immunohistochemical survey using tissue microarrays. Mod Pathol. 16(9):913–919, 2003. 64. Barbareschi M, Murer B, Colby TV, et al: CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs. Am J Surg Pathol. 27(2):141–149, 2003. 65. Li MK, Folpe AL: CDX-2, a new marker for adenocarcinoma of gastrointestinal origin. Adv Anat Pathol. 11(2):101–105, 2004. 66. Simeone DM, Ji B, Banerjee M, et al: CEACAM1, a novel serum biomarker for pancreatic cancer. Pancreas. 34(4):436–443, 2007. 67. Hoorens A, Lemoine NR, McLellan E, et al: Pancreatic acinar cell carcinoma. An analysis of cell lineage markers, p53 expression, and Ki-ras mutation. Am J Pathol. 143(3):685–698, 1993. 68. Morohoshi T, Kanda M, Horie A, et al: Immunocytochemical markers of uncommon pancreatic tumors. Acinar cell carcinoma, pancreatoblastoma, and solid cystic (papillary-cystic) tumor. Cancer. 59(4):739–747, 1987. 69. Kamisawa T, Fukayama M, Tabata I, et al: Neuroendocrine differentiation in pancreatic duct carcinoma special emphasis on duct-endocrine cell carcinoma of the pancreas. Pathol Res Pract. 192(9):901–908, 1996. 70. Pour PM, Permert J, Mogaki M, et al: Endocrine aspects of exocrine cancer of the pancreas. Their patterns and suggested biologic significance. Am J Clin Pathol. 100(3):223–230, 1993. 71. Bommer G, Friedl U, Heitz PU, et al: Pancreatic PP cell distribution and hyperplasia. Immunocytochemical morphology in the normal human pancreas, in chronic pancreatitis and pancreatic carcinoma. Virchows Arch A Pathol Anat Histol. 387(3):319–331, 1980. 72. Ademmer K, Ebert M, Muller-Ostermeyer F, et al: Effector T lymphocyte subsets in human pancreatic cancer: detection of CD8+CD18+ cells and CD8+CD103+ cells by multi-epitope imaging. Clin Exp Immunol. 112(1):21–26, 1998. 73. Yen TW, Aardal NP, Bronner MP, et al: Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas. Surgery. 131(2):129–134, 2002. 74. Koshiba T, Hosotani R, Wada M, et al: Involvement of matrix metalloproteinase-2 activity in invasion and metastasis of pancreatic carcinoma. Cancer. 82(4):642–650, 1998. 75. Maitra A, Iacobuzio-Donahue C, Rahman A, et al: Immunohistochemical validation of a novel epithelial and a novel stromal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47. Am J Clin Pathol. 118(1):52–59, 2002. 76. Yamamoto H, Itoh F, Iku S, et al: Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human pancreatic adenocarcinomas: clinicopathologic and prognostic significance of matrilysin expression. J Clin Oncol. 19(4):1118–1127, 2001. 77. Zhou W, Sokoll LJ, Bruzek DJ, et al: Identifying markers for pancreatic cancer by gene expression analysis. Cancer Epidemiol Biomarkers Prev. 7(2):109–112, 1998. 78. Furukawa T, Horii A: Molecular pathology of pancreatic cancer: in quest of tumor suppressor genes. Pancreas. 28(3):253–256, 2004. 79. Hruban RH, Iacobuzio-Donahue C, Wilentz RE, et al: Molecular pathology of pancreatic cancer. Cancer J. 7(4):251–258, 2001. 80. Rozenblum E, Schutte M, Goggins M, et al: Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res. 57(9):1731– 1734, 1997. 81. Moore PS, Orlandini S, Zamboni G, et al: Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis. Br J Cancer. 84(2):253–262, 2001. 82. Moore PS, Sipos B, Orlandini S, et al: Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4. Virchows Arch. 439(6):798–802, 2001. 83. DiGiuseppe JA, Hruban RH, Offerhaus GJ, et al: Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pancreatic carcinoma. Am J Pathol. 144(5):889–895, 1994. 84. van Heek T, Rader AE, Offerhaus GJ, et al: K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: a molecular panel correlates with and supplements cytologic diagnosis. Am J Clin Pathol. 117(5):755–765, 2002.
References 85. Wilentz RE, Geradts J, Maynard R, et al: Inactivation of the p16 (INK4A) tumor-suppressor gene in pancreatic duct lesions: loss of intranuclear expression. Cancer Res. 58(20):4740–4744, 1998. 86. Boschman CR, Stryker S, Reddy JK, et al: Expression of p53 protein in precursor lesions and adenocarcinoma of human pancreas. Am J Pathol. 145(6):1291–1295, 1994. 87. DiGiuseppe JA, Hruban RH, Goodman SN, et al: Overexpression of p53 protein in adenocarcinoma of the pancreas. Am J Clin Pathol. 101(6):684–688, 1994. 88. Li Y, Bhuiyan M, Vaitkevicius VK, et al: Molecular analysis of the p53 gene in pancreatic adenocarcinoma. Diagn Mol Pathol. 7(1):4–9, 1998. 89. Redston MS, Caldas C, Seymour AB, et al: p53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions. Cancer Res. 54(11):3025–3033, 1994. 90. Schutte M: DPC4/SMAD4 gene alterations in human cancer, and their functional implications. Ann Oncol. 10(Suppl 4):56– 59, 1999. 91. Hahn SA, Schutte M, Hoque AT, et al: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 271(5247):350–353, 1996. 92. Wilentz RE, Su GH, Dai JL, et al: Immunohistochemical labeling for dpc4 mirrors genetic status in pancreatic adenocarcinomas: a new marker of DPC4 inactivation. Am J Pathol. 156(1):37–43, 2000. 93. Iacobuzio-Donahue CA, Song J, Parmiagiani G, et al: Missense mutations of MADH4: characterization of the mutational hot spot and functional consequences in human tumors. Clin Cancer Res. 10(5):1597–1604, 2004. 94. Wilentz RE, Iacobuzio-Donahue CA, Argani P, et al: Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression. Cancer Res. 60(7):2002–2006, 2000. 95. Tascilar M, Offerhaus GJ, Altink R, et al: Immunohistochemical labeling for the Dpc4 gene product is a specific marker for adenocarcinoma in biopsy specimens of the pancreas and bile duct. Am J Clin Pathol. 116(6):831–837, 2001. 96. Ali S, Cohen C, Little JV, et al: The utility of SMAD4 as a diagnostic immunohistochemical marker for pancreatic adenocarcinoma, and its expression in other solid tumors. Diagn Cytopathol. 35(10):644–648, 2007. 97. Bartsch D, Shevlin DW, Callery MP, et al: Reduced survival in patients with ductal pancreatic adenocarcinoma associated with CDKN2 mutation. J Natl Cancer Inst. 88(10):680–682, 1996. 98. Schutte M, Hruban RH, Geradts J, et al: Abrogation of the Rb/ p16 tumor-suppressive pathway in virtually all pancreatic carcinomas. Cancer Res. 57(15):3126–3130, 1997. 99. Caldas C, Hahn SA, da Costa LT, et al: Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet. 8(1):27–32, 1994. 100. Geradts J, Hruban RH, Schutte M, et al: Immunohistochemical p16INK4a analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene. A comparison of four commercial antibodies. Appl Immunohistochem Mol Morphol. 8(1):71–79, 2000. 101. Almoguera C, Shibata D, Forrester K, et al: Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 53(4):549–554, 1988. 102. Satoh K, Sawai T, Shimosegawa T, et al: The point mutation of c-Ki-ras at codon 12 in carcinoma of the pancreatic head region and in intraductal mucin-hypersecreting neoplasm of the pancreas. Int J Pancreatol. 14(2):135–143, 1993. 103. Day JD, Digiuseppe JA, Yeo C, et al: Immunohistochemical evaluation of HER-2/neu expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasms. Hum Pathol. 27(2):119–124, 1996. 104. Yamanaka Y, Friess H, Kobrin MS, et al: Overexpression of HER2/neu oncogene in human pancreatic carcinoma. Hum Pathol. 24(10):1127–1134, 1993. 105. Dancer J, Takei H, Ro JY, et al: Coexpression of EGFR and HER-2 in pancreatic ductal adenocarcinoma: a comparative study using immunohistochemistry correlated with gene amplification by fluorescencent in situ hybridization. Oncol Rep. 18(1):151–155, 2007.
583.e3
106. Cheng JQ, Ruggeri B, Klein WM, et al: Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A. 93(8):3636–3641, 1996. 107. Ruggeri BA, Huang L, Wood M, et al: Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Mol Carcinog. 21(2):81–86, 1998. 108. Iacobuzio-Donahue CA, Maitra A, Shen-Ong GL, et al: Discovery of novel tumor markers of pancreatic cancer using global gene expression technology. Am J Pathol. 160(4):1239–1249, 2002. 109. Iacobuzio-Donahue CA, Ryu B, Hruban RH, et al: Exploring the host desmoplastic response to pancreatic carcinoma: gene expression of stromal and neoplastic cells at the site of primary invasion. Am J Pathol. 160(1):91–99, 2002. 110. Ryu B, Jones J, Hollingsworth MA, et al: Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers. Cancer Res. 61(5):1833–1838, 2001. 111. Ryu B, Jones J, Blades NJ, et al: Relationships and differentially expressed genes among pancreatic cancers examined by largescale serial analysis of gene expression. Cancer Res. 62(3):819– 826, 2002. 112. Argani P, Iacobuzio-Donahue C, Ryu B, et al: Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE). Clin Cancer Res. 7(12):3862–3868, 2001. 113. Argani P, Rosty C, Reiter RE, et al: Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma. Cancer Res. 61(11):4320–4324, 2001. 114. Armengol G, Knuutila S, Lluis F, et al: DNA copy number changes and evaluation of MYC, IGF1R, and FES amplification in xenografts of pancreatic adenocarcinoma. Cancer Genet Cytogenet. 116(2):133–141, 2000. 115. Nichols LS, Ashfaq R, Iacobuzio-Donahue CA: Claudin 4 protein expression in primary and metastatic pancreatic cancer: support for use as a therapeutic target. Am J Clin Pathol. 121(2):226–230, 2004. 116. Crnogorac-Jurcevic T, Missiaglia E, Blaveri E, et al: Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent. J Pathol. 201(1):63–74, 2003. 117. Rosty C, Christa L, Kuzdzal S, et al: Identification of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein I as a biomarker for pancreatic ductal adenocarcinoma by protein biochip technology. Cancer Res. 62(6):1868–1875, 2002. 118. Vimalachandran D, Greenhalf W, Thompson C, et al: High nuclear S100A6 (Calcyclin) is significantly associated with poor survival in pancreatic cancer patients. Cancer Res. 65(8):3218– 3225, 2005. 119. Argani P, Shaukat A, Kaushal M, et al: Differing rates of loss of DPC4 expression and of p53 overexpression among carcinomas of the proximal and distal bile ducts. Cancer. 91(7):1332–1341, 2001. 120. Ligato S, Zhao H, Mandich D, et al: KOC (K homology domain containing protein overexpressed in cancer) and S100A4protein immunoreactivity improves the diagnostic sensitivity of biliary brushing cytology for diagnosing pancreaticobiliary malignancies. Diagn Cytopathol. 36(8):561–567, 2008. 121. Iacobuzio-Donahue CA, Hruban RH: Gene expression in neoplasms of the pancreas: applications to diagnostic pathology. Adv Anat Pathol. 10(3):125–134, 2003. 122. Porschen R, Remy U, Bevers G, et al: Prognostic significance of DNA ploidy in adenocarcinoma of the pancreas. A flow cytometric study of paraffin-embedded specimens. Cancer. 71(12):3846–3850, 1993. 123. Yoshimura T, Manabe T, Imamura T, et al: Flow cytometric analysis of nuclear DNA content of duct cell carcinoma of the pancreas. Cancer. 70(5):1069–1074, 1992. 124. Allison DC, Piantadosi S, Hruban RH, et al: DNA content and other factors associated with ten-year survival after resection of pancreatic carcinoma. J Surg Oncol. 67(3):151–159, 1998.
583.e4
Immunohistology of Pancreas and Hepatobiliary Tract
125. Borg A, Sandberg T, Nilsson K, et al: High frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive melanoma families. J Natl Cancer Inst. 92(15):1260–1266, 2000. 126. Griffin CA, Hruban RH, Long PP, et al: Chromosome abnormalities in pancreatic adenocarcinoma. Genes Chromosomes Cancer. 9(2):93–100, 1994. 127. Griffin CA, Hruban RH, Morsberger LA, et al: Consistent chromosome abnormalities in adenocarcinoma of the pancreas. Cancer Res. 55(11):2394–2399, 1995. 128. Johansson B, Bardi G, Pandis N, et al: Karyotypic pattern of pancreatic adenocarcinomas correlates with survival and tumour grade. Int J Cancer. 58(1):8–13, 1994. 129. Wood LD, Adsay V, Hruban RH: Molecular Pathology of Pancreatic Cancer. In Chen L, editor: Molecular Genetic Pathology, ed 2, in press. 130. Coppola D, Lu L, Fruehauf JP, et al: Analysis of p53, p21WAF1, and TGF-beta1 in human ductal adenocarcinoma of the pancreas: TGF-beta1 protein expression predicts longer survival. Am J Clin Pathol. 110(1):16–23, 1998. 131. van der Heijden MS, Yeo CJ, Hruban RH, et al: Fanconi anemia gene mutations in young-onset pancreatic cancer. Cancer Res. 63(10):2585–2588, 2003. 132. Su GH, Hruban RH, Bansal RK, et al: Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol. 154(6):1835–1840, 1999. 133. Su GH, Bansal R, Murphy KM, et al: ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma. Proc Natl Acad Sci U S A. 98(6):3254–3257, 2001. 134. Westra WH, Sturm P, Drillenburg P, et al: K-ras oncogene mutations in osteoclast-like giant cell tumors of the pancreas and liver: genetic evidence to support origin from the duct epithelium. Am J Surg Pathol. 22(10):1247–1254, 1998. 135. Goggins M, Offerhaus GJ, Hilgers W, et al: Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+. Am J Pathol. 152(6):1501–1507, 1998. 136. Shi C, Daniels JA, Hruban RH: Molecular characterization of pancreatic neoplasms. Adv Anat Pathol. 15(4):185–195, 2008. 137. Hruban RH, Adsay NV: Molecular Classification of Neoplasms of the Pancreas. Hum Pathol. 40(5):612–623, 2009. 138. Hruban RH, van Mansfeld AD, Offerhaus GJ, et al: K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol. 143(2):545–554, 1993. 139. Luttges J, Diederichs A, Menke MA, et al: Ductal lesions in patients with chronic pancreatitis show K-ras mutations in a frequency similar to that in the normal pancreas and lack nuclear immunoreactivity for p53. Cancer. 88(11):2495–2504, 2000. 140. Terhune PG, Phifer DM, Tosteson TD, et al: K-ras mutation in focal proliferative lesions of human pancreas. Cancer Epidemiol Biomarkers Prev. 7(6):515–521, 1998. 141. Moskaluk CA, Hruban RH, Kern SE: p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma. Cancer Res. 57(11):2140–2143, 1997. 142. Aguirre AJ, Brennan C, Bailey G, et al: High-resolution characterization of the pancreatic adenocarcinoma genome. Proc Natl Acad Sci U S A. 101(24):9067–9072, 2004. 143. Calhoun ES, Jones JB, Ashfaq R, et al: BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets. Am J Pathol. 163(4):1255–1260, 2003. 144. Miwa W, Yasuda J, Murakami Y, et al: Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer. Biochem Biophys Res Commun. 225(3):968–974, 1996. 145. Wallrapp C, Muller-Pillasch F, Solinas-Toldo S, et al: Characterization of a high copy number amplification at 6q24 in pancreatic cancer identifies c-myb as a candidate oncogene. Cancer Res. 57(15):3135–3139, 1997. 146. Montgomery E, Wilentz RE, Argani P, et al: Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies. Cancer Biol Ther. 2(3):248–252, 2003.
147. Yamamoto H, Itoh F, Nakamura H, et al: Genetic and clinical features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability. Cancer Res. 61(7):3139– 3144, 2001. 148. Wilentz RE, Goggins M, Redston M, et al: Genetic, immunohistochemical, and clinical features of medullary carcinoma of the pancreas: A newly described and characterized entity. Am J Pathol. 156(5):1641–1651, 2000. 149. Ali-Fehmi R, Basturk O, Cheng JD, et al: Immunohistochemistry as a helpful adjunct in the differential diagnosis of intraabdominal carcinomatosis originating from the pancreas versus ovary. Mod Pathol. 18(1):273A, 2005. 150. Goldstein NS, Bassi D, Uzieblo A: WT1 is an integral component of an antibody panel to distinguish pancreaticobiliary and some ovarian epithelial neoplasms. Am J Clin Pathol. 116(2):246–252, 2001. 151. Alguacil-Garcia A, Weiland LH: The histologic spectrum, prognosis, and histogenesis of the sarcomatoid carcinoma of the pancreas. Cancer. 39(3):1181–1189, 1977. 152. Paal E, Thompson LD, Frommelt RA, et al: A clinicopathologic and immunohistochemical study of 35 anaplastic carcinomas of the pancreas with a review of the literature. Ann Diagn Pathol. 5(3):129–140, 2001. 153. Motoo Y, Kawashima A, Watanabe H, et al: Undifferentiated (anaplastic) carcinoma of the pancreas showing sarcomatous change and neoplastic cyst formation. Int J Pancreatol. 21(3):243– 248, 1997. 154. Goldstein NS, Bosler DS: Immunohistochemistry of the Gastrointestinal Tract, Pancreas, Bile Ducts, Gall Bladder and Liver. In Dabbs D, editor: Diagnostic Immunohistchemistry, ed 2, 2006, Elsevier, pp 442–508. 155. Nishihara K, Katsumoto F, Kurokawa Y, et al: Anaplastic carcinoma showing rhabdoid features combined with mucinous cystadenocarcinoma of the pancreas. Arch Pathol Lab Med. 121(10):1104–1107, 1997. 156. Gatteschi B, Saccomanno S, Bartoli FG, et al: Mixed pleomorphicosteoclast-like tumor of the pancreas. Light microscopical, immunohistochemical, and molecular biological studies. Int J Pancreatol. 18(2):169–175, 1995. 157. Deckard-Janatpour K, Kragel S, Teplitz RL, et al: Tumors of the pancreas with osteoclast-like and pleomorphic giant cells: an immunohistochemical and ploidy study. Arch Pathol Lab Med. 122(3):266–272, 1998. 158. Klimstra DS, Adsay NV: Benign and malignant tumors of the pancreas. In Odze RD, Goldblum JR, Crawford JM, editors: Surgical pathology of the GI tract, liver, biliary tract and pancreas, Philadelphia, 2004, Saunders, pp 699–731. 159. Winter JM, Ting AH, Vilardell F, et al: Absence of E-cadherin expression distinguishes noncohesive from cohesive pancreatic cancer. Clin Cancer Res. 14(2):412–418, 2008. 160. Dhall D, Klimstra DS: The cellular composition of osteoclastlike giant cell-containing tumors of the pancreatobiliary tree. Am J Surg Pathol. 32(2):335–337; author response 7, 2008. 161. Berendt RC, Shnitka TK, Wiens E, et al: The osteoclast-type giant cell tumor of the pancreas. Arch Pathol Lab Med. 111(1):43–48, 1987. 162. Dizon MA, Multhaupt HA, Paskin DL, et al: Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study. Arch Pathol Lab Med. 120(3):306–309, 1996. 163. Lukas Z, Dvorak K, Kroupova I, et al: Immunohistochemical and genetic analysis of osteoclastic giant cell tumor of the pancreas. Pancreas. 32(3):325–329, 2006. 164. Molberg KH, Heffess C, Delgado R, et al: Undifferentiated carcinoma with osteoclast-like giant cells of the pancreas and periampullary region. Cancer. 82(7):1279–1287, 1998. 165. Sakai Y, Kupelioglu AA, Yanagisawa A, et al: Origin of giant cells in osteoclast-like giant cell tumors of the pancreas. Hum Pathol. 31(10):1223–1229, 2000. 166. Shiozawa M, Imada T, Ishiwa N, et al: Osteoclast-like giant cell tumor of the pancreas. Int J Clin Oncol. 7(6):376–380, 2002. 167. Gocke CD, Dabbs DJ, Benko FA, et al: KRAS oncogene mutations suggest a common histogenetic origin for pleomorphic giant cell tumor of the pancreas, osteoclastoma of the pancreas, and pancreatic duct adenocarcinoma. Hum Pathol. 28(1):80–83, 1997.
References 168. Imai Y, Morishita S, Ikeda Y, et al: Immunohistochemical and molecular analysis of giant cell carcinoma of the pancreas: a report of three cases. Pancreas. 18(3):308–315, 1999. 169. Goggins M, Shekher M, Turnacioglu K, et al: Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas. Cancer Res. 58(23):5329– 5332, 1998. 170. Ueki T, Toyota M, Sohn T, et al: Hypermethylation of multiple genes in pancreatic adenocarcinoma. Cancer Res. 60(7):1835– 1839, 2000. 171. Kardon DE, Thompson LD, Przygodzki RM, et al: Adenosquamous carcinoma of the pancreas: a clinicopathologic series of 25 cases. Mod Pathol. 14(5):443–451, 2001. 172. Ylagan LR, Scholes J, Demopoulos R: Cd44: a marker of squamous differentiation in adenosquamous neoplasms. Arch Pathol Lab Med. 124(2):212–215, 2000. 173. Adsay NV, Hasteh F, Sarkar F, et al: Squamous Cell And Adenosquamous Carcinomas Of The Pancreas: A Clinicopathologic Analysis Of 11 Cases. Mod Pathol. 13:179a, 2000. 174. Basturk O, Khanani F, Sarkar F, et al: DeltaNp63 expression in pancreas and pancreatic neoplasia. Mod Pathol. 18(9):1193– 1198, 2005. 175. Brody JR, Costantino CL, Potoczek M, et al: Adenosquamous carcinoma of the pancreas harbors KRAS2, DPC4 and TP53 molecular alterations similar to pancreatic ductal adenocarcinoma. Mod Pathol. 22(5):651–659, 2009. 176. Madura JA, Jarman BT, Doherty MG, et al: Adenosquamous carcinoma of the pancreas. Arch Surg. 134(6):599–603, 1999. 177. Andea A, Sarkar F, Adsay VN: Clinicopathological correlates of pancreatic intraepithelial neoplasia: a comparative analysis of 82 cases with and 152 cases without pancreatic ductal adenocarcinoma. Mod Pathol. 16(10):996–1006, 2003. 178. Adsay NV, Pierson C, Sarkar F, et al: Colloid (mucinous noncystic) carcinoma of the pancreas. Am J Surg Pathol. 25(1):26–42, 2001. 179. Adsay NV, Merati K, Nassar H, et al: Pathogenesis of colloid (pure mucinous) carcinoma of exocrine organs: Coupling of gel-forming mucin (MUC2) production with altered cell polarity and abnormal cell-stroma interaction may be the key factor in the morphogenesis and indolent behavior of colloid carcinoma in the breast and pancreas. Am J Surg Pathol. 27(5):571– 578, 2003. 180. Iacobuzio-Donahue CA, Wilentz RE, Argani P, et al: Dpc4 protein in mucinous cystic neoplasms of the pancreas: frequent loss of expression in invasive carcinomas suggests a role in genetic progression. Am J Surg Pathol. 24(11):1544–1548, 2000. 181. Hruban RH, Takaori K, Klimstra DS, et al: An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms. Am J Surg Pathol. 28(8):977–987, 2004. 182. Maitra A, Adsay NV, Argani P, et al: Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray. Mod Pathol. 16(9):902–912, 2003. 183. Klein WM, Hruban RH, Klein-Szanto AJ, et al: Direct correlation between proliferative activity and dysplasia in pancreatic intraepithelial neoplasia (PanIN): additional evidence for a recently proposed model of progression. Mod Pathol. 15(4):441– 447, 2002. 184. Kanda M, Matthaei H, Wu J, et al: Presence of somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology. 142(4):730–733 e9, 2012. 185. Adsay NV, Basturk O, Cheng JD, et al: Ductal neoplasia of the pancreas: nosologic, clinicopathologic, and biologic aspects. Semin Radiat Oncol. 15(4):254–264, 2005. 186. Prasad NB, Biankin AV, Fukushima N, et al: Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells. Cancer Res. 65(5):1619–1626, 2005. 187. Basturk O, Coban I, Adsay NV: Pancreatic cysts: pathologic classification, differential diagnosis, and clinical implications. Arch Pathol Lab Med. 133(3):423–438, 2009. 188. Adsay NV, Adair CF, Heffess CS, et al: Intraductal oncocytic papillary neoplasms of the pancreas. Am J Surg Pathol. 20(8):980– 994, 1996.
583.e5
189. Bosman FT, Carneiro F, Hruban RH, et al, editors: WHO Classification of Tumors of the Digestive System, Lyon, 2010, International Agency for Research on Cancer. 190. Mukawa K, Kawa S, Aoki Y, et al: Reduced expression of p53 and cyclin A in intraductal mucin-hypersecreting neoplasm of the pancreas compared with usual pancreatic ductal adenocarcinoma. Am J Gastroenterol. 94(8):2263–2267, 1999. 191. Terada T, Ohta T, Kitamura Y, et al: Cell proliferative activity in intraductal papillary-mucinous neoplasms and invasive ductal adenocarcinomas of the pancreas: an immunohistochemical study. Arch Pathol Lab Med. 122(1):42–46, 1998. 192. Tomaszewska R, Okon K, Nowak K, et al: HER-2/Neu expression as a progression marker in pancreatic intraepithelial neoplasia. Pol J Pathol. 49(2):83–92, 1998. 193. Terris B, Blaveri E, Crnogorac-Jurcevic T, et al: Characterization of gene expression profiles in intraductal papillary-mucinous tumors of the pancreas. Am J Pathol. 160(5):1745–1754, 2002. 194. Fukushima N, Sato N, Sahin F, et al: Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer. 89(2):338–343, 2003. 195. Adsay NV, Merati K, Basturk O, et al: Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an “intestinal” pathway of carcinogenesis in the pancreas. Am J Surg Pathol. 28(7):839–848, 2004. 196. Terris B, Dubois S, Buisine MP, et al: Mucin gene expression in intraductal papillary-mucinous pancreatic tumours and related lesions. J Pathol. 197(5):632–637, 2002. 197. Chung SM, Hruban RH, Iacobuzio-Donahue CA, et al: Analysis Of Molecular Alterations And Differentiation Pathways In Intraductal Oncocytic Papillary Neoplasm Of The Pancreas. Mod Pathol. 18(Supp 1):277A, 2005. 198. Paal E, Thompson LD, Przygodzki RM, et al: A clinicopathologic and immunohistochemical study of 22 intraductal papillary mucinous neoplasms of the pancreas, with a review of the literature. Mod Pathol. 12(5):518–528, 1999. 199. Nagai E, Ueki T, Chijiiwa K, et al: Intraductal papillary mucinous neoplasms of the pancreas associated with so-called “mucinous ductal ectasia”. Histochemical and immunohistochemical analysis of 29 cases. Am J Surg Pathol. 19(5):576–589, 1995. 200. Nagasaka T, Nakashima N: Problems in histological diagnosis of intraductal papillary-mucinous tumor (IPMT). Hepatogastroenterology. 48(40):972–976, 2001. 201. Adsay NV, Pierson C, Sarkar F, et al: Comparative Immunohistochemical Analysis of Oncoprotein Expression in Various Components of IPMNs. Pancreatology. 28(2):103A, 2000. 202. Tobi M, Hatfield J, Adsay V, et al: Prognostic Significance of the Labeling of Adnab-9 in Pancreatic Intraductal Papillary Mucinous Neoplasms. Int J Gastrointest Cancer. 29(3):141–150, 2001. 203. Satoh K, Sasano H, Shimosegawa T, et al: An immunohistochemical study of the c-erbB-2 oncogene product in intraductal mucin-hypersecreting neoplasms and in ductal cell carcinomas of the pancreas. Cancer. 72(1):51–56, 1993. 204. Terada T, Ohta T, Kitamura Y, et al: Endocrine cells in intraductal papillary-mucinous neoplasms of the pancreas. A histochemical and immunohistochemical study. Virchows Arch. 431(1):31–36, 1997. 205. Niijima M, Yamaguchi T, Ishihara T, et al: Immunohistochemical analysis and in situ hybridization of cyclooxygenase-2 expression in intraductal papillary-mucinous tumors of the pancreas. Cancer. 94(5):1565–1573, 2002. 206. Patel SA, Adams R, Goldstein M, et al: Genetic analysis of invasive carcinoma arising in intraductal oncocytic papillary neoplasm of the pancreas. Am J Surg Pathol. 26(8):1071–1077, 2002. 207. Fujii H, Inagaki M, Kasai S, et al: Genetic progression and heterogeneity in intraductal papillary-mucinous neoplasms of the pancreas. Am J Pathol. 151(5):1447–1454, 1997. 208. Hoshi T, Imai M, Ogawa K: Frequent K-ras mutations and absence of p53 mutations in mucin-producing tumors of the pancreas. J Surg Oncol. 55(2):84–91, 1994. 209. Izawa T, Obara T, Tanno S, et al: Clonality and field cancerization in intraductal papillary-mucinous tumors of the pancreas. Cancer. 92(7):1807–1817, 2001.
583.e6
Immunohistology of Pancreas and Hepatobiliary Tract
210. Nakata B, Yashiro M, Nishioka N, et al: Very low incidence of microsatellite instability in intraductal papillary-mucinous neoplasm of the pancreas. Int J Cancer. 102(6):655–659, 2002. 211. Satoh K, Shimosegawa T, Moriizumi S, et al: K-ras mutation and p53 protein accumulation in intraductal mucin-hypersecreting neoplasms of the pancreas. Pancreas. 12(4):362–368, 1996. 212. Sessa F, Solcia E, Capella C, et al: Intraductal papillary-mucinous tumours represent a distinct group of pancreatic neoplasms: an investigation of tumour cell differentiation and K-ras, p53 and c-erbB-2 abnormalities in 26 patients. Virchows Arch. 425(4):357–367, 1994. 213. Tada M, Omata M, Ohto M: Ras gene mutations in intraductal papillary neoplasms of the pancreas. Analysis in five cases. Cancer. 67(3):634–637, 1991. 214. Kaino M, Kondoh S, Okita S, et al: Detection of K-ras and p53 gene mutations in pancreatic juice for the diagnosis of intraductal papillary mucinous tumors. Pancreas. 18(3):294–299, 1999. 215. Yanagisawa A, Kato Y, Ohtake K, et al: c-Ki-ras point mutations in ductectatic-type mucinous cystic neoplasms of the pancreas. Jpn J Cancer Res. 82(10):1057–1060, 1991. 216. Yoshizawa K, Nagai H, Sakurai S, et al: Clonality and K-ras mutation analyses of epithelia in intraductal papillary mucinous tumor and mucinous cystic tumor of the pancreas. Virchows Arch. 441(5):437–443, 2002. 217. Wu J, Matthaei H, Maitra A, et al: Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development. Sci Transl Med. 3(92):92ra66, 2011. 218. Biankin AV, Biankin SA, Kench JG, et al: Aberrant p16(INK4A) and DPC4/Smad4 expression in intraductal papillary mucinous tumours of the pancreas is associated with invasive ductal adenocarcinoma. Gut. 50(6):861–868, 2002. 219. Flejou JF, Boulange B, Bernades P, et al: p53 protein expression and DNA ploidy in cystic tumors of the pancreas. Pancreas. 13(3):247–252, 1996. 220. Islam HK, Fujioka Y, Tomidokoro T, et al: Immunohistochemical analysis of expression of molecular biologic factors in intraductal papillary-mucinous tumors of pancreas–diagnostic and biologic significance. Hepatogastroenterology. 46(28):2599–2605, 1999. 221. Islam HK, Fujioka Y, Tomidokoro T, et al: Immunohistochemical study of genetic alterations in intraductal and invasive ductal tumors of the pancreas. Hepatogastroenterology. 48(39):879–883, 2001. 222. Kawahira H, Kobayashi S, Kaneko K, et al: p53 protein expression in intraductal papillary mucinous tumors (IPMT) of the pancreas as an indicator of tumor malignancy. Hepatogastroenterology. 47(34):973–977, 2000. 223. Kitagawa Y, Unger TA, Taylor S, et al: Mucus is a predictor of better prognosis and survival in patients with intraductal papillary mucinous tumor of the pancreas. J Gastrointest Surg. 7(1):12–18, discussion 8–9, 2003. 224. Sasaki S, Yamamoto H, Kaneto H, et al: Differential roles of alterations of p53, p16, and SMAD4 expression in the progression of intraductal papillary-mucinous tumors of the pancreas. Oncol Rep. 10(1):21–25, 2003. 225. Iacobuzio-Donahue CA, Klimstra DS, Adsay NV, et al: Dpc-4 protein is expressed in virtually all human intraductal papillary mucinous neoplasms of the pancreas: comparison with conventional ductal adenocarcinomas. Am J Pathol. 157(3):755–761, 2000. 226. Hong SP, Lee EK, Park JY, et al: Cripto-1 overexpression is involved in the tumorigenesis of gastric-type and pancreatobiliary-type intraductal papillary mucinous neoplasms of the pancreas. Oncol Rep. 21(1):19–24, 2009. 227. Kloppel G, Kosmahl M: Cystic lesions and neoplasms of the pancreas. The features are becoming clearer. Pancreatology. 1(6):648–655, 2001. 228. Fukushima N, Mukai K: “Ovarian-type” stroma of pancreatic mucinous cystic tumor expresses smooth muscle phenotype. Pathol Int. 47(11):806–808, 1997. 229. Sarr MG, Carpenter HA, Prabhakar LP, et al: Clinical and pathologic correlation of 84 mucinous cystic neoplasms of the pancreas: can one reliably differentiate benign from malignant (or premalignant) neoplasms? Ann Surg. 231(2):205–212, 2000. 230. Yamaguchi K, Enjoji M: Cystic neoplasms of the pancreas. Gastroenterology. 92(6):1934–1943, 1987.
231. Thompson LD, Becker RC, Przygodzki RM, et al: Mucinous cystic neoplasm (mucinous cystadenocarcinoma of low-grade malignant potential) of the pancreas: a clinicopathologic study of 130 cases. Am J Surg Pathol. 23(1):1–16, 1999. 232. Luttges J, Feyerabend B, Buchelt T, et al: The mucin profile of noninvasive and invasive mucinous cystic neoplasms of the pancreas. Am J Surg Pathol. 26(4):466–471, 2002. 233. Ohta T, Nagakawa T, Fukushima W, et al: Immunohistochemical study of carcinoembryonic antigen in mucinous cystic neoplasm of the pancreas. Eur Surg Res. 24(1):37–44, 1992. 234. van den Berg W, Tascilar M, Offerhaus GJ, et al: Pancreatic mucinous cystic neoplasms with sarcomatous stroma: molecular evidence for monoclonal origin with subsequent divergence of the epithelial and sarcomatous components. Mod Pathol. 13(1):86–91, 2000. 235. Batge B, Bosslet K, Sedlacek HH, et al: Monoclonal antibodies against CEA-related components discriminate between pancreatic duct type carcinomas and nonneoplastic duct lesions as well as nonduct type neoplasias. Virchows Arch A Pathol Anat Histopathol. 408(4):361–374, 1986. 236. Sessa F, Bonato M, Frigerio B, et al: Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells. Gastroenterology. 98(6):1655–1665, 1990. 237. Yu HC, Shetty J: Mucinous cystic neoplasm of the pancreas with high carcinoembryonic antigen. Arch Pathol Lab Med. 109(4):375–377, 1985. 238. Khalifeh I, Basturk O, Zamboni G, et al: Villous-Intestinal Differentiation and Progression to Colloid Carcinoma, Characteristic of a Major Subset of IPMNs, Are Not Features of Mucinous Cystic Neoplasms. Mod Pathol. 18(1):281A, 2005. 239. Albores-Saavedra J, Nadji M, Henson DE, et al: Entero-endocrine cell differentiation in carcinomas of the gallbladder and mucinous cystadenocarcinomas of the pancreas. Pathol Res Pract. 183(2):169–175, 1988. 240. Terada T, Kitamura Y, Ohta T, et al: Endocrine cells in hepatobiliary cystadenomas and cystadenocarcinomas. Virchows Arch. 430(1):37–40, 1997. 241. Albores-Saavedra J, Angeles-Angeles A, Nadji M, et al: Mucinous cystadenocarcinoma of the pancreas. Morphologic and immunocytochemical observations. Am J Surg Pathol. 11(1):11–20, 1987. 242. Khalifeh I, Qureshi F, Jacques S, et al: The nature of “ovarianlike” mesenchyme of pancreatic and hepatic mucinous cystic neoplasms: A recapitulation of the periductal fetal mesenchme? Mod Pathol. 17(1):304A, 2004. 243. Wilentz RE, Albores-Saavedra J, Hruban RH: Mucinous cystic neoplasms of the pancreas. Semin Diagn Pathol. 17(1):31–42, 2000. 244. Zamboni G, Scarpa A, Bogina G, et al: Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and relationship to other mucinous cystic tumors. Am J Surg Pathol. 23(4):410–422, 1999. 245. Izumo A, Yamaguchi K, Eguchi T, et al: Mucinous cystic tumor of the pancreas: immunohistochemical assessment of “ovariantype stroma”. Oncol Rep. 10(3):515–525, 2003. 246. Weihing RR, Shintaku IP, Geller SA, et al: Hepatobiliary and pancreatic mucinous cystadenocarcinomas with mesenchymal stroma: analysis of estrogen receptors/progesterone receptors and expression of tumor-associated antigens. Mod Pathol. 10(4):372–379, 1997. 247. Ridder GJ, Maschek H, Klempnauer J: Favourable prognosis of cystadeno- over adenocarcinoma of the pancreas after curative resection. Eur J Surg Oncol. 22(3):232–236, 1996. 248. Zheng W, Sung CJ, Hanna I, et al: Alpha and beta subunits of inhibin/activin as sex cord-stromal differentiation markers. Int J Gynecol Pathol. 16(3):263–271, 1997. 249. Bartsch D, Bastian D, Barth P, et al: K-ras oncogene mutations indicate malignancy in cystic tumors of the pancreas. Ann Surg. 228(1):79–86, 1998. 250. Jimenez RE, Warshaw AL, Z’Graggen K, et al: Sequential accumulation of K-ras mutations and p53 overexpression in the progression of pancreatic mucinous cystic neoplasms to malignancy. Ann Surg. 230(4):501–509; discussion 9–11, 1999. 251. Kim SG, Wu TT, Lee JH, et al: Comparison of epigenetic and genetic alterations in mucinous cystic neoplasm and serous microcystic adenoma of pancreas. Mod Pathol. 16(11):1086– 1094, 2003.
References 252. Tajiri T, Tate G, Inagaki T, et al: Intraductal tubular neoplasms of the pancreas: histogenesis and differentiation. Pancreas. 30(2):115–121, 2005. 253. Tajiri T, Tate G, Kunimura T, et al: Histologic and immunohistochemical comparison of intraductal tubular carcinoma, intraductal papillary-mucinous carcinoma, and ductal adenocarcinoma of the pancreas. Pancreas. 29(2):116–122, 2004. 254. Klimstra DS: Nonductal neoplasms of the pancreas. Mod Pathol. 20(Suppl 1):S94–S112, 2007. 255. Albores-Saavedra J, Sheahan K, O’Riain C, et al: Intraductal tubular adenoma, pyloric type, of the pancreas: additional observations on a new type of pancreatic neoplasm. Am J Surg Pathol. 28(2):233–238, 2004. 256. Bakotic BW, Robinson MJ, Sturm PD, et al: Pyloric gland adenoma of the main pancreatic duct. Am J Surg Pathol. 23(2):227–231, 1999. 257. Kato N, Akiyama S, Motoyama T: Pyloric gland-type tubular adenoma superimposed on intraductal papillary mucinous tumor of the pancreas. Pyloric gland adenoma of the pancreas. Virchows Arch. 440(2):205–208, 2002. 258. Nakayama Y, Inoue H, Hamada Y, et al: Intraductal tubular adenoma of the pancreas, pyloric gland type: a clinicopathologic and immunohistochemical study of 6 cases. Am J Surg Pathol. 29(5):607–616, 2005. 259. Compton CC: Serous cystic tumors of the pancreas. Semin Diagn Pathol. 17(1):43–55, 2000. 260. Alpert LC, Truong LD, Bossart MI, et al: Microcystic adenoma (serous cystadenoma) of the pancreas. A study of 14 cases with immunohistochemical and electron-microscopic correlation. Am J Surg Pathol. 12(4):251–263, 1988. 261. Egawa N, Maillet B, Schroder S, et al: Serous oligocystic and ill-demarcated adenoma of the pancreas: a variant of serous cystic adenoma. Virchows Arch. 424(1):13–17, 1994. 262. Ohike N, Kosmahl M, Kloppel G: Mixed acinar-endocrine carcinoma of the pancreas. A clinicopathological study and comparison with acinar-cell carcinoma. Virchows Arch. 445(3):231–235, 2004. 263. Shorten SD, Hart WR, Petras RE: Microcystic adenomas (serous cystadenomas) of pancreas. A clinicopathologic investigation of eight cases with immunohistochemical and ultrastructural studies. Am J Surg Pathol. 10(6):365–372, 1986. 264. Sperti C, Pasquali C, Perasole A, et al: Macrocystic serous cystadenoma of the pancreas: clinicopathologic features in seven cases. Int J Pancreatol. 28(1):1–7, 2000. 265. Yasuhara Y, Sakaida N, Uemura Y, et al: Serous microcystic adenoma (glycogen-rich cystadenoma) of the pancreas: study of 11 cases showing clinicopathological and immunohistochemical correlations. Pathol Int. 52(4):307–312, 2002. 266. Perez-Ordonez B, Naseem A, Lieberman PH, et al: Solid serous adenoma of the pancreas. The solid variant of serous cystadenoma? Am J Surg Pathol. 20(11):1401–1405, 1996. 267. Ishikawa T, Nakao A, Nomoto S, et al: Immunohistochemical and molecular biological studies of serous cystadenoma of the pancreas. Pancreas. 16(1):40–44, 1998. 268. Kosmahl M, Wagner J, Peters K, et al: Serous cystic neoplasms of the pancreas: an immunohistochemical analysis revealing alpha-inhibin, neuron-specific enolase, and MUC6 as new markers. Am J Surg Pathol. 28(3):339–346, 2004. 269. Helpap B, Vogel J: Immunohistochemical studies on cystic pancreatic neoplasms. Pathol Res Pract. 184(1):39–45, 1988. 270. Solcia E, Capella C, Kloppel G: Tumors of the Pancreas, ed 3, Washington, DC, 1997, American Registry of Pathology. 271. Thirabanjasak D, Basturk O, Altinel D, et al: Is Serous Cystadenoma of Pancreas A Model of Clear Cell Associated Angiogenesis and Tumorigenesis? Pancreatology. 9(1-2):182–188, 2008. 272. Mohr VH, Vortmeyer AO, Zhuang Z, et al: Histopathology and molecular genetics of multiple cysts and microcystic (serous) adenomas of the pancreas in von Hippel-Lindau patients. Am J Pathol. 157(5):1615–1621, 2000. 273. Vortmeyer AO, Lubensky IA, Fogt F, et al: Allelic deletion and mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in pancreatic microcystic adenomas. Am J Pathol. 151(4):951–956, 1997. 274. Moore PS, Missiaglia E, Antonello D, et al: Role of diseasecausing genes in sporadic pancreatic endocrine tumors: MEN1 and VHL. Genes Chromosomes Cancer. 32(2):177–181, 2001.
583.e7
275. Santos LD, Chow C, Henderson CJ, et al: Serous oligocystic adenoma of the pancreas: a clinicopathological and immunohistochemical study of three cases with ultrastructural findings. Pathology. 34(2):148–156, 2002. 276. Ordonez NG: Pancreatic acinar cell carcinoma. Adv Anat Pathol. 8(3):144–159, 2001. 277. Hartman GG, Ni H, Pickleman J: Acinar cell carcinoma of the pancreas. Arch Pathol Lab Med. 125(8):1127–1128, 2001. 278. La Rosa S, Adsay V, Albarello L, et al: Clinicopathologic study of 62 acinar cell carcinomas of the pancreas: insights into the morphology and immunophenotype and search for prognostic markers. Am J Surg Pathol. 36(12):1782–1795, 2012. 279. Klimstra DS, Heffess CS, Oertel JE, et al: Acinar cell carcinoma of the pancreas. A clinicopathologic study of 28 cases. Am J Surg Pathol. 16(9):815–837, 1992. 280. Skacel M, Ormsby AH, Petras RE, et al: Immunohistochemistry in the differential diagnosis of acinar and endocrine pancreatic neoplasms. Appl Immunohistochem Mol Morphol. 8(3):203–209, 2000. 281. Kuerer H, Shim H, Pertsemlidis D, et al: Functioning pancreatic acinar cell carcinoma: immunohistochemical and ultrastructural analyses. Am J Clin Oncol. 20(1):101–107, 1997. 282. Ishihara A, Sanda T, Takanari H, et al: Elastase-1-secreting acinar cell carcinoma of the pancreas. A cytologic, electron microscopic and histochemical study. Acta Cytol. 33(2):157–163, 1989. 283. La Rosa S, Franzi F, Marchet S, et al: The monoclonal antiBCL10 antibody (clone 331.1) is a sensitive and specific marker of pancreatic acinar cell carcinoma and pancreatic metaplasia. Virchows Arch. 454(2):133–142, 2009. 284. Basturk O, Zamboni G, Klimstra DS, et al: Intraductal and papillary variants of acinar cell carcinomas: a new addition to the challenging differential diagnosis of intraductal neoplasms. Am J Surg Pathol. 31(3):363–370, 2007. 285. Pellegata NS, Sessa F, Renault B, et al: K-ras and p53 gene mutations in pancreatic cancer: ductal and nonductal tumors progress through different genetic lesions. Cancer Res. 54(6):1556–1560, 1994. 286. Terhune PG, Heffess CS, Longnecker DS: Only wild-type c-Kiras codons 12, 13, and 61 in human pancreatic acinar cell carcinomas. Mol Carcinog. 10(2):110–114, 1994. 287. Terhune PG, Memoli VA, Longnecker DS: Evaluation of p53 mutation in pancreatic acinar cell carcinomas of humans and transgenic mice. Pancreas. 16(1):6–12, 1998. 288. de Wilde RF, Ottenhof NA, Jansen M, et al: Analysis of LKB1 mutations and other molecular alterations in pancreatic acinar cell carcinoma. Mod Pathol. 24(9):1229–1236, 2011. 289. Abraham SC, Wu TT, Hruban RH, et al: Genetic and immunohistochemical analysis of pancreatic acinar cell carcinoma: frequent allelic loss on chromosome 11p and alterations in the APC/beta-catenin pathway. Am J Pathol. 160(3):953–962, 2002. 290. Longnecker DS: Molecular pathology of invasive carcinoma. Ann N Y Acad Sci. 880:74–82, 1999. 291. Moore PS, Beghelli S, Zamboni G, et al: Genetic abnormalities in pancreatic cancer. Mol Cancer. 2:7, 2003. 292. Rigaud G, Moore PS, Zamboni G, et al: Allelotype of pancreatic acinar cell carcinoma. Int J Cancer. 88(5):772–777, 2000. 293. Taruscio D, Paradisi S, Zamboni G, et al: Pancreatic acinar carcinoma shows a distinct pattern of chromosomal imbalances by comparative genomic hybridization. Genes Chromosomes Cancer. 28(3):294–299, 2000. 294. Henke AC, Kelley CM, Jensen CS, et al: Fine-needle aspiration cytology of pancreatoblastoma. Diagn Cytopathol. 25(2):118– 121, 2001. 295. Kawamoto K, Matsuo T, Jubashi T, et al: Primary pancreatic carcinoma in childhood. Pancreatoblastoma. Acta Pathol Jpn. 35(1):137–143, 1985. 296. Klimstra DS, Wenig BM, Adair CF, et al: Pancreatoblastoma. A clinicopathologic study and review of the literature. Am J Surg Pathol. 19(12):1371–1389, 1995. 297. Nishimata S, Kato K, Tanaka M, et al: Expression pattern of keratin subclasses in pancreatoblastoma with special emphasis on squamoid corpuscles. Pathol Int. 55(6):297–302, 2005. 298. Silverman JF, Holbrook CT, Pories WJ, et al: Fine needle aspiration cytology of pancreatoblastoma with immunocytochemical and ultrastructural studies. Acta Cytol. 34(5):632–640, 1990.
583.e8
Immunohistology of Pancreas and Hepatobiliary Tract
299. Ohaki Y, Misugi K, Fukuda J, et al: Immunohistochemical study of pancreatoblastoma. Acta Pathol Jpn. 37(10):1581–1590, 1987. 300. Cooper JE, Lake BD: Use of enzyme histochemistry in the diagnosis of pancreatoblastoma. Histopathology. 15(4):407–414, 1989. 301. He L, Li P, Liu S, et al: [A clinicopathological study of pancreatoblastoma]. Zhonghua Bing Li Xue Za Zhi. 28(5):337–339, 1999. 302. Iseki M, Suzuki T, Koizumi Y, et al: Alpha-fetoprotein-producing pancreatoblastoma. A case report. Cancer. 57(9):1833–1835, 1986. 303. Abraham SC, Wu TT, Klimstra DS, et al: Distinctive molecular genetic alterations in sporadic and familial adenomatous polyposis-associated pancreatoblastomas : frequent alterations in the APC/beta-catenin pathway and chromosome 11p. Am J Pathol. 159(5):1619–1627, 2001. 304. Tanaka Y, Kato K, Notohara K, et al: Significance of aberrant (cytoplasmic/nuclear) expression of beta-catenin in pancreatoblastoma. J Pathol. 199(2):185–190, 2003. 305. Kerr NJ, Chun YH, Yun K, et al: Pancreatoblastoma is associated with chromosome 11p loss of heterozygosity and IGF2 overexpression. Med Pediatr Oncol. 39(1):52–54, 2002. 306. Abraham SC, Klimstra DS, Wilentz RE, et al: Solidpseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations. Am J Pathol. 160(4):1361–1369, 2002. 307. Wiley J, Posekany K, Riley R, et al: Cytogenetic and flow cytometric analysis of a pancreatoblastoma. Cancer Genet Cytogenet. 79(2):115–118, 1995. 308. Balercia G, Zamboni G, Bogina G, et al: Solid-cystic tumor of the pancreas. An extensive ultrastructural study of fourteen cases. J Submicrosc Cytol Pathol. 27(3):331–340, 1995. 309. Martin RC, Klimstra DS, Brennan MF, et al: Solid-pseudopapillary tumor of the pancreas: a surgical enigma? Ann Surg Oncol. 9(1):35–40, 2002. 310. Miettinen M, Partanen S, Fraki O, et al: Papillary cystic tumor of the pancreas. An analysis of cellular differentiation by electron microscopy and immunohistochemistry. Am J Surg Pathol. 11(11):855–865, 1987. 311. Yamaguchi K, Miyagahara T, Tsuneyoshi M, et al: Papillary cystic tumor of the pancreas: an immunohistochemical and ultrastructural study of 14 patients. Jpn J Clin Oncol. 19(2):102–111, 1989. 312. Wunsch LP, Flemming P, Werner U, et al: Diagnosis and treatment of papillary cystic tumor of the pancreas in children. Eur J Pediatr Surg. 7(1):45–47, 1997. 313. von Herbay A, Sieg B, Otto HF: Solid-cystic tumour of the pancreas. An endocrine neoplasm? Virchows Arch A Pathol Anat Histopathol. 416(6):535–538, 1990. 314. Pettinato G, Manivel JC, Ravetto C, et al: Papillary cystic tumor of the pancreas. A clinicopathologic study of 20 cases with cytologic, immunohistochemical, ultrastructural, and flow cytometric observations, and a review of the literature. Am J Clin Pathol. 98(5):478–488, 1992. 315. Lieber MR, Lack EE, Roberts JR, Jr, et al: Solid and papillary epithelial neoplasm of the pancreas. An ultrastructural and immunocytochemical study of six cases. Am J Surg Pathol. 11(2):85–93, 1987. 316. Stommer P, Kraus J, Stolte M, et al: Solid and cystic pancreatic tumors. Clinical, histochemical, and electron microscopic features in ten cases. Cancer. 67(6):1635–1641, 1991. 317. Yoon DY, Hines OJ, Bilchik AJ, et al: Solid and papillary epithelial neoplasms of the pancreas: aggressive resection for cure. Am Surg. 67(12):1195–1199, 2001. 318. Lam KY, Lo CY, Fan ST: Pancreatic solid-cystic-papillary tumor: clinicopathologic features in eight patients from Hong Kong and review of the literature. World J Surg. 23(10):1045–1050, 1999. 319. Kosmahl M, Seada LS, Janig U, et al: Solid-pseudopapillary tumor of the pancreas: its origin revisited. Virchows Arch. 436(5):473–480, 2000. 320. Lee WY, Tzeng CC, Chen RM, et al: Papillary cystic tumors of the pancreas: assessment of malignant potential by analysis of progesterone receptor, flow cytometry, and ras oncogene mutation. Anticancer Res. 17(4A):2587–2591, 1997.
321. Notohara K, Hamazaki S, Tsukayama C, et al: Solidpseudopapillary tumor of the pancreas: immunohistochemical localization of neuroendocrine markers and CD10. Am J Surg Pathol. 24(10):1361–1371, 2000. 322. Zamboni G, Bonetti F, Scarpa A, et al: Expression of progesterone receptors in solid-cystic tumour of the pancreas: a clinicopathological and immunohistochemical study of ten cases. Virchows Arch A Pathol Anat Histopathol. 423(6):425–431, 1993. 323. Muller-Hocker J, Zietz CH, Sendelhofert A: Deregulated expression of cell cycle-associated proteins in solid pseudopapillary tumor of the pancreas. Mod Pathol. 14(2):47–53, 2001. 324. Chott A, Kloppel G, Buxbaum P, et al: Neuron specific enolase demonstration in the diagnosis of a solid-cystic (papillary cystic) tumour of the pancreas. Virchows Arch A Pathol Anat Histopathol. 410(5):397–402, 1987. 325. Kloppel G, Maurer R, Hofmann E, et al: Solid-cystic (papillarycystic) tumours within and outside the pancreas in men: report of two patients. Virchows Arch A Pathol Anat Histopathol. 418(2):179–183, 1991. 326. Kim MJ, Jang SJ, Yu E: Loss of E-cadherin and cytoplasmicnuclear expression of beta-catenin are the most useful immunoprofiles in the diagnosis of solid-pseudopapillary neoplasm of the pancreas. Hum Pathol. 39(2):251–258, 2008. 327. Tien YW, Ser KH, Hu RH, et al: Solid pseudopapillary neoplasms of the pancreas: is there a pathologic basis for the observed gender differences in incidence? Surgery. 137(6):591– 596, 2005. 328. Nishihara K, Tsuneyoshi M, Ohshima A, et al: Papillary cystic tumor of the pancreas. Is it a hormone-dependent neoplasm? Pathol Res Pract. 189(5):521–526, 1993. 329. Cao D, Antonescu C, Wong G, et al: Positive immunohistochemical staining of KIT in solid-pseudopapillary neoplasms of the pancreas is not associated with KIT/PDGFRA mutations. Mod Pathol. 19(9):1157–1163, 2006. 330. Tiemann K, Heitling U, Kosmahl M, et al: Solid pseudopapillary neoplasms of the pancreas show an interruption of the Wntsignaling pathway and express gene products of 11q. Mod Pathol. 20(9):955–960, 2007. 331. Doglioni C, Tos AP, Laurino L, et al: Calretinin: a novel immunocytochemical marker for mesothelioma. Am J Surg Pathol. 20(9):1037–1046, 1996. 332. Tanaka Y, Kato K, Notohara K, et al: Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm. Cancer Res. 61(23):8401–8404, 2001. 333. Yamaue H, Tanimura H, Shono Y, et al: Solid and cystic tumor of the pancreas: clinicopathologic and genetic studies of four cases. Int J Pancreatol. 27(1):69–76, 2000. 334. Tiemann K, Kosmahl M, Ohlendorf J, et al: Solid pseudopapillary neoplasms of the pancreas are associated with FLI-1 expression, but not with EWS/FLI-1 translocation. Mod Pathol. 19(11):1409–1413, 2006. 335. Lloyd RV, Mervak T, Schmidt K, et al: Immunohistochemical detection of chromogranin and neuron-specific enolase in pancreatic endocrine neoplasms. Am J Surg Pathol. 8(8):607–614, 1984. 336. Thomas RM, Baybick JH, Elsayed AM, et al: Gastric carcinoids. An immunohistochemical and clinicopathologic study of 104 patients. Cancer. 73(8):2053–2058, 1994. 337. Al-Khafaji B, Noffsinger AE, Miller MA, et al: Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors. Hum Pathol. 29(9):992–999, 1998. 338. Bordi C: Gastric carcinoids: an immunohistochemical and clinicopathologic study of 104 patients. Cancer. 75(1):129–130, 1995. 339. Bordi C, Yu JY, Baggi MT, et al: Gastric carcinoids and their precursor lesions. A histologic and immunohistochemical study of 23 cases. Cancer. 67(3):663–672, 1991. 340. Le Gall F, Vallet VS, Thomas D, et al: Immunohistochemical study of secretogranin II in 62 neuroendocrine tumours of the digestive tract and of the pancreas in comparison with other granins. Pathol Res Pract. 193(3):179–185, 1997. 341. Burke AP, Federspiel BH, Sobin LH, et al: Carcinoids of the duodenum. A histologic and immunohistochemical study of 65 tumors. Am J Surg Pathol. 13(10):828–837, 1989.
References 342. Makhlouf HR, Burke AP, Sobin LH: Carcinoid tumors of the ampulla of Vater: a comparison with duodenal carcinoid tumors. Cancer. 85(6):1241–1249, 1999. 343. Lam KY, Lo CY: Pancreatic endocrine tumour: a 22-year clinicopathological experience with morphological, immunohistochemical observation and a review of the literature. Eur J Surg Oncol. 23(1):36–42, 1997. 344. Azzoni C, Doglioni C, Viale G, et al: Involvement of BCL-2 oncoprotein in the development of enterochromaffin-like cell gastric carcinoids. Am J Surg Pathol. 20(4):433–441, 1996. 345. Hayashi H, Nakagawa M, Kitagawa S, et al: Immunohistochemical analysis of gastrointestinal carcinoids. Gastroenterol Jpn. 28(4):483–490, 1993. 346. Rindi G, Ubiali A, Villanacci V: The phenotype of gut endocrine tumours. Dig Liver Dis. 36(Suppl 1):S26–S30, 2004. 347. Erickson LA, Lloyd RV: Practical markers used in the diagnosis of endocrine tumors. Adv Anat Pathol. 11(4):175–189, 2004. 348. Chetty R, Asa SL: Pancreatic endocrine tumors: an update. Adv Anat Pathol. 11(4):202–210, 2004. 349. Bostwick DG, Null WE, Holmes D, et al: Expression of opioid peptides in tumors. N Engl J Med. 317(23):1439–1443, 1987. 350. Chejfec G, Falkmer S, Grimelius L, et al: Synaptophysin. A new marker for pancreatic neuroendocrine tumors. Am J Surg Pathol. 11(4):241–247, 1987. 351. Simpson S, Vinik AI, Marangos PJ, et al: Immunohistochemical localization of neuron-specific enolase in gastroenteropancreatic neuroendocrine tumors. Correlation with tissue and serum levels of neuron-specific enolase. Cancer. 54(7):1364–1369, 1984. 352. Heitz PU, Kasper M, Polak JM, et al: Pancreatic endocrine tumors. Hum Pathol. 13(3):263–271, 1982. 353. Mukai K: Functional pathology of pancreatic islets: immunocytochemical exploration. Pathol Annu. 18(Pt 2):87–107, 1983. 354. Mukai K, Grotting JC, Greider MH, et al: Retrospective study of 77 pancreatic endocrine tumors using the immunoperoxidase method. Am J Surg Pathol. 6(5):387–399, 1982. 355. Clark ES, Carney JA: Pancreatic islet cell tumor associated with Cushing’s syndrome. Am J Surg Pathol. 8(12):917–924, 1984. 356. Schmid KW, Brink M, Freytag G, et al: Expression of chromogranin A and B and secretoneurin immunoreactivity in neoplastic and nonneoplastic pancreatic alpha cells. Virchows Arch. 425(2):127–132, 1994. 357. Delis S, Bakoyiannis A, Giannakou N, et al: Asymptomatic calcitonin-secreting tumor of the pancreas. A case report. Jop. 7(1):70–73, 2006. 358. Miraliakbari BA, Asa SL, Boudreau SF: Parathyroid hormonelike peptide in pancreatic endocrine carcinoma and adenocarcinoma associated with hypercalcemia. Hum Pathol. 23(8):884–887, 1992. 359. Berger G, Trouillas J, Bloch B, et al: Multihormonal carcinoid tumor of the pancreas. Secreting growth hormone-releasing factor as a cause of acromegaly. Cancer. 54(10):2097–2108, 1984. 360. Bostwick DG, Quan R, Hoffman AR, et al: Growth-hormonereleasing factor immunoreactivity in human endocrine tumors. Am J Pathol. 117(2):167–170, 1984. 361. Dayal Y, Lin HD, Tallberg K, et al: Immunocytochemical demonstration of growth hormone-releasing factor in gastrointestinal and pancreatic endocrine tumors. Am J Clin Pathol. 85(1):13– 20, 1986. 362. La Rosa S, Uccella S, Billo P, et al: Immunohistochemical localization of alpha- and betaA-subunits of inhibin/activin in human normal endocrine cells and related tumors of the digestive system. Virchows Arch. 434(1):29–36, 1999. 363. Kimura N, Pilichowska M, Okamoto H, et al: Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors. Mod Pathol. 13(2):140–146, 2000. 364. Tomita T: Metallothionein in pancreatic endocrine neoplasms. Mod Pathol. 13(4):389–395, 2000. 365. Papotti M, Bongiovanni M, Volante M, et al: Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reverse-transcriptase polymerase chain reaction analysis. Virchows Arch. 440(5):461–475, 2002.
583.e9
366. Larsson LI, Grimelius L, Hakanson R, et al: Mixed endocrine pancreatic tumors producing several peptide hormones. Am J Pathol. 79(2):271–284, 1975. 367. Liu TH, Tseng HC, Zhu Y, et al: Insulinoma. An immunocytochemical and morphologic analysis of 95 cases. Cancer. 56(6):1420–1429, 1985. 368. Deshpande V, Fernandez-del Castillo C, Muzikansky A, et al: Cytokeratin 19 is a powerful predictor of survival in pancreatic endocrine tumors. Am J Surg Pathol. 28(9):1145–1153, 2004. 369. Schmitt AM, Anlauf M, Rousson V, et al: WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors. Am J Surg Pathol. 31(11):1677–1682, 2007. 370. Ali A, Serra S, Asa SL, et al: The predictive value of CK19 and CD99 in pancreatic endocrine tumors. Am J Surg Pathol. 30(12):1588–1594, 2006. 371. Cai YC, Banner B, Glickman J, et al: Cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors. Hum Pathol. 32(10):1087–1093, 2001. 372. d’Amore ES, Manivel JC, Pettinato G, et al: Intestinal ganglioneuromatosis: mucosal and transmural types. A clinicopathologic and immunohistochemical study of six cases. Hum Pathol. 22(3):276–286, 1991. 373. Hochwald SN, Zee S, Conlon KC, et al: Prognostic factors in pancreatic endocrine neoplasms: an analysis of 136 cases with a proposal for low-grade and intermediate-grade groups. J Clin Oncol. 20(11):2633–2642, 2002. 374. Kamisawa T, Tu Y, Egawa N, et al: Ductal and acinar differentiation in pancreatic endocrine tumors. Dig Dis Sci. 47(10):2254– 2261, 2002. 375. Yantiss RK, Chang HK, Farraye FA, et al: Prevalence and prognostic significance of acinar cell differentiation in pancreatic endocrine tumors. Am J Surg Pathol. 26(7):893–901, 2002. 376. Klimstra DS, Rosai J, Heffess CS: Mixed acinar-endocrine carcinomas of the pancreas. Am J Surg Pathol. 18(8):765–778, 1994. 377. Arihiro K, Inai K: Malignant islet cell tumor of the pancreas with multiple hormone production and expression of CEA and CA19-9. Report of an autopsy case. Acta Pathol Jpn. 41(2):150– 157, 1991. 378. Hussain S, Arwini A, Chetty R, et al: Oncocytic Pancreatic Endocrine Neoplasms: A Clinicopathologic and Immnunohistochemical Analysis of 21 Cases. Mod Pathol. 18:279A, 2005. 379. Goto A, Niki T, Terado Y, et al: Prevalence of CD99 protein expression in pancreatic endocrine tumours (PETs). Histopathology. 45(4):384–392, 2004. 380. Viale G, Doglioni C, Gambacorta M, et al: Progesterone receptor immunoreactivity in pancreatic endocrine tumors. An immunocytochemical study of 156 neuroendocrine tumors of the pancreas, gastrointestinal and respiratory tracts, and skin. Cancer. 70(9):2268–2277, 1992. 381. Basturk O, Yang Z, Tang LH, et al: Increased (>20%) Ki67 proliferation index in morphologically well differentiated pancreatic neuroendocrine tumors (PanNETs) correlates with decreased overall survival. Mod Pathol. 2013; in press. 382. Klimstra DS, Modlin IR, Coppola D, et al: The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems. Pancreas. 39(6):707–712, 2010. 383. Tang LH, Gonen M, Hedvat C, et al: Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods. Am J Surg Pathol. 36(12):1761– 1770, 2012. 384. Bagci P, Ohike N, Dursun N, et al: Comparative analysis of different counting methodologies for Ki-67 in pancreatic neuroendocrine tumors. Mod Pathol. 25(2):441A, 2012. 385. Sofocleous CT, Garg S, Petrovic LM, et al: Ki-67 is a prognostic biomarker of survival after radiofrequency ablation of liver malignancies. Ann Surg Oncol. 19(13):4262–4269, 2012. 386. Yang Z, Tang LH, Klimstra DS: Effect of tumor heterogeneity on the assessment of Ki67 labeling index in well-differentiated neuroendocrine tumors metastatic to the liver: implications for prognostic stratification. Am J Surg Pathol. 35(6):853–860, 2011. 387. Adsay V: Ki67 labeling index in neuroendocrine tumors of the gastrointestinal and pancreatobiliary tract: to count or not to count is not the question, but rather how to count. Am J Surg Pathol. 36(12):1743–1746, 2012.
583.e10
Immunohistology of Pancreas and Hepatobiliary Tract
388. Speel EJ, Richter J, Moch H, et al: Genetic differences in endocrine pancreatic tumor subtypes detected by comparative genomic hybridization. Am J Pathol. 155(6):1787–1794, 1999. 389. Stumpf E, Aalto Y, Hoog A, et al: Chromosomal alterations in human pancreatic endocrine tumors. Genes Chromosomes Cancer. 29(1):83–87, 2000. 390. Zhao J, Moch H, Scheidweiler AF, et al: Genomic imbalances in the progression of endocrine pancreatic tumors. Genes Chromosomes Cancer. 32(4):364–372, 2001. 391. Amato E, Barbi S, Malpeli G, et al: Chromosome 3p alterations in pancreatic endocrine neoplasia. Virchows Arch. 458(1):39–45, 2011. 392. Asteria C, Anagni M, Fugazzola L, et al: MEN1 gene mutations are a rare event in patients with sporadic neuroendocrine tumors. Eur J Intern Med. 13(5):319–323, 2002. 393. Cupisti K, Hoppner W, Dotzenrath C, et al: Lack of MEN1 gene mutations in 27 sporadic insulinomas. Eur J Clin Invest. 30(4):325–329, 2000. 394. Gortz B, Roth J, Krahenmann A, et al: Mutations and allelic deletions of the MEN1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms. Am J Pathol. 154(2):429– 436, 1999. 395. Hessman O, Lindberg D, Einarsson A, et al: Genetic alterations on 3p, 11q13, and 18q in nonfamilial and MEN 1-associated pancreatic endocrine tumors. Genes Chromosomes Cancer. 26(3):258–264, 1999. 396. Komminoth P: Review: multiple endocrine neoplasia type 1, sporadic neuroendocrine tumors, and MENIN. Diagn Mol Pathol. 8(3):107–112, 1999. 397. Shan L, Nakamura Y, Nakamura M, et al: Somatic mutations of multiple endocrine neoplasia type 1 gene in the sporadic endocrine tumors. Lab Invest. 78(4):471–475, 1998. 398. Wang EH, Ebrahimi SA, Wu AY, et al: Mutation of the MENIN gene in sporadic pancreatic endocrine tumors. Cancer Res. 58(19):4417–4420, 1998. 399. Zhuang Z, Vortmeyer AO, Pack S, et al: Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res. 57(21):4682–4686, 1997. 400. Lubensky IA, Pack S, Ault D, et al: Multiple neuroendocrine tumors of the pancreas in von Hippel-Lindau disease patients: histopathological and molecular genetic analysis. Am J Pathol. 153(1):223–231, 1998. 401. Goebel SU, Iwamoto M, Raffeld M, et al: Her-2/neu expression and gene amplification in gastrinomas: correlations with tumor biology, growth, and aggressiveness. Cancer Res. 62(13):3702– 3710, 2002. 402. Chung DC, Smith AP, Louis DN, et al: A novel pancreatic endocrine tumor suppressor gene locus on chromosome 3p with clinical prognostic implications. J Clin Invest. 100(2):404–410, 1997. 403. Scarpa A, Orlandini S, Moore PS, et al: Dpc4 is expressed in virtually all primary and metastatic pancreatic endocrine carcinomas. Virchows Arch. 440(2):155–159, 2002. 404. Srivastava A, Hornick JL: Immunohistochemical Staining for CDX-2, PDX-1, NESP-55, and TTF-1 can Help Distinguish Gastrointestinal Carcinoid Tumors From Pancreatic Endocrine and Pulmonary Carcinoid Tumors. Am J Surg Pathol. 2008. 405. Sellner F, Sobhian B, De Santis M, et al: Well or poorly differentiated nonfunctioning neuroendocrine carcinoma of the pancreas: a single institution experience with 17 cases. Eur J Surg Oncol. 34(2):191–195, 2008. 406. Nilsson O, Van Cutsem E, Delle Fave G, et al: Poorly differentiated carcinomas of the foregut (gastric, duodenal and pancreatic). Neuroendocrinology. 84(3):212–215, 2006. 407. Stukavec J, Jirasek T, Mandys V, et al: Poorly differentiated endocrine carcinoma and intraductal papillary-mucinous neoplasm of the pancreas: Description of an unusual case. Pathol Res Pract. 203(12):879–884, 2007. 408. Kaufmann O, Georgi T, Dietel M: Utility of 123C3 monoclonal antibody against CD56 (NCAM) for the diagnosis of small cell carcinomas on paraffin sections. Hum Pathol. 28(12):1373–1378, 1997. 409. Capella C, Heitz PU, Hofler H, et al: Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virchows Arch. 425(6):547–560, 1995.
410. Kaifi JT, Zinnkann U, Yekebas EF, et al: L1 is a potential marker for poorly-differentiated pancreatic neuroendocrine carcinoma. World J Gastroenterol. 12(1):94–98, 2006. 411. Kaifi JT, Heidtmann S, Schurr PG, et al: Absence of L1 in pancreatic masses distinguishes adenocarcinomas from poorly differentiated neuroendocrine carcinomas. Anticancer Res. 26(2A):1167–1170, 2006. 412. Movahedi-Lankarani S, Hruban RH, Westra WH, et al: Primitive neuroectodermal tumors of the pancreas: a report of seven cases of a rare neoplasm. Am J Surg Pathol. 26(8):1040–1047, 2002. 413. Basturk O, Hruban R, Adsay V, et al: High-grade neuroendocrine carcinomas of the pancreas: A clinicopathologic analysis of 60 cases. Mod Pathol. 2013. 414. Adsay V, Jang K-T, Ohike N, et al: International consensus study on the terminology and diagnosis of tumoral intraepithelial neoplasms (adenomas and intracystic papillary neoplasms of WHO2010) of the gallbladder. Mod Pathol. in press. 415. Adsay V, Jang KT, Roa JC, et al: Intracholecystic papillarytubular neoplasms (ICPN) of the gallbladder (neoplastic polyps, adenomas, and papillary neoplasms that are >/=1.0 cm): clinicopathologic and immunohistochemical analysis of 123 cases. Am J Surg Pathol. 36(9):1279–1301, 2012. 416. Nakatani Y, Masudo K, Miyagi Y, et al: Aberrant nuclear localization and gene mutation of beta-catenin in low-grade adenocarcinoma of fetal lung type: up-regulation of the Wnt signaling pathway may be a common denominator for the development of tumors that form morules. Mod Pathol. 15(6):617–624, 2002. 417. Rouzbahman M, Serra S, Adsay NV, et al: Oncocytic papillary neoplasms of the biliary tract: a clinicopathological, mucin core and Wnt pathway protein analysis of four cases. Pathology. 39(4):413–418, 2007. 418. Ji Y, Fan J, Zhou J, et al: Intraductal papillary neoplasms of bile duct. A distinct entity like its counterpart in pancreas. Histol Histopathol. 23(1):41–50, 2008. 419. Tanaka M, Fukushima N, Noda N, et al: Intraductal oncocytic papillary neoplasm of the bile duct: clinicopathologic and immunohistochemical characteristics of 6 cases. Hum Pathol. 40(11):1543–1552, 2009. 420. Furukawa T, Hatori T, Fujita I, et al: Prognostic relevance of morphological types of intraductal papillary mucinous neoplasms of the pancreas. Gut. 60(4):509–516, 2011. 421. Wistuba II, Miquel JF, Gazdar AF, et al: Gallbladder adenomas have molecular abnormalities different from those present in gallbladder carcinomas. Hum Pathol. 30(1):21–25, 1999. 422. Matthaei H, Wu J, Dal Molin M, et al: GNAS codon 201 mutations are uncommon in intraductal papillary neoplasms of the bile duct. HPB (Oxford). 14(10):677–683, 2012. 423. Devaney K, Goodman ZD, Ishak KG: Hepatobiliary cystadenoma and cystadenocarcinoma. A light microscopic and immunohistochemical study of 70 patients. Am J Surg Pathol. 18(11):1078–1091, 1994. 424. Ojeda VJ, Shilkin KB, Walters MN: Premalignant epithelial lesions of the gall bladder: a prospective study of 120 cholecystectomy specimens. Pathology. 17(3):451–454, 1985. 425. Chan KW: Review of 253 cases of significant pathology in 7,910 cholecystectomies in Hong Kong. Pathology. 20(1):20–23, 1988. 426. Wistuba II, Albores-Saavedra J: Genetic abnormalities involved in the pathogenesis of gallbladder carcinoma. J Hepatobiliary Pancreat Surg. 6(3):237–244, 1999. 427. Adsay NV, Klimstra DS: Benign and Malignant Tumors of the Gallbladder and Extrahepatic Biliary Tract. In Odze RD, Goldblum JR, editors: Surgical Pathology of the GI tract, Liver, Biliary Tract, and Pancreas, ed 3, Philadelphia, Elsevier, in press. 428. Hansel DE, Meeker AK, Hicks J, et al: Telomere length variation in biliary tract metaplasia, dysplasia, and carcinoma. Mod Pathol. 19(6):772–779, 2006. 429. Stolzenberg-Solomon R, Fraumeni JF, Wideroff L: New Malignancies Following Cancer of the Digestive Tract, Excluding Colorectal Cancer (Chapter 4). New Malignancies Among Cancer Survivors: SEER Cancer Registries, 1973-2000, 2000. 430. Albores-Saavedra J, Henson DE, Klimstra DS: Tumors of the gallbladder, extrahepatic bile ducts and ampulla of Vater, ed 3, Washington D.C, 2000, American Registry of Pathology. 431. Roa I, Ibacache G, Roa J, et al: Gallstones and gallbladder cancervolume and weight of gallstones are associated with gallbladder cancer: a case-control study. J Surg Oncol. 93(8):624–628, 2006.
References 432. Kumar S: Infection as a risk factor for gallbladder cancer. J Surg Oncol. 93(8):633–639, 2006. 433. Esterly JR, Spicer SS: Mucin histochemistry of human gallbladder: changes in adenocarcinoma, cystic fibrosis, and cholecystitis. J Natl Cancer Inst. 40(1):1–12, 1968. 434. Lack EE: Pathology of the pancreas, gallbladder, extrahepatic biliary tract and ampullary region, New York, 2003, Oxford University Press. 435. Yamamoto M, Nakajo S, Tahara E: Immunohistochemical analysis of estrogen receptors in human gallbladder. Acta Pathol Jpn. 40(1):14–21, 1990. 436. Tian Y, Ding RY, Zhi YH, et al: Analysis of p53 and vascular endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity. World J Gastroenterol. 12(3):415–419, 2006. 437. Rashid A, Ueki T, Gao YT, et al: K-ras mutation, p53 overexpression, and microsatellite instability in biliary tract cancers: a population-based study in China. Clin Cancer Res. 8(10):3156– 3163, 2002. 438. Parwani AV, Geradts J, Caspers E, et al: Immunohistochemical and genetic analysis of non-small cell and small cell gallbladder carcinoma and their precursor lesions. Mod Pathol. 16(4):299– 308, 2003. 439. Kim YT, Kim J, Jang YH, et al: Genetic alterations in gallbladder adenoma, dysplasia and carcinoma. Cancer Lett. 169(1):59–68, 2001. 440. Shi YZ, Hui AM, Li X, et al: Overexpression of retinoblastoma protein predicts decreased survival and correlates with loss of p16INK4 protein in gallbladder carcinomas. Clin Cancer Res. 6(10):4096–4100, 2000. 441. Quan ZW, Wu K, Wang J, et al: Association of p53, p16, and vascular endothelial growth factor protein expressions with the prognosis and metastasis of gallbladder cancer. J Am Coll Surg. 193(4):380–383, 2001. 442. Rashid A: Cellular and molecular biology of biliary tract cancers. Surg Oncol Clin N Am. 11(4):995–1009, 2002. 443. Masuhara S, Kasuya K, Aoki T, et al: Relation between K-ras codon 12 mutation and p53 protein overexpression in gallbladder cancer and biliary ductal epithelia in patients with pancreaticobiliary maljunction. Pancreat Surg. 7(2):198–205, 2000. 444. Imai M, Hoshi T, Ogawa K: K-ras codon 12 mutations in biliary tract tumors detected by polymerase chain reaction denaturing gradient gel electrophoresis. Cancer. 73(11):2727–2733, 1994. 445. Roa JC, Roa I, de Aretxabala X, et al: [K-ras gene mutation in gallbladder carcinoma]. Rev Med Chil. 132(8):955–960, 2004. 446. Ajiki T, Fujimori T, Onoyama H, et al: K-ras gene mutation in gall bladder carcinomas and dysplasia. Gut. 38(3):426–429, 1996. 447. Kim YW, Huh SH, Park YK, et al: Expression of the c-erb-B2 and p53 protein in gallbladder carcinomas. Oncol Rep. 8(5):1127– 1132, 2001. 448. Jan YY, Yeh TS, Yeh JN, et al: Expression of epidermal growth factor receptor, apomucins, matrix metalloproteinases, and p53 in rat and human cholangiocarcinoma: appraisal of an animal model of cholangiocarcinoma. Ann Surg. 240(1):89–94, 2004. 449. Dursun N, Escalona OT, Roa JC, et al: Mucinous carcinomas of the gallbladder: clinicopathologic analysis of 15 cases identified in 606 carcinomas. Arch Pathol Lab Med. 136(11):1347–1358, 2012. 450. Watanabe M, Hori Y, Nojima T, et al: Alpha-fetoproteinproducing carcinoma of the gallbladder. Dig Dis Sci. 38(3):561– 564, 1993. 451. Gakiopoulou H, Givalos N, Liapis G, et al: Hepatoid adenocarcinoma of the gallbladder. Dig Dis Sci. 52(12):3358–3362, 2007. 452. Terracciano LM, Glatz K, Mhawech P, et al: Hepatoid adenocarcinoma with liver metastasis mimicking hepatocellular carcinoma: an immunohistochemical and molecular study of eight cases. Am J Surg Pathol. 27(10):1302–1312, 2003. 453. Kim KW, Kim SH, Kim MA, et al: Adenosquamous carcinoma of the extrahepatic bile duct: clinicopathologic and radiologic features. Abdom Imaging. 2008. 454. Oohashi Y, Shirai Y, Wakai T, et al: Adenosquamous carcinoma of the gallbladder warrants resection only if curative resection is feasible. Cancer. 94(11):3000–3005, 2002. 455. Roa JC, Tapia O, Cakir A, et al: Squamous cell and adenosquamous carcinomas of the gallbladder: clinicopathological analysis
583.e11
of 34 cases identified in 606 carcinomas. Mod Pathol. 24(8):1069– 1078, 2011. 456. Chan S, Currie J, Malik AI, et al: Paediatric cholecystectomy: Shifting goalposts in the laparoscopic era. Surg Endosc. 22(5):1392–1395, 2008. 457. Albores-Saavedra J, Molberg K, Henson DE: Unusual malignant epithelial tumors of the gallbladder. Semin Diagn Pathol. 13(4):326–338, 1996. 458. Nishihara K, Tsuneyoshi M: Undifferentiated spindle cell carcinoma of the gallbladder: a clinicopathologic, immunohistochemical, and flow cytometric study of 11 cases. Hum Pathol. 24(12):1298–1305, 1993. 459. Modlin IM, Sandor A: An analysis of 8305 cases of carcinoid tumors. Cancer. 79(4):813–829, 1997. 460. Barron-Rodriguez LP, Manivel JC, Mendez-Sanchez N, et al: Carcinoid tumor of the common bile duct: evidence for its origin in metaplastic endocrine cells. Am J Gastroenterol. 86(8):1073– 1076, 1991. 461. Anjaneyulu V, Shankar-Swarnalatha G, Rao SC: Carcinoid tumor of the gall bladder. Ann Diagn Pathol. 11(2):113–116, 2007. 462. Jun SR, Lee JM, Han JK, et al: High-grade neuroendocrine carcinomas of the gallbladder and bile duct: Report of four cases with pathological correlation. J Comput Assist Tomogr. 30(4):604–609, 2006. 463. Papotti M, Cassoni P, Sapino A, et al: Large cell neuroendocrine carcinoma of the gallbladder: report of two cases. Am J Surg Pathol. 24(10):1424–1428, 2000. 464. Bal A, Joshi K, Vaiphei K, et al: Primary duodenal neoplasms: a retrospective clinico-pathological analysis. World J Gastroenterol. 13(7):1108–1111, 2007. 465. Noffsinger A: Epithelial neoplasms of the small intestine. In Odze RD, Goldblum JR, editors: Surgical Pathology of the GI Tract, Liver, Biliary Tract and Pancreas, Philadelphia, 2009, Saunders, pp 581–596. 466. Cavallini M, Cavaniglia D, Felicioni F, et al: Large periampullary villous tumor of the duodenum. J Hepatobiliary Pancreat Surg. 14(5):526–528, 2007. 467. Albores-Saavedra J, Schwartz AM, Batich K, et al: Cancers of the ampulla of vater: demographics, morphology, and survival based on 5,625 cases from the SEER program. J Surg Oncol. 100(7):598–605, 2009. 468. Takashima M, Ueki T, Nagai E, et al: Carcinoma of the ampulla of Vater associated with or without adenoma: a clinicopathologic analysis of 198 cases with reference to p53 and Ki-67 immunohistochemical expressions. Mod Pathol. 13(12):1300– 1307, 2000. 469. Ohike N, Kim GE, Tajiri T, et al: Intra-ampullary papillarytubular neoplasm (IAPN): characterization of tumoral intraepithelial neoplasia occurring within the ampulla: a clinicopathologic analysis of 82 cases. Am J Surg Pathol. 34(12):1731–1748, 2010. 470. Kimura W, Futakawa N, Yamagata S, et al: Different clinicopathologic findings in two histologic types of carcinoma of papilla of Vater. Jpn J Cancer Res. 85(2):161–166, 1994. 471. Fathallah L, Vyas P, Klimstra D, et al: MUC1 and MUC2 Expression in Carcinomas of Ampullary Region and Their Diagnostic and Clinical Significance. Mod Pathol. 15(Supp 1):284A, 2002. 472. Chu PG, Schwarz RE, Lau SK, et al: Immunohistochemical staining in the diagnosis of pancreatobiliary and ampulla of Vater adenocarcinoma: application of CDX2, CK17, MUC1, and MUC2. Am J Surg Pathol. 29(3):359–367, 2005. 473. Hansel DE, Maitra A, Lin JW, et al: Expression of the caudaltype homeodomain transcription factors CDX 1/2 and outcome in carcinomas of the ampulla of Vater. J Clin Oncol. 23(9):1811– 1818, 2005. 474. Paulsen FP, Varoga D, Paulsen AR, et al: Prognostic value of mucins in the classification of ampullary carcinomas. Hum Pathol. 37(2):160–167, 2006. 475. Zhou H, Schaefer N, Wolff M, et al: Carcinoma of the ampulla of Vater: comparative histologic/immunohistochemical classification and follow-up. Am J Surg Pathol. 28(7):875–882, 2004. 476. Sessa F, Furlan D, Zampatti C, et al: Prognostic factors for ampullary adenocarcinomas: tumor stage, tumor histology, tumor location, immunohistochemistry and microsatellite instability. Virchows Arch. 451(3):649–657, 2007.
583.e12
Immunohistology of Pancreas and Hepatobiliary Tract
477. Kamisawa T, Fukayama M, Koike M, et al: Carcinoma of the ampulla of Vater: expression of cancer-associated antigens inversely correlated with prognosis. Am J Gastroenterol. 83(10):1118–1123, 1988. 478. Scarpa A, Capelli P, Zamboni G, et al: Neoplasia of the ampulla of Vater. Ki-ras and p53 mutations. Am J Pathol. 142(4):1163– 1172, 1993. 479. McCarthy DM, Hruban RH, Argani P, et al: Role of the DPC4 tumor suppressor gene in adenocarcinoma of the ampulla of Vater: analysis of 140 cases. Mod Pathol. 16(3):272–278, 2003. 480. Howe JR, Klimstra DS, Cordon-Cardo C, et al: K-ras mutation in adenomas and carcinomas of the ampulla of vater. Clin Cancer Res. 3(1):129–133, 1997. 481. Thirabanjasak D, Tang L, Klimstra DS, et al: Glandular carcinoids of the ampulla with psammoma bodies (so-called ampullary somatostatinomas): analysis of 12 cases of an underrecognized entity. Mod Pathol. 20(1):623A, 2008. 482. Nassar H, Albores-Saavedra J, Klimstra DS: High-grade neuroendocrine carcinoma of the ampulla of vater: a clinicopathologic and immunohistochemical analysis of 14 cases. Am J Surg Pathol. 29(5):588–594, 2005. 483. Rindi G, Kloppel G, Alhman H, et al: TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 449(4):395–401, 2006. 484. Deshpande V, Chicano S, Finkelberg D, et al: Autoimmune pancreatitis: a systemic immune complex mediated disease. Am J Surg Pathol. 30(12):1537–1545, 2006. 485. Deshpande V, Mino-Kenudson M, Brugge W, et al: Autoimmune pancreatitis: more than just a pancreatic disease? A contemporary review of its pathology. Arch Pathol Lab Med. 129(9):1148– 1154, 2005. 486. Ball FM: Calcareous pancreatitis and diabetes, report of a case. J S C Med Assoc. 44(10):325–328, 1948. 487. Finkelberg DL, Sahani D, Deshpande V, et al: Autoimmune pancreatitis. N Engl J Med. 355(25):2670–2676, 2006. 488. Sarles H, Sarles JC, Camatte R, et al: Observations on 205 confirmed cases of acute pancreatitis, recurring pancreatitis, and chronic pancreatitis. Gut. 6(6):545–559, 1965. 489. Yoshida S, Todoroki T, Ichikawa Y, et al: Mutations of p16Ink4/ CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers. Cancer Res. 55(13):2756–2760, 1995. 490. Kloppel G, Sipos B, Zamboni G, et al: Autoimmune pancreatitis: histo- and immunopathological features. J Gastroenterol. 42 (Suppl 18):28–31, 2007. 491. Cornell LD, Chicano SL, Deshpande V, et al: Pseudotumors due to IgG4 immune-complex tubulointerstitial nephritis associated with autoimmune pancreatocentric disease. Am J Surg Pathol. 31(10):1586–1597, 2007. 492. Aalberse RC, Schuurman J: IgG4 breaking the rules. Immunology. 105(1):9–19, 2002. 493. Deshpande V, Sainani NI, Chung RT, et al: IgG4-associated cholangitis: a comparative histological and immunophenotypic study with primary sclerosing cholangitis on liver biopsy material. Mod Pathol. 22(10):1287–1295, 2009. 494. Wang WL, Farris AB, Lauwers GY, et al: Autoimmune pancreatitis-related cholecystitis: a morphologically and immunologically distinctive form of lymphoplasmacytic sclerosing cholecystitis. Histopathology. 54(7):829–836, 2009. 495. Geyer JT, Ferry JA, Harris NL, et al: Chronic sclerosing sialadenitis (Kuttner tumor) is an IgG4-associated disease. Am J Surg Pathol. 34(2):202–210, 2010. 496. Mino-Kenudson M, Lauwers GY: Histopathology of autoimmune pancreatitis: recognized features and unsolved issues. J Gastrointest Surg. 9(1):6–10, 2005. 497. Deshpande V, Zen Y, Chan JK, et al: Consensus statement on the pathology of IgG4-related disease. Mod Pathol. 25(9):1181– 1192, 2012. 498. Hamano H, Kawa S, Horiuchi A, et al: High serum IgG4 concentrations in patients with sclerosing pancreatitis. N Engl J Med. 344(10):732–738, 2001. 499. Okazaki K, Chiba T: [Autoimmune-related pancreatitis, autoimmune pancreatitis]. Ryoikibetsu shokogun shirizu. (31):230–235, 2000. 500. Okazaki K, Uchida K, Ohana M, et al: Autoimmune-related pancreatitis is associated with autoantibodies and a Th1/
Th2-type cellular immune response. Gastroenterology. 118(3):573–581, 2000. 501. Ito T, Nakano I, Koyanagi S, et al: Autoimmune pancreatitis as a new clinical entity. Three cases of autoimmune pancreatitis with effective steroid therapy. Dig Dis Sci. 42(7):1458–1468, 1997. 502. Deshpande SB, Hida H, Takei-Io N, et al: Involvement of nitric oxide in 3-nitropropionic acid-induced striatal toxicity in rats. Brain Res. 1108(1):205–215, 2006. 503. Uchida K, Satoi S, Miyoshi H, et al: Inflammatory pseudotumors of the pancreas and liver with infiltration of IgG4-positive plasma cells. Intern Med. 46(17):1409–1412, 2007. 504. Uchida K, Okazaki K, Asada M, et al: Case of chronic pancreatitis involving an autoimmune mechanism that extended to retroperitoneal fibrosis. Pancreas. 26(1):92–94, 2003. 505. Chutaputti A, Burrell MI, Boyer JL: Pseudotumor of the pancreas associated with retroperitoneal fibrosis: a dramatic response to corticosteroid therapy. Am J Gastroenterol. 90(7):1155–1158, 1995. 506. Mizukami H, Yajima N, Wada R, et al: Pancreatic malignant fibrous histiocytoma, inflammatory myofibroblastic tumor, and inflammatory pseudotumor related to autoimmune pancreatitis: characterization and differential diagnosis. Virchows Arch. 448(5):552–560, 2006. 507. Nonomura A, Minato H, Shimizu K, et al: Hepatic hilar inflammatory pseudotumor mimicking cholangiocarcinoma with cholangitis and phlebitis–a variant of primary sclerosing cholangitis? Pathol Res Pract. 193(7):519–525; discussion 26, 1997. 508. Kojima M, Sipos B, Klapper W, et al: Autoimmune pancreatitis: frequency, IgG4 expression, and clonality of T and B cells. Am J Surg Pathol. 31(4):521–528, 2007. 509. Chari ST, Kloeppel G, Zhang L, et al: Histopathologic and clinical subtypes of autoimmune pancreatitis: the Honolulu consensus document. Pancreas. 39(5):549–554, 2010. 510. Park DH, Kim MH, Chari ST: Recent advances in autoimmune pancreatitis. Gut. 58(12):1680–1689, 2009. 511. Zhang L, Notohara K, Levy MJ, et al: IgG4-positive plasma cell infiltration in the diagnosis of autoimmune pancreatitis. Mod Pathol. 20(1):23–28, 2007. 512. Chari ST: Diagnosis of autoimmune pancreatitis using its five cardinal features: introducing the Mayo Clinic’s HISORt criteria. J Gastroenterol. 42(Suppl 18):39–41, 2007. 513. Kwon S, Kim MH, Choi EK: The diagnostic criteria for autoimmune chronic pancreatitis: it is time to make a consensus. Pancreas. 34(3):279–286, 2007. 514. Sepehr A, Mino-Kenudson M, Ogawa F, et al: IgG4+ to IgG+ plasma cells ratio of ampulla can help differentiate autoimmune pancreatitis from other “mass forming” pancreatic lesions. Am J Surg Pathol. 32(12):1770–1779, 2008. 515. Van Eyken P, Sciot R, Desmet VJ: Immunocytochemistry of cytokeratins in primary human liver tumors. APMIS Suppl. 23:77–85, 1991. 516. Fischer HP, Altmannsberger M, Weber K, et al: Keratin polypeptides in malignant epithelial liver tumors. Differential diagnostic and histogenetic aspects. Am J Pathol. 127(3):530–537, 1987. 517. van Eyken P, Sciot R, van Damme B, et al: Keratin immunohistochemistry in normal human liver. Cytokeratin pattern of hepatocytes, bile ducts and acinar gradient. Virchows Arch A Pathol Anat Histopathol. 412(1):63–72, 1987. 518. Moll R, Franke WW, Schiller DL, et al: The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 31(1):11–24, 1982. 519. Zatloukal K, Stumptner C, Fuchsbichler A, et al: The keratin cytoskeleton in liver diseases. The diagnostic value of hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: a review. J Pathol. 204(4):367–376, 2004. 520. Suriawinata AA, Thung SN: Liver. In Mills SE, editor: Histology for Pathologists, ed 3, Philadelphia, PA, 2007, Lippincott Williams and Wilkins, pp 685–703. 521. Theise ND, Fiel IM, Hytiroglou P, et al: Macroregenerative nodules in cirrhosis are not associated with elevated serum or stainable tissue alpha-fetoprotein. Liver. 15(1):30–34, 1995. 522. Dodson A, Campbell F: Biotin inclusions: a potential pitfall in immunohistochemistry avoided. Histopathology. 34(2):178–179, 1999.
References 523. Iezzoni JC, Mills SE, Pelkey TJ, et al: Inhibin is not an immunohistochemical marker for hepatocellular carcinoma. An example of the potential pitfall in diagnostic immunohistochemistry caused by endogenous biotin. Am J Clin Pathol. 111(2):229– 234, 1999. 524. Bussolati G, Gugliotta P, Volante M, et al: Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Histopathology. 31(5):400–407, 1997. 525. Wennerberg AE, Nalesnik MA, Coleman WB: Hepatocyte paraffin 1: a monoclonal antibody that reacts with hepatocytes and can be used for differential diagnosis of hepatic tumors. Am J Pathol. 143(4):1050–1054, 1993. 526. Leong AS, Sormunen RT, Tsui WM, et al: Hep Par 1 and selected antibodies in the immunohistological distinction of hepatocellular carcinoma from cholangiocarcinoma, combined tumours and metastatic carcinoma. Histopathology. 33(4):318–324, 1998. 527. Wieczorek TJ, Pinkus JL, Glickman JN, et al: Comparison of thyroid transcription factor-1 and hepatocyte antigen immunohistochemical analysis in the differential diagnosis of hepatocellular carcinoma, metastatic adenocarcinoma, renal cell carcinoma, and adrenal cortical carcinoma. Am J Clin Pathol. 118(6):911–921, 2002. 528. Borscheri N, Roessner A, Rocken C: Canalicular immunostaining of neprilysin (CD10) as a diagnostic marker for hepatocellular carcinomas. Am J Surg Pathol. 25(10):1297–1303, 2001. 529. Lu ZH, Chen W, Ju CX, et al: CD25 Is a Novel Marker of Hepatic Bile Canaliculus. Int J Surg Pathol. 2012. 530. Lai YS, Thung SN, Gerber MA, et al: Expression of cytokeratins in normal and diseased livers and in primary liver carcinomas. Arch Pathol Lab Med. 113(2):134–138, 1989. 531. Nakajima T, Kondo Y: Well-differentiated cholangiocarcinoma: diagnostic significance of morphologic and immunohistochemical parameters. Am J Surg Pathol. 13(7):569–573, 1989. 532. Rusu MC, Pop F, Hostiuc S, et al: Extrahepatic and intrahepatic human portal interstitial Cajal cells. Anat Rec (Hoboken). 294(8):1382–1392, 2011. 533. Tiniakos DG, Lee JA, Burt AD: Innervation of the liver: morphology and function. Liver. 16(3):151–160, 1996. 534. Terayama N, Terada T, Nakanuma Y: An immunohistochemical study of tumour vessels in metastatic liver cancers and the surrounding liver tissue. Histopathology. 29(1):37–43, 1996. 535. Haratake J, Scheuer PJ: An immunohistochemical and ultrastructural study of the sinusoids of hepatocellular carcinoma. Cancer. 65(9):1985–1993, 1990. 536. Ruck P, Xiao JC, Kaiserling E: Immunoreactivity of sinusoids in hepatocellular carcinoma. An immunohistochemical study using lectin UEA-1 and antibodies against endothelial markers, including CD34. Arch Pathol Lab Med. 119(2):173–178, 1995. 537. Terayama N, Terada T, Nakanuma Y: A morphometric and immunohistochemical study on angiogenesis of human metastatic carcinomas of the liver. Hepatology. 24(4):816–819, 1996. 538. Mak KM, Chu E, Lau KH, et al: Liver Fibrosis in Elderly Cadavers: Localization of Collagen Types I, III, and IV, alpha-Smooth Muscle Actin, and Elastic Fibers. Anat Rec (Hoboken). 295(7):1159–1167, 2012. 539. Afdhal NH, Nunes D: Evaluation of liver fibrosis: a concise review. Am J Gastroenterol. 99(6):1160–1174, 2004. 540. James J, Lygidakis NJ, van Eyken P, et al: Application of keratin immunocytochemistry and sirius red staining in evaluating intrahepatic changes with acute extrahepatic cholestasis due to hepatic duct carcinoma. Hepatogastroenterology. 36(3):151–155, 1989. 541. Manning DS, Afdhal NH: Diagnosis and quantitation of fibrosis. Gastroenterology. 134(6):1670–1681, 2008. 542. Desmet VJ: Histopathology of chronic cholestasis and adult ductopenic syndrome. Clin Liver Dis. 2(2):249–264, viii, 1998. 543. Halfon P, Cacoub P: [Viral quantitation of hepatitis C virus: present and future]. Rev Med Interne. 21(2):174–181, 2000. 544. Mardini H, Record C: Detection assessment and monitoring of hepatic fibrosis: biochemistry or biopsy? Ann Clin Biochem. 42(Pt 6):441–447, 2005. 545. Lockwood DS, Yeadon TM, Clouston AD, et al: Tumor progression in hepatocellular carcinoma: relationship with tumor stroma and parenchymal disease. J Gastroenterol Hepatol. 18(6):666–672, 2003.
583.e13
546. Gulubova M: Immunohistochemical localization of collagen type III and type IV, laminin, tenascin and alpha-smooth muscle actin (alphaSMA) in the human liver in peliosis. Pathol Res Pract. 198(12):803–812, 2002. 547. Torimura T, Ueno T, Inuzuka S, et al: The extracellular matrix in hepatocellular carcinoma shows different localization patterns depending on the differentiation and the histological pattern of tumors: immunohistochemical analysis. J Hepatol. 21(1):37–46, 1994. 548. Schuppan D: Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis. 10(1):1–10, 1990. 549. Schuppan D, Ruehl M, Somasundaram R, et al: Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis. 21(3):351– 372, 2001. 550. Schuppan D, Porov Y: Hepatic fibrosis: from bench to bedside. J Gastroenterol Hepatol. 17(Suppl 3):S300–S305, 2002. 551. Bataller R, Brenner DA: Liver fibrosis. J Clin Invest. 115(2):209– 218, 2005. 552. Friedman SL: Mechanisms of hepatic fibrogenesis. Gastroenterology. 134(6):1655–1669, 2008. 553. Uyama N, Iimuro Y, Kawada N, et al: Fascin, a novel marker of human hepatic stellate cells, may regulate their proliferation, migration, and collagen gene expression through the FAK-PI3KAkt pathway. Lab Invest. 92(1):57–71, 2012. 554. Knittel T, Aurisch S, Neubauer K, et al: Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair. Am J Pathol. 149(2):449–462, 1996. 555. Neubauer K, Knittel T, Aurisch S, et al: Glial fibrillary acidic protein–a cell type specific marker for Ito cells in vivo and in vitro. J Hepatol. 24(6):719–730, 1996. 556. Cassiman D, van Pelt J, De Vos R, et al: Synaptophysin: A novel marker for human and rat hepatic stellate cells. Am J Pathol. 155(6):1831–1839, 1999. 557. Ogawa K, Suzuki J, Mukai H, et al: Sequential changes of extracellular matrix and proliferation of Ito cells with enhanced expression of desmin and actin in focal hepatic injury. Am J Pathol. 125(3):611–619, 1986. 558. Abdeen SM, Olusi SO: Peptidyl arginine deiminase: A novel immunohistochemical marker for liver fibrosis in patients with chronic hepatitis. Acta Histochem. 112(6):592–603, 2010. 559. Soderberg C, Marmur J, Eckes K, et al: Microvesicular fat, inter cellular adhesion molecule-1 and regulatory T-lymphocytes are of importance for the inflammatory process in livers with nonalcoholic steatohepatitis. Apmis. 119(7):412–420, 2011. 560. Stanton MC, Chen SC, Jackson JV, et al: Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice. J Inflamm (Lond). 8:8, 2011. 561. Zatloukal K, French SW, Stumptner C, et al: From Mallory to Mallory-Denk bodies: what, how and why? Exp Cell Res. 313(10):2033–2049, 2007. 562. Snover D: Immunohistochemical analysis in steatohepatitis: does it have a role in diagnosis and management? Am J Clin Pathol. 123(4):491–493, 2005. 563. Van Eyken P, Sciot R, Desmet VJ: A cytokeratin immunohistochemical study of alcoholic liver disease: evidence that hepatocytes can express ‘bile duct-type’ cytokeratins. Histopathology. 13(6):605–617, 1988. 564. Hazan R, Denk H, Franke WW, et al: Change of cytokeratin organization during development of Mallory bodies as revealed by a monoclonal antibody. Lab Invest. 54(5):543–553, 1986. 565. Nobili V, Carpino G, Alisi A, et al: Hepatic progenitor cells activation, fibrosis and adipokines production in pediatric nonalcoholic fatty liver disease. Hepatology. 2012. 566. Van Eyken P, Sciot R, Desmet VJ: A cytokeratin immunohistochemical study of cholestatic liver disease: evidence that hepatocytes can express “bile duct-type” cytokeratins. Histopathology. 15(2):125–135, 1989. 567. van Eyken P, Sciot R, Callea F, et al: A cytokeratinimmunohistochemical study of focal nodular hyperplasia of the liver: further evidence that ductular metaplasia of hepatocytes contributes to ductular “proliferation”. Liver. 9(6):372–377, 1989.
583.e14
Immunohistology of Pancreas and Hepatobiliary Tract
568. Cabibi D, Licata A, Barresi E, et al: Expression of cytokeratin 7 and 20 in pathological conditions of the bile tract. Pathol Res Pract. 199(2):65–70, 2003. 569. Nakanuma Y, Ohta G: Immunohistochemical study on bile ductular proliferation in various hepatobiliary diseases. Liver. 6(4):205–211, 1986. 570. Bateman AC, Hubscher SG: Cytokeratin expression as an aid to diagnosis in medical liver biopsies. Histopathology. 56(4):415– 425, 2010. 571. Turanyi E, Dezso K, Csomor J, et al: Immunohistochemical classification of ductular reactions in human liver. Histopathology. 57(4):607–614, 2010. 572. Washington K, Clavien PA, Killenberg P: Peribiliary vascular plexus in primary sclerosing cholangitis and primary biliary cirrhosis. Hum Pathol. 28(7):791–795, 1997. 573. Vertemati M, Minola E, Goffredi M, et al: Computerized morphometry of the cirrhotic liver: comparative analysis in primary biliary cirrhosis, alcoholic cirrhosis, and posthepatitic cirrhosis. Microsc Res Tech. 65(3):113–121, 2004. 574. Kobayashi S, Nakanuma Y, Matsui O: Intrahepatic peribiliary vascular plexus in various hepatobiliary diseases: a histological survey. Hum Pathol. 25(9):940–946, 1994. 575. Moreira RK, Lee H, Stapp R, et al: Immunohistochemical staining of inflammatory cells in liver biopsy specimens of patients with autoimmune hepatitis, primary biliary cirrhosis, and overlap syndromes. Am J Clin Pathol. 134(5):852–853, 2010. 576. Moreira RK, Revetta F, Koehler E, et al: Diagnostic utility of IgG and IgM immunohistochemistry in autoimmune liver disease. World J Gastroenterol. 16(4):453–457, 2010. 577. Lee H, Stapp RT, Ormsby AH, et al: The usefulness of IgG and IgM immunostaining of periportal inflammatory cells (plasma cells and lymphocytes) for the distinction of autoimmune hepatitis and primary biliary cirrhosis and their staining pattern in autoimmune hepatitis-primary biliary cirrhosis overlap syndrome. Am J Clin Pathol. 133(3):430–437, 2010. 578. Tsuda M, Ambrosini YM, Zhang W, et al: Fine phenotypic and functional characterization of effector cluster of differentiation 8 positive T cells in human patients with primary biliary cirrhosis. Hepatology. 54(4):1293–1302, 2011. 579. Spee B, Carpino G, Schotanus BA, et al: Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling. Gut. 59(2):247–257, 2010. 580. Daniels JA, Torbenson M, Anders RA, et al: Immunostaining of plasma cells in primary biliary cirrhosis. Am J Clin Pathol. 131(2):243–249, 2009. 581. Umemura T, Zen Y, Hamano H, et al: Immunoglobin G4-hepatopathy: association of immunoglobin G4-bearing plasma cells in liver with autoimmune pancreatitis. Hepatology. 46(2):463–471, 2007. 582. Umemura T, Zen Y, Hamano H, et al: IgG4 associated autoimmune hepatitis: a differential diagnosis for classical autoimmune hepatitis. Gut. 56(10):1471–1472, 2007. 583. Zen Y, Fujii T, Sato Y, et al: Pathological classification of hepatic inflammatory pseudotumor with respect to IgG4-related disease. Mod Pathol. 20(8):884–894, 2007. 584. Zen Y, Fujii T, Harada K, et al: Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis and cholangitis. Hepatology. 45(6):1538–1546, 2007. 585. Lefkowitch JH: Hepatobiliary pathology. Curr Opin Gastroenterol. 24(3):269–277, 2008. 586. Bjornsson E, Chari ST, Smyrk TC, et al: Immunoglobulin G4 associated cholangitis: description of an emerging clinical entity based on review of the literature. Hepatology. 45(6):1547–1554, 2007. 587. Zhang L, Lewis JT, Abraham SC, et al: IgG4+ plasma cell infiltrates in liver explants with primary sclerosing cholangitis. Am J Surg Pathol. 34(1):88–94, 2010. 588. Koyabu M, Uchida K, Miyoshi H, et al: Analysis of regulatory T cells and IgG4-positive plasma cells among patients of IgG4related sclerosing cholangitis and autoimmune liver diseases. J Gastroenterol. 45(7):732–741, 2010. 589. Topazian M, Witzig TE, Smyrk TC, et al: Rituximab therapy for refractory biliary strictures in immunoglobulin G4-associated cholangitis. Clin Gastroenterol Hepatol. 6(3):364–366, 2008.
590. Osterreicher CH, Penz-Osterreicher M, Grivennikov SI, et al: Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver. Proc Natl Acad Sci U S A. 108(1):308–313, 2011. 591. Rygiel KA, Robertson H, Marshall HL, et al: Epithelialmesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease. Lab Invest. 88(2):112–123, 2008. 592. Rygiel KA, Robertson H, Willet JD, et al: T cell-mediated biliary epithelial-to-mesenchymal transition in liver allograft rejection. Liver Transpl. 16(5):567–576, 2010. 593. Lau SK, Prakash S, Geller SA, et al: Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma, and metastatic adenocarcinoma. Hum Pathol. 33(12):1175–1181, 2002. 594. Hurlimann J, Gardiol D: Immunohistochemistry in the differential diagnosis of liver carcinomas. Am J Surg Pathol. 15(3):280– 288, 1991. 595. Sell S, Leffert HL: Liver cancer stem cells. J Clin Oncol. 26(17):2800–2805, 2008. 596. Kamiya A, Gonzalez FJ, Nakauchi H: Identification and differentiation of hepatic stem cells during liver development. Front Biosci. 11:1302–1310, 2006. 597. Gerber MA, Thung SN: Liver stem cells and development. Lab Invest. 68(3):253–254, 1993. 598. Sell S: Is there a liver stem cell? Cancer Res. 50(13):3811–3815, 1990. 599. Goldstein NS, Blue DE, Hankin R, et al: Serum alpha-fetoprotein levels in patients with chronic hepatitis C. Relationships with serum alanine aminotransferase values, histologic activity index, and hepatocyte MIB-1 scores. Am J Clin Pathol. 111(6):811– 816, 1999. 600. Davies SE, Portmann BC, O’Grady JG, et al: Hepatic histological findings after transplantation for chronic hepatitis B virus infection, including a unique pattern of fibrosing cholestatic hepatitis. Hepatology. 13(1):150–157, 1991. 601. Pan JJ, Oh SH, Soldevila-Pico C, et al: Low prevalence of HBV DNA in the liver allograft from anti-HBc-positive donors: a single-center experience. Clin Transplant. 25(1):164–170, 2011. 602. Ray MB, Desmet VJ, Fevery J, et al: Hepatitis B surface antigen (HBsAg) in the liver of patients with hepatitis; a comparison with serological detection. J Clin Pathol. 29(2):89–93, 1976. 603. Callea F: Immunohistochemical techniques for the demonstration of viral antigens in liver tissue. Ric Clin Lab. 18(2-3):223– 231, 1988. 604. Roskams T: The role of immunohistochemistry in diagnosis. Clin Liver Dis. 6(2):571–589, x, 2002. 605. Booth JC, Goldin RD, Brown JL, et al: Fibrosing cholestatic hepatitis in a renal transplant recipient associated with the hepatitis B virus precore mutant. J Hepatol. 22(4):500–503, 1995. 606. Chen CH, Chen PJ, Chu JS, et al: Fibrosing cholestatic hepatitis in a hepatitis B surface antigen carrier after renal transplantation. Gastroenterology. 107(5):1514–1518, 1994. 607. Fang JW, Wright TL, Lau JY: Fibrosing cholestatic hepatitis in patient with HIV and hepatitis B. Lancet. 342(8880):1175, 1993. 608. Fischer HP, Willsch E, Bierhoff E, et al: Histopathologic findings in chronic hepatitis C. J Hepatol. 24(2 Suppl):35–42, 1996. 609. Kasprzak A, Biczysko W, Adamek A, et al: Morphological lesions detected by light and electron microscopies in chronic type B hepatitis. Pol J Pathol. 53(3):103–115, 2002. 610. Sansonno D, Iacobelli AR, Cornacchiulo V, et al: Immunohistochemical detection of hepatitis C virus-related proteins in liver tissue. Clin Exp Rheumatol. 13(Suppl 13):S29–S32, 1995. 611. Lau JY, Krawczynski K, Negro F, et al: In situ detection of hepatitis C virus–a critical appraisal. J Hepatol. 24(2 Suppl):43–51, 1996. 612. Scheuer PJ, Krawczynski K, Dhillon AP: Histopathology and detection of hepatitis C virus in liver. Springer Semin Immunopathol. 19(1):27–45, 1997. 613. Gowans EJ: Distribution of markers of hepatitis C virus infection throughout the body. Semin Liver Dis. 20(1):85–102, 2000. 614. Brody RI, Eng S, Melamed J, et al: Immunohistochemical detection of hepatitis C antigen by monoclonal antibody TORDJI-22
References compared with PCR viral detection. Am J Clin Pathol. 110(1):32– 37, 1998. 615. Uchida T, Shikata T, Tanaka E, et al: Immunoperoxidase staining of hepatitis C virus in formalin-fixed, paraffin-embedded needle liver biopsies. Virchows Arch. 424(5):465–469, 1994. 616. Li T, Li D, Cheng L, et al: Epithelial-mesenchymal transition induced by hepatitis C virus core protein in cholangiocarcinoma. Ann Surg Oncol. 17(7):1937–1944, 2010. 617. Sturm N, Thelu MA, Camous X, et al: Characterization and role of intra-hepatic regulatory T cells in chronic hepatitis C pathogenesis. J Hepatol. 53(1):25–35, 2010. 618. Negro F, Pacchioni D, Mondardini A, et al: In situ hybridization in viral hepatitis. Liver. 12(4 Pt 2):217–226, 1992. 619. Negro F, Papotti M, Taraglio S, et al: Relationship between hepatocyte proliferation and hepatitis delta virus replication in neoplastic and non-neoplastic liver tissues. J Viral Hepat. 4(2):93–98, 1997. 620. Raymond E, Tricottet V, Samuel D, et al: Epstein-Barr virusrelated localized hepatic lymphoproliferative disorders after liver transplantation. Cancer. 76(8):1344–1351, 1995. 621. Chen CJ, Jeng LB, Huang SF: Lymphoepithelioma-like hepatocellular carcinoma. Chang Gung Med J. 30(2):172–177, 2007. 622. Chen TC, Ng KF, Kuo T: Intrahepatic cholangiocarcinoma with lymphoepithelioma-like component. Mod Pathol. 14(5):527– 532, 2001. 623. Vortmeyer AO, Kingma DW, Fenton RG, et al: Hepatobiliary lymphoepithelioma-like carcinoma associated with Epstein-Barr virus. Am J Clin Pathol. 109(1):90–95, 1998. 624. Vanstapel MJ, Desmet VJ: Cytomegalovirus hepatitis: a histological and immunohistochemical study. Appl Pathol. 1(1):41– 49, 1983. 625. Lautenschlager I, Halme L, Hockerstedt K, et al: Cytomegalovirus infection of the liver transplant: virological, histological, immunological, and clinical observations. Transpl Infect Dis. 8(1):21–30, 2006. 626. Humphrey DM, Weiner MH: Mycobacterial antigen detection by immunohistochemistry in pulmonary tuberculosis. Hum Pathol. 18(7):701–708, 1987. 627. Saad RS, Demetris AJ, Lee RG, et al: Adenovirus hepatitis in the adult allograft liver. Transplantation. 64(10):1483–1485, 1997. 628. Makhlouf HR, Abdul-Al HM, Goodman ZD: Diagnosis of focal nodular hyperplasia of the liver by needle biopsy. Hum Pathol. 36(11):1210–1216, 2005. 629. Nguyen BN, Flejou JF, Terris B, et al: Focal nodular hyperplasia of the liver: a comprehensive pathologic study of 305 lesions and recognition of new histologic forms. Am J Surg Pathol. 23(12):1441–1454, 1999. 630. Ferrell LD: Chapter 47: Benign and Malignant Tumors of the Liver. In Odze RD, Goldblum JR, editors: Surgical Pathology of the GI Tract, Liver, Biliary Tract, and Pancrea, Philadelphia, PA, 2009, Saunders, an imprint of Elsevier Inc.. 631. Shafizadeh N, Kakar S: Diagnosis of well-differentiated hepatocellular lesions: role of immunohistochemistry and other ancillary techniques. Adv Anat Pathol. 18(6):438–445, 2011. 632. Bioulac-Sage P, Laumonier H, Cubel G, et al: Hepatic resection for inflammatory hepatocellular adenomas: pathological identification of micronodules expressing inflammatory proteins. Liver Int. 30(1):149–154, 2010. 633. Malone M, Mieli-Vergani G, Mowat AP, et al: The fetal liver in PiZZ alpha-1-antitrypsin deficiency: a report of five cases. Pediatr Pathol. 9(6):623–631, 1989. 634. Fairbanks KD, Tavill AS: Liver disease in alpha 1-antitrypsin deficiency: a review. Am J Gastroenterol. 103(8):2136–2141; quiz 42, 2008. 635. Portmann BC, Thompson RJ, Roberts EA, et al: Genetic and metabolic liver disease. In Burt AD, Portmann BC, Ferrell LD, editors: MacSween’s Pathology of the Liver, ed 5, Philadelphia, PA, 2007, Churchill Livingstone Elsevier, pp 199–326. 636. Medicina D, Fabbretti G, Brennan SO, et al: Genetic and immunological characterization of fibrinogen inclusion bodies in patients with hepatic fibrinogen storage and liver disease. Ann N Y Acad Sci. 936:522–525, 2001. 637. Demetris AJ, Adeyi O, Bellamy CO, et al: Liver biopsy interpretation for causes of late liver allograft dysfunction. Hepatology. 44(2):489–501, 2006.
583.e15
638. Haga H, Egawa H, Fujimoto Y, et al: Acute humoral rejection and C4d immunostaining in ABO blood type-incompatible liver transplantation. Liver Transpl. 12(3):457–464, 2006. 639. Colvin RB: C4d in liver allografts: a sign of antibody-mediated rejection? Am J Transplant. 6(3):447–448, 2006. 640. Schmeding M, Dankof A, Krenn V, et al: C4d in acute rejection after liver transplantation–a valuable tool in differential diagnosis to hepatitis C recurrence. Am J Transplant. 6(3):523–530, 2006. 641. Bu X, Zheng Z, Yu Y, et al: Significance of C4d deposition in the diagnosis of rejection after liver transplantation. Transplant Proc. 38(5):1418–1421, 2006. 642. Lorho R, Turlin B, Aqodad N, et al: C4d: a marker for hepatic transplant rejection. Transplant Proc. 38(7):2333–2334, 2006. 643. Troxell ML, Higgins JP, Kambham N: Evaluation of C4d staining in liver and small intestine allografts. Arch Pathol Lab Med. 130(10):1489–1496, 2006. 644. Jain A, Ryan C, Mohanka R, et al: Characterization of CD4, CD8, CD56 positive lymphocytes and C4d deposits to distinguish acute cellular rejection from recurrent hepatitis C in postliver transplant biopsies. Clin Transplant. 20(5):624–633, 2006. 645. Bellamy CO, Herriot MM, Harrison DJ, et al: C4d immunopositivity is uncommon in ABO-compatible liver allografts, but correlates partially with lymphocytotoxic antibody status. Histopathology. 50(6):739–749, 2007. 646. Sakashita H, Haga H, Ashihara E, et al: Significance of C4d staining in ABO-identical/compatible liver transplantation. Mod Pathol. 20(6):676–684, 2007. 647. Terasaki PI: Deduction of the fraction of immunologic and nonimmunologic failure in cadaver donor transplants. Clinical transplants. 449–452, 2003. 648. Dankof A, Schmeding M, Morawietz L, et al: Portal capillary C4d deposits and increased infiltration by macrophages indicate humorally mediated mechanisms in acute cellular liver allograft rejection. Virchows Arch. 447(1):87–93, 2005. 649. Michaels PJ, Fishbein MC, Colvin RB: Humoral rejection of human organ transplants. Springer Semin Immunopathol. 25(2):119–140, 2003. 650. Knechtle SJ: Guidance for liver transplant immunosuppression. Am J Transplant. 11(5):886–887, 2011. 651. Musat AI, Agni RM, Wai PY, et al: The significance of donorspecific HLA antibodies in rejection and ductopenia development in ABO compatible liver transplantation. Am J Transplant. 11(3):500–510, 2011. 652. Sawada T, Shimizu A, Kubota K, et al: Lobular damage caused by cellular and humoral immunity in liver allograft rejection. Clin Transplant. 19(1):110–114, 2005. 653. Kozlowski T, Andreoni K, Schmitz J, et al: Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: Diagnostic considerations in the evaluation of liver allografts. Liver Transpl. 18(6):641–658, 2012. 654. Krukemeyer MG, Moeller J, Morawietz L, et al: Description of B lymphocytes and plasma cells, complement, and chemokines/ receptors in acute liver allograft rejection. Transplantation. 78(1):65–70, 2004. 655. Watson R, Kozlowski T, Nickeleit V, et al: Isolated donor specific alloantibody-mediated rejection after ABO compatible liver transplantation. Am J Transplant. 6(12):3022–3029, 2006. 656. Drachenberg CB, Torrealba JR, Nankivell BJ, et al: Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema. Am J Transplant. 11(9):1792–1802, 2011. 657. MacQuillan GC, de Boer WB, Allan JE, et al: Hepatocellular MxA protein expression supports the differentiation of recurrent hepatitis C disease from acute cellular rejection after liver transplantation. Clin Transplant. 24(2):252–258, 2010. 658. Pai RK, Hart JA: Aberrant expression of cytokeratin 7 in perivenular hepatocytes correlates with a cholestatic chemistry profile in patients with heart failure. Mod Pathol. 23(12):1650– 1656, 2010. 659. Roels F, Espeel M, De Craemer D: Liver pathology and immunocytochemistry in congenital peroxisomal diseases: a review. J Inherit Metab Dis. 14(6):853–875, 1991. 660. Yu S, Wang F, Zhang K, et al: Immunocytochemical and immunohistochemical application of monoclonal antibodies against
583.e16
Immunohistology of Pancreas and Hepatobiliary Tract
peroxisomal biogenesis factor 14. Hybridoma (Larchmt). 31(2):142–145, 2012. 661. Depreter M, Espeel M, Roels F: Human peroxisomal disorders. Microsc Res Tech. 61(2):203–223, 2003. 662. Rubbia-Brandt L, Neerman-Arbez M, Rougemont AL, et al: Fibrinogen gamma375 arg–>trp mutation (fibrinogen aguadilla) causes hereditary hypofibrinogenemia, hepatic endoplasmic reticulum storage disease and cirrhosis. Am J Surg Pathol. 30(7):906–911, 2006. 663. Chan ES, Yeh MM: The use of immunohistochemistry in liver tumors. Clin Liver Dis. 14(4):687–703, 2010. 664. Geller SA, Dhall D, Alsabeh R: Application of immunohistochemistry to liver and gastrointestinal neoplasms: liver, stomach, colon, and pancreas. Arch Pathol Lab Med. 132(3):490–499, 2008. 665. Kakar S, Gown AM, Goodman ZD, et al: Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus metastatic neoplasms. Arch Pathol Lab Med. 131(11):1648– 1654, 2007. 666. Varma V, Cohen C: Immunohistochemical and molecular markers in the diagnosis of hepatocellular carcinoma. Adv Anat Pathol. 11(5):239–249, 2004. 667. Hirohashi S, Ishak KG, Kojiro M, et al: Hepatocellular carcinoma. In Hamilton SR, Aaltonen LA, editors: Tumours of the Digestive System, Lyon, 2000, International Agency for Research on Cancer (IARC), pp 159–172. 668. Ronot M, Bahrami S, Calderaro J, et al: Hepatocellular adenomas: accuracy of magnetic resonance imaging and liver biopsy in subtype classification. Hepatology. 53(4):1182–1191, 2011. 669. Zucman-Rossi J, Jeannot E, Nhieu JT, et al: Genotype-phenotype correlation in hepatocellular adenoma: new classification and relationship with HCC. Hepatology. 43(3):515–524, 2006. 670. Bioulac-Sage P, Balabaud C, Bedossa P, et al: Pathological diagnosis of liver cell adenoma and focal nodular hyperplasia: Bordeaux update. J Hepatol. 46(3):521–527, 2007. 671. Huang H, Fujii H, Sankila A, et al: Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection. Am J Pathol. 155(6):1795–1801, 1999. 672. Libbrecht L, Severi T, Cassiman D, et al: Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules. Am J Surg Pathol. 30(11):1405–1411, 2006. 673. Shafizadeh N, Ferrell LD, Kakar S: Utility and limitations of glypican-3 expression for the diagnosis of hepatocellular carcinoma at both ends of the differentiation spectrum. Mod Pathol. 21(8):1011–1018, 2008. 674. Coston WM, Loera S, Lau SK, et al: Distinction of hepatocellular carcinoma from benign hepatic mimickers using Glypican-3 and CD34 immunohistochemistry. Am J Surg Pathol. 32(3):433– 444, 2008. 675. Roncalli M, Borzio M, Di Tommaso L: Hepatocellular dysplastic nodules. Hepatol Res. 37(Suppl 2):S125–S134, 2007. 676. Roncalli M, Borzio M, Di Tommaso L: Hepatocellular dysplastic nodules. Ann Ital Chir. 79(2):81–89, 2008. 677. Roskams T, Kojiro M: Pathology of early hepatocellular carcinoma: conventional and molecular diagnosis. Semin Liver Dis. 30(1):17–25, 2010. 678. Sakamoto M, Mori T, Masugi Y, et al: Candidate molecular markers for histological diagnosis of early hepatocellular carcinoma. Intervirology. 51(Suppl 1):42–45, 2008. 679. Walther Z, Jain D: Molecular pathology of hepatic neoplasms: classification and clinical significance. Patholog Res Int. 2011:403929, 2011. 680. Bioulac-Sage P, Balabaud C, Zucman-Rossi J: Focal nodular hyperplasia, hepatocellular adenomas: past, present, future. Gastroenterol Clin Biol. 34(6-7):355–358, 2010. 681. Jeannot E, Poussin K, Chiche L, et al: Association of CYP1B1 germ line mutations with hepatocyte nuclear factor 1alphamutated hepatocellular adenoma. Cancer Res. 67(6):2611–2616, 2007. 682. Rebouissou S, Imbeaud S, Balabaud C, et al: HNF1alpha inactivation promotes lipogenesis in human hepatocellular adenoma independently of SREBP-1 and carbohydrateresponse element-binding protein (ChREBP) activation. J Biol Chem. 282(19):14437–14446, 2007.
683. Bacq Y, Jacquemin E, Balabaud C, et al: Familial liver adenomatosis associated with hepatocyte nuclear factor 1alpha inactivation. Gastroenterology. 125(5):1470–1475, 2003. 684. van Aalten SM, Verheij J, Terkivatan T, et al: Validation of a liver adenoma classification system in a tertiary referral centre: implications for clinical practice. J Hepatol. 55(1):120–125, 2011. 685. Moll R, Divo M, Langbein L: The human keratins: biology and pathology. Histochem Cell Biol. 129(6):705–733, 2008. 686. Strnad P, Stumptner C, Zatloukal K, et al: Intermediate filament cytoskeleton of the liver in health and disease. Histochem Cell Biol. 129(6):735–749, 2008. 687. Abdul-Al HM, Wang G, Makhlouf HR, et al: Fibrolamellar hepatocellular carcinoma: an immunohistochemical comparison with conventional hepatocellular carcinoma. Int J Surg Pathol. 18(5):313–318, 2010. 688. Yoneda N, Sato Y, Kitao A, et al: Epidermal growth factor induces cytokeratin 19 expression accompanied by increased growth abilities in human hepatocellular carcinoma. Lab Invest. 91(2):262–272, 2011. 689. Yuan RH, Jeng YM, Hu RH, et al: Role of p53 and beta-catenin mutations in conjunction with CK19 expression on early tumor recurrence and prognosis of hepatocellular carcinoma. J Gastrointest Surg. 15(2):321–329, 2011. 690. Rishi M, Kovatich A, Ehya H: Utility of polyclonal and monoclonal antibodies against carcinoembryonic antigen in hepatic fine-needle aspirates. Diagn Cytopathol. 11(4):358–361; discussion 61–62, 1994. 691. Wang L, Vuolo M, Suhrland MJ, et al: HepPar1, MOC-31, pCEA, mCEA and CD10 for distinguishing hepatocellular carcinoma vs. metastatic adenocarcinoma in liver fine needle aspirates. Acta Cytol. 50(3):257–262, 2006. 692. Wang FH, Yip YC, Zhang M, et al: Diagnostic utility of glypican-3 for hepatocellular carcinoma on liver needle biopsy. J Clin Pathol. 63(7):599–603, 2010. 693. Llovet JM, Chen Y, Wurmbach E, et al: A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology. 131(6):1758–1767, 2006. 694. Wang XY, Degos F, Dubois S, et al: Glypican-3 expression in hepatocellular tumors: diagnostic value for preneoplastic lesions and hepatocellular carcinomas. Hum Pathol. 37(11):1435–1441, 2006. 695. Yamauchi N, Watanabe A, Hishinuma M, et al: The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol. 18(12):1591–1598, 2005. 696. Yao M, Yao DF, Bian YZ, et al: Oncofetal antigen glypican-3 as a promising early diagnostic marker for hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int. 10(3):289–294, 2011. 697. Yasuda E, Kumada T, Toyoda H, et al: Evaluation for clinical utility of GPC3, measured by a commercially available ELISA kit with Glypican-3 (GPC3) antibody, as a serological and histological marker for hepatocellular carcinoma. Hepatol Res. 40(5):477–485, 2010. 698. Yorita K, Takahashi N, Takai H, et al: Prognostic significance of circumferential cell surface immunoreactivity of glypican-3 in hepatocellular carcinoma. Liver Int. 31(1):120–131, 2011. 699. Ning S, Bin C, Na H, et al: Glypican-3, a novel prognostic marker of hepatocellular cancer, is related with postoperative metastasis and recurrence in hepatocellular cancer patients. Mol Biol Rep. 39(1):351–357, 2012. 700. Yan B, Wei JJ, Qian YM, et al: Expression and clinicopathologic significance of glypican 3 in hepatocellular carcinoma. Ann Diagn Pathol. 15(3):162–169, 2011. 701. McKnight R, Nassar A, Cohen C, et al: Arginase-1: A novel immunohistochemical marker of hepatocellular differentiation in fine needle aspiration cytology. Cancer Cytopathol. 2012. 702. Krishna M, Lloyd RV, Batts KP: Detection of albumin messenger RNA in hepatic and extrahepatic neoplasms. A marker of hepatocellular differentiation. Am J Surg Pathol. 21(2):147–152, 1997. 703. Ruck P, Xiao JC, Pietsch T, et al: Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6. Histopathology. 31(4):324–329, 1997. 704. D’Errico A, Deleonardi G, Fiorentino M, et al: Diagnostic implications of albumin messenger RNA detection and cytokeratin
References pattern in benign hepatic lesions and biliary cystadenocarcinoma. Diagn Mol Pathol. 7(6):289–294, 1998. 705. Papotti M, Pacchioni D, Negro F, et al: Albumin gene expression in liver tumors: diagnostic interest in fine needle aspiration biopsies. Mod Pathol. 7(3):271–275, 1994. 706. Papotti M, Sambataro D, Marchesa P, et al: A combined hepatocellular/cholangiocellular carcinoma with sarcomatoid features. Liver. 17(1):47–52, 1997. 707. Kakar S, Muir T, Murphy LM, et al: Immunoreactivity of Hep Par 1 in hepatic and extrahepatic tumors and its correlation with albumin in situ hybridization in hepatocellular carcinoma. Am J Clin Pathol. 119(3):361–366, 2003. 708. Oliveira AM, Erickson LA, Burgart LJ, et al: Differentiation of primary and metastatic clear cell tumors in the liver by in situ hybridization for albumin messenger RNA. Am J Surg Pathol. 24(2):177–182, 2000. 709. Supriatna Y, Kishimoto T, Uno T, et al: Evidence for hepatocellular differentiation in alpha-fetoprotein-negative gastric adenocarcinoma with hepatoid morphology: a study with in situ hybridisation for albumin mRNA. Pathology. 37(3):211–215, 2005. 710. Lopez-Beltran A, Luque RJ, Quintero A, et al: Hepatoid adenocarcinoma of the urinary bladder. Virchows Arch. 442(4):381– 387, 2003. 711. Tanigawa H, Kida Y, Kuwao S, et al: Hepatoid adenocarcinoma in Barrett’s esophagus associated with achalasia: first case report. Pathol Int. 52(2):141–146, 2002. 712. Roberts CC, Colby TV, Batts KP: Carcinoma of the stomach with hepatocyte differentiation (hepatoid adenocarcinoma). Mayo Clin Proc. 72(12):1154–1160, 1997. 713. Minervini MI, Demetris AJ, Lee RG, et al: Utilization of hepatocyte-specific antibody in the immunocytochemical evaluation of liver tumors. Mod Pathol. 10(7):686–692, 1997. 714. Murakata LA, Ishak KG, Nzeako UC: Clear cell carcinoma of the liver: a comparative immunohistochemical study with renal clear cell carcinoma. Mod Pathol. 13(8):874–881, 2000. 715. Lazaro J, Rubio D, Repolles M, et al: Hepatoid carcinoma of the ovary and management. Acta Obstet Gynecol Scand. 86(4):498– 499, 2007. 716. Hameed O, Xu H, Saddeghi S, et al: Hepatoid carcinoma of the pancreas: a case report and literature review of a heterogeneous group of tumors. Am J Surg Pathol. 31(1):146–152, 2007. 717. Yigit S, Uyaroglu MA, Kus Z, et al: Hepatoid carcinoma of the ovary: immunohistochemical finding of one case and literature review. Int J Gynecol Cancer. 16(3):1439–1441, 2006. 718. Pan CC, Chen PC, Tsay SH, et al: Cytoplasmic immunoreactivity for thyroid transcription factor-1 in hepatocellular carcinoma: a comparative immunohistochemical analysis of four commercial antibodies using a tissue array technique. Am J Clin Pathol. 121(3):343–349, 2004. 719. Lei JY, Bourne PA, diSant’Agnese PA, et al: Cytoplasmic staining of TTF-1 in the differential diagnosis of hepatocellular carcinoma vs cholangiocarcinoma and metastatic carcinoma of the liver. Am J Clin Pathol. 125(4):519–525, 2006. 720. Gaffey MJ, Traweek ST, Mills SE, et al: Cytokeratin expression in adrenocortical neoplasia: an immunohistochemical and biochemical study with implications for the differential diagnosis of adrenocortical, hepatocellular, and renal cell carcinoma. Hum Pathol. 23(2):144–153, 1992. 721. Tsuji M, Kashihara T, Terada N, et al: An immunohistochemical study of hepatic atypical adenomatous hyperplasia, hepatocellular carcinoma, and cholangiocarcinoma with alpha-fetoprotein, carcinoembryonic antigen, CA19-9, epithelial membrane antigen, and cytokeratins 18 and 19. Pathol Int. 49(4):310–317, 1999. 722. Pinkus GS, Kurtin PJ: Epithelial membrane antigen–a diagnostic discriminant in surgical pathology: immunohistochemical profile in epithelial, mesenchymal, and hematopoietic neoplasms using paraffin sections and monoclonal antibodies. Hum Pathol. 16(9):929–940, 1985. 723. Christensen WN, Boitnott JK, Kuhajda FP: Immunoperoxidase staining as a diagnostic aid for hepatocellular carcinoma. Mod Pathol. 2(1):8–12, 1989. 724. Morrison C, Marsh W, Jr, Frankel WL: A comparison of CD10 to pCEA, MOC-31, and hepatocyte for the distinction of malignant tumors in the liver. Mod Pathol. 15(12):1279–1287, 2002.
583.e17
725. Proca DM, Niemann TH, Porcell AI, et al: MOC31 immunoreactivity in primary and metastatic carcinoma of the liver. Report of findings and review of other utilized markers. Appl Immunohistochem Mol Morphol. 8(2):120–125, 2000. 726. Niemann TH, Hughes JH, De Young BR: MOC-31 aids in the differentiation of metastatic adenocarcinoma from hepatocellular carcinoma. Cancer. 87(5):295–298, 1999. 727. Porcell AI, De Young BR, Proca DM, et al: Immunohistochemical analysis of hepatocellular and adenocarcinoma in the liver: MOC31 compares favorably with other putative markers. Mod Pathol. 13(7):773–778, 2000. 728. Fucich LF, Cheles MK, Thung SN, et al: Primary vs metastatic hepatic carcinoma. An immunohistochemical study of 34 cases. Arch Pathol Lab Med. 118(9):927–930, 1994. 729. Gottschalk-Sabag S, Ron N, Glick T: Use of CD34 and factor VIII to diagnose hepatocellular carcinoma on fine needle aspirates. Acta Cytol. 42(3):691–696, 1998. 730. Tanigawa N, Lu C, Mitsui T, et al: Quantitation of sinusoid-like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology. 26(5):1216–1223, 1997. 731. Terada T, Nakanuma Y: Expression of ABH blood group antigens, receptors of Ulex europaeus agglutinin I, and factor VIII-related antigen on sinusoidal endothelial cells in adenomatous hyperplasia in human cirrhotic livers. Hum Pathol. 22(5):486–493, 1991. 732. Terada T, Nakanuma Y: Expression of ABH blood group antigens, Ulex europaeus agglutinin I, and type IV collagen in the sinusoids of hepatocellular carcinoma. Arch Pathol Lab Med. 115(1):50–55, 1991. 733. Dhillon AP, Colombari R, Savage K, et al: An immunohistochemical study of the blood vessels within primary hepatocellular tumours. Liver. 12(5):311–318, 1992. 734. Chen H, Jia WD, Li JS, et al: Extracellular matrix protein 1, a novel prognostic factor, is associated with metastatic potential of hepatocellular carcinoma. Med Oncol. 28(Suppl 1):S318– S325, 2011. 735. Park JO, Yeh MM: Clinical significance and implication of neoangiogenesis in hepatocellular carcinoma. J Gastroenterol Hepatol. 26(5):792–793, 2011. 736. Sakaguchi T, Suzuki S, Higashi H, et al: Expression of tight junction protein claudin-5 in tumor vessels and sinusoidal endothelium in patients with hepatocellular carcinoma. J Surg Res. 147(1):123–131, 2008. 737. Huang GW, Ding X, Chen SL, et al: Expression of claudin 10 protein in hepatocellular carcinoma: impact on survival. J Cancer Res Clin Oncol. 137(8):1213–1218, 2011. 738. Ohashi M, Wakai T, Korita PV, et al: Histological evaluation of intracapsular venous invasion for discrimination between portal and hepatic venous invasion in hepatocellular carcinoma. J Gastroenterol Hepatol. 25(1):143–149, 2010. 739. Kradin RL, Lynch GW, Kurnick JT, et al: Factor XIII A is synthesized and expressed on the surface of U937 cells and alveolar macrophages. Blood. 69(3):778–785, 1987. 740. Sciot R, Paterson AC, van Eyken P, et al: Transferrin receptor expression in human hepatocellular carcinoma: an immunohistochemical study of 34 cases. Histopathology. 12(1):53–63, 1988. 741. Yoshitake K, Tanaka S, Mogushi K, et al: Importin-alpha1 as a novel prognostic target for hepatocellular carcinoma. Ann Surg Oncol. 18(7):2093–2103, 2011. 742. Guzman G, Alagiozian-Angelova V, Layden-Almer JE, et al: p53, Ki-67, and serum alpha feto-protein as predictors of hepatocellular carcinoma recurrence in liver transplant patients. Mod Pathol. 18(11):1498–1503, 2005. 743. Jing Z, Nan KJ, Hu ML: Cell proliferation, apoptosis and the related regulators p27, p53 expression in hepatocellular carcinoma. World J Gastroenterol. 11(13):1910–1916, 2005. 744. Sugo H, Takamori S, Kojima K, et al: The significance of p53 mutations as an indicator of the biological behavior of recurrent hepatocellular carcinomas. Surg Today. 29(9):849–855, 1999. 745. Csepregi A, Ebert MP, Rocken C, et al: Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma. BMC Cancer. 10:317, 2010. 746. Quint K, Agaimy A, Di Fazio P, et al: Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC. Virchows Arch. 459(2):129–139, 2011.
583.e18
Immunohistology of Pancreas and Hepatobiliary Tract
747. Song MJ, Jung CK, Park CH, et al: RPL36 as a prognostic marker in hepatocellular carcinoma. Pathol Int. 61(11):638–644, 2011. 748. Takatori H, Yamashita T, Honda M, et al: dUTP pyrophosphatase expression correlates with a poor prognosis in hepatocellular carcinoma. Liver Int. 30(3):438–446, 2010. 749. Zhou P, Chen Z, Chang RM, et al: Growth arrest-specific homeobox is associated with poor survival in patients with hepatocellular carcinoma. Med Oncol. 2012. 750. Wang C, Zhang L, He Q, et al: Differences in Yes-associated protein and mRNA levels in regenerating liver and hepatocellular carcinoma. Mol Med Report. 5(2):410–414, 2012. 751. Yue X, Zhang Z, Liang X, et al: Zinc fingers and homeoboxes 2 inhibits hepatocellular carcinoma cell proliferation and represses expression of cyclins a and e. Gastroenterology. 142(7):1559– 1570 e2, 2012. 752. Zhang C, Tu Z, Du S, et al: Expression of matrix metalloproteinase 2 and extracellular matrix metalloproteinase inducer are unfavorable postoperative prognostic factors in intrahepatic cholangiocarcinoma. Pathol Oncol Res. 16(1):47–53, 2010. 753. Wang Q, Tan YX, Ren YB, et al: Zinc finger protein ZBTB20 expression is increased in hepatocellular carcinoma and associated with poor prognosis. BMC Cancer. 11:271, 2011. 754. Zhang SH, Qian YM, Liu AW, et al: Clinicopathologic significance and function of S-phase kinase-associated protein 2 overexpression in hepatocellular carcinoma. Hum Pathol. 43(7):1084–1093, 2012. 755. Lu HC, Chou FP, Yeh KT, et al: Expression of protein kinase C family in human hepatocellular carcinoma. Pathol Oncol Res. 16(3):385–391, 2010. 756. Zhong XY, Xu XX, Yu JH, et al: Clinical and biological significance of Cdk10 in hepatocellular carcinoma. Gene. 498(1):68– 74, 2012. 757. Ueda S, Shirabe K, Morita K, et al: Evaluation of ERCC1 expression for cisplatin sensitivity in human hepatocellular carcinoma. Ann Surg Oncol. 18(4):1204–1211, 2011. 758. Cho S, Lee JH, Cho SB, et al: Epigenetic methylation and expression of caspase 8 and survivin in hepatocellular carcinoma. Pathol Int. 60(3):203–211, 2010. 759. Yang X, Wang D, Dong W, et al: Expression and modulation of Na(+)/H(+) exchanger 1 gene in hepatocellular carcinoma: A potential therapeutic target. J Gastroenterol Hepatol. 26(2):364– 370, 2011. 760. Peroukides S, Bravou V, Alexopoulos A, et al: Survivin overexpression in HCC and liver cirrhosis differentially correlates with p-STAT3 and E-cadherin. Histol Histopathol. 25(3):299–307, 2010. 761. Huang H, Zhang XF, Zhou HJ, et al: Expression and prognostic significance of osteopontin and caspase-3 in hepatocellular carcinoma patients after curative resection. Cancer Sci. 101(5):1314– 1319, 2010. 762. Tan SM, Li F, Rajendran P, et al: Identification of beta-escin as a novel inhibitor of signal transducer and activator of transcription 3/Janus-activated kinase 2 signaling pathway that suppresses proliferation and induces apoptosis in human hepatocellular carcinoma cells. J Pharmacol Exp Ther. 334(1): 285–293, 2010. 763. Tomuleasa C, Soritau O, Rus-Ciuca D, et al: Isolation and characterization of hepatic cancer cells with stem-like properties from hepatocellular carcinoma. J Gastrointestin Liver Dis. 19(1):61–67, 2010. 764. Sun YL, Yin SY, Xie HY, et al: Stem-like cells in hepatitis B virus-associated cirrhotic livers and adjacent tissue to hepatocellular carcinomas possess the capacity of tumorigenicity. J Gastroenterol Hepatol. 23(8 Pt 1):1280–1286, 2008. 765. Shibuya M, Kondo F, Sano K, et al: Immunohistochemical study of hepatocyte, cholangiocyte and stem cell markers of hepatocellular carcinoma. J Hepatobiliary Pancreat Sci. 18(4):537–543, 2011. 766. Tanaka M, Miyajima A: Identification and isolation of adult liver stem/progenitor cells. Methods Mol Biol. 826:25–32, 2012. 767. Terris B, Cavard C, Perret C: EpCAM, a new marker for cancer stem cells in hepatocellular carcinoma. J Hepatol. 52(2):280– 281, 2010.
768. Lingala S, Cui YY, Chen X, et al: Immunohistochemical staining of cancer stem cell markers in hepatocellular carcinoma. Exp Mol Pathol. 89(1):27–35, 2010. 769. Zhang C, Xu Y, Zhao J, et al: Elevated expression of the stem cell marker CD133 associated with Line-1 demethylation in hepatocellular carcinoma. Ann Surg Oncol. 18(8):2373–2380, 2011. 770. Lu JW, Chang JG, Yeh KT, et al: Overexpression of Thy1/CD90 in human hepatocellular carcinoma is associated with HBV infection and poor prognosis. Acta Histochem. 113(8):833–838, 2011. 771. Yin X, Li YW, Zhang BH, et al: Coexpression of Stemness Factors Oct4 and Nanog Predict Liver Resection. Ann Surg Oncol. 2012. 772. Nakata T, Takayama E, Magari H, et al: Simultaneous detection of T lymphocyte-related antigens (CD4/CD8, CD57, TCRbeta) with nuclei by fluorescence-based immunohistochemistry in paraffin-embedded human lymph node, liver cancer and stomach cancer. Acta Cytol. 55(4):357–363, 2011. 773. Shi F, Shi M, Zeng Z, et al: PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients. Int J Cancer. 128(4):887– 896, 2011. 774. Lai TY, Su CC, Kuo WW, et al: beta-catenin plays a key role in metastasis of human hepatocellular carcinoma. Oncol Rep. 26(2):415–422, 2011. 775. Togni R, Bagla N, Muiesan P, et al: Microsatellite instability in hepatocellular carcinoma in non-cirrhotic liver in patients older than 60 years. Hepatol Res. 39(3):266–273, 2009. 776. Chiappini F, Gross-Goupil M, Saffroy R, et al: Microsatellite instability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers. Carcinogenesis. 25(4):541–547, 2004. 777. Wang L, Bani-Hani A, Montoya DP, et al: hMLH1 and hMSH2 expression in human hepatocellular carcinoma. Int J Oncol. 19(3):567–570, 2001. 778. Zhou L, Huang Y, Li J, et al: The mTOR pathway is associated with the poor prognosis of human hepatocellular carcinoma. Med Oncol. 27(2):255–261, 2010. 779. Ren F, Wu H, Lei Y, et al: Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target in hepatocellular carcinoma. Mol Cancer. 9:81, 2010. 780. Yan BC, Gong C, Song J, et al: Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol. 34(8):1147–1154, 2010. 781. Sharma D, Wang J, Fu PP, et al: Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis. Hepatology. 52(5):1713–1722, 2010. 782. Ogunwobi OO, Liu C: Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways. Clin Exp Metastasis. 28(8):721–731, 2011. 783. Ng KT, Lee TK, Cheng Q, et al: Suppression of tumorigenesis and metastasis of hepatocellular carcinoma by shRNA interference targeting on homeoprotein Six1. Int J Cancer. 127(4):859– 872, 2010. 784. Shin E, Ryu HS, Kim SH, et al: The clinicopathological significance of heat shock protein 70 and glutamine synthetase expression in hepatocellular carcinoma. J Hepatobiliary Pancreat Sci. 18(4):544–550, 2011. 785. Sun H, Teng M, Liu J, et al: FOXM1 expression predicts the prognosis in hepatocellular carcinoma patients after orthotopic liver transplantation combined with the Milan criteria. Cancer Lett. 306(2):214–222, 2011. 786. Takeishi K, Taketomi A, Shirabe K, et al: Diacylglycerol kinase alpha enhances hepatocellular carcinoma progression by activation of Ras-Raf-MEK-ERK pathway. J Hepatol. 57(1):77–83, 2012. 787. Tomimaru Y, Eguchi H, Wada H, et al: IGFBP7 downregulation is associated with tumor progression and clinical outcome in hepatocellular carcinoma. Int J Cancer. 130(2):319–327, 2012. 788. Xu C, Chen X, Chang C, et al: Genome-wide analysis of gene expression in dendritic cells from rat regenerating liver after partial hepatectomy. Cell Biochem Funct. 29(3):255–264, 2011.
References 789. Xu HX, Zhu XD, Zhuang PY, et al: Expression and prognostic significance of placental growth factor in hepatocellular carcinoma and peritumoral liver tissue. Int J Cancer. 128(7):1559– 1569, 2011. 790. Zhan DQ, Wei S, Liu C, et al: Reduced N-cadherin expression is associated with metastatic potential and poor surgical outcomes of hepatocellular carcinoma. J Gastroenterol Hepatol. 27(1):173–180, 2012. 791. Zhao JJ, Pan K, Li JJ, et al: Identification of LZAP as a new candidate tumor suppressor in hepatocellular carcinoma. PLoS One. 6(10):e26608, 2011. 792. Yu DC, Chen J, Sun XT, et al: Mechanism of endothelial progenitor cell recruitment into neo-vessels in adjacent non-tumor tissues in hepatocellular carcinoma. BMC Cancer. 10:435, 2010. 793. Zeng W, Gouw AS, van den Heuvel MC, et al: Hepatocellular carcinomas in cirrhotic and noncirrhotic human livers share angiogenic characteristics. Ann Surg Oncol. 17(6):1564–1571, 2010. 794. Patel SH, Kneuertz PJ, Delgado M, et al: Clinically relevant biomarkers to select patients for targeted inhibitor therapy after resection of hepatocellular carcinoma. Ann Surg Oncol. 18(12):3384–3390, 2011. 795. Shan C, Zhang S, Cui W, et al: Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement attack. Carcinogenesis. 32(8):1190–1197, 2011. 796. Takeuchi T, Adachi Y, Nagayama T: Expression of a secretory protein C1qTNF6, a C1qTNF family member, in hepatocellular carcinoma. Anal Cell Pathol (Amst). 34(3):113–121, 2011. 797. Wang Q, Tian X, Zhang C: Upregulation of vasohibin-1 expression with angiogenesis and poor prognosis of hepatocellular carcinoma after curative surgery. Med Oncol. 2011. 798. Villanueva A, Hernandez-Gea V, Llovet JM: Medical therapies for hepatocellular carcinoma: a critical view of the evidence. Nat Rev Gastroenterol Hepatol. 2012. 799. Noda T, Yamamoto H, Takemasa I, et al: PLOD2 induced under hypoxia is a novel prognostic factor for hepatocellular carcinoma after curative resection. Liver Int. 32(1):110–118, 2012. 800. Xiang ZL, Zeng ZC, Fan J, et al: The expression of HIF-1alpha in primary hepatocellular carcinoma and its correlation with radiotherapy response and clinical outcome. Mol Biol Rep. 39(2):2021–2029, 2012. 801. Tak E, Lee S, Lee J, et al: Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells. J Hepatol. 54(2):328–339, 2011. 802. Nart D, Yaman B, Yilmaz F, et al: Expression of matrix metalloproteinase-9 in predicting prognosis of hepatocellular carcinoma after liver transplantation. Liver Transpl. 16(5):621– 630, 2010. 803. Tao K, Qian N, Tang Y, et al: Increased expression of a disintegrin and metalloprotease-9 in hepatocellular carcinoma: implications for tumor progression and prognosis. Jpn J Clin Oncol. 40(7):645– 651, 2010. 804. Ke AW, Shi GM, Zhou J, et al: CD151 amplifies signaling by integrin alpha6beta1 to PI3K and induces the epithelialmesenchymal transition in HCC cells. Gastroenterology. 140(5):1629–1641 e15, 2011. 805. Woo HY, Min AL, Choi JY, et al: Clinicopathologic significance of the expression of Snail in hepatocellular carcinoma. Korean J Hepatol. 17(1):12–18, 2011. 806. Riener MO, Fritzsche FR, Soll C, et al: Expression of the extracellular matrix protein periostin in liver tumours and bile duct carcinomas. Histopathology. 56(5):600–606, 2010. 807. Pecic V, Stankovic-Djordjevic D, Nestorovic M, et al: Hepatitis C virus-related hepatocellular carcinoma and liver cirrhosis. J Buon. 16(2):277–281, 2011. 808. Soares JB, Pimentel-Nunes P, Afonso L, et al: Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammationfibrosis-carcinoma sequence. Innate Immun. 2012. 809. Tanaka J, Sugimoto K, Shiraki K, et al: Functional cell surface expression of toll-like receptor 9 promotes cell proliferation and survival in human hepatocellular carcinomas. Int J Oncol. 37(4):805–814, 2010. 810. Spangenberg HC, Thimme R, Blum HE: Serum markers of hepatocellular carcinoma. Semin Liver Dis. 26(4):385–390, 2006.
583.e19
811. Suzuki H, Komatsu A, Fujioka Y, et al: Angiosarcoma-like metastatic carcinoma of the liver. Pathol Res Pract. 206(7):484–488, 2010. 812. Tangkijvanich P, Chanmee T, Komtong S, et al: Diagnostic role of serum glypican-3 in differentiating hepatocellular carcinoma from non-malignant chronic liver disease and other liver cancers. J Gastroenterol Hepatol. 25(1):129–137, 2010. 813. Zhong C, Wei W, Su XK, et al: Serum and tissue vascular endothelial growth factor predicts prognosis in hepatocellular carcinoma patients after partial liver resection. Hepatogastroenterology. 59(113):93–97, 2012. 814. Sun S, Poon RT, Lee NP, et al: Proteomics of hepatocellular carcinoma: serum vimentin as a surrogate marker for small tumors (
583.e20
Immunohistology of Pancreas and Hepatobiliary Tract
833. Pan CC, Chen PC, Tsay SH, et al: Differential immunoprofiles of hepatocellular carcinoma, renal cell carcinoma, and adrenocortical carcinoma: a systemic immunohistochemical survey using tissue array technique. Appl Immunohistochem Mol Morphol. 13(4):347–352, 2005. 834. Henderson-Jackson E, Nasir NA, Hakam A, et al: Primary mixed lymphoepithelioma-like carcinoma and intra-hepatic cholangiocarcinoma: a case report and review of literature. Int J Clin Exp Pathol. 3(7):736–741, 2010. 835. Van Eyken P, Sciot R, Paterson A, et al: Cytokeratin expression in hepatocellular carcinoma: an immunohistochemical study. Hum Pathol. 19(5):562–568, 1988. 836. Ma CK, Zarbo RJ, Frierson HF, Jr, et al: Comparative immunohistochemical study of primary and metastatic carcinomas of the liver. Am J Clin Pathol. 99(5):551–557, 1993. 837. Wu PC, Fang JW, Lau VK, et al: Classification of hepatocellular carcinoma according to hepatocellular and biliary differentiation markers. Clinical and biological implications. Am J Pathol. 149(4):1167–1175, 1996. 838. Kanamoto M, Yoshizumi T, Ikegami T, et al: Cholangiolocellular carcinoma containing hepatocellular carcinoma and cholangiocellular carcinoma, extremely rare tumor of the liver:a case report. J Med Invest. 55(1-2):161–165, 2008. 839. Yeh MM: Pathology of combined hepatocellularcholangiocarcinoma. J Gastroenterol Hepatol. 25(9):1485–1492, 2010. 840. Yu XH, Xu LB, Zeng H, et al: Clinicopathological analysis of 14 patients with combined hepatocellular carcinoma and cholangiocarcinoma. Hepatobiliary Pancreat Dis Int. 10(6):620–625, 2011. 841. Zhang F, Chen XP, Zhang W, et al: Combined hepatocellular cholangiocarcinoma originating from hepatic progenitor cells: immunohistochemical and double-fluorescence immunostaining evidence. Histopathology. 52(2):224–232, 2008. 842. Cazals-Hatem D, Rebouissou S, Bioulac-Sage P, et al: Clinical and molecular analysis of combined hepatocellularcholangiocarcinomas. J Hepatol. 41(2):292–298, 2004. 843. Wakasa T, Wakasa K, Shutou T, et al: A histopathological study on combined hepatocellular and cholangiocarcinoma: cholangiocarcinoma component is originated from hepatocellular carcinoma. Hepatogastroenterology. 54(74):508–513, 2007. 844. Taguchi J, Nakashima O, Tanaka M, et al: A clinicopathological study on combined hepatocellular and cholangiocarcinoma. J Gastroenterol Hepatol. 11(8):758–764, 1996. 845. Tickoo SK, Zee SY, Obiekwe S, et al: Combined hepatocellularcholangiocarcinoma: a histopathologic, immunohistochemical, and in situ hybridization study. Am J Surg Pathol. 26(8):989– 997, 2002. 846. Jarnagin WR, Weber S, Tickoo SK, et al: Combined hepatocellular and cholangiocarcinoma: demographic, clinical, and prognostic factors. Cancer. 94(7):2040–2046, 2002. 847. Tatrai P, Somoracz A, Batmunkh E, et al: Agrin and CD34 immunohistochemistry for the discrimination of benign versus malignant hepatocellular lesions. Am J Surg Pathol. 33(6):874– 885, 2009. 848. Karabork A, Kaygusuz G, Ekinci C: The best immunohistochemical panel for differentiating hepatocellular carcinoma from metastatic adenocarcinoma. Pathol Res Pract. 206(8):572– 577, 2010. 849. Somoracz A, Tatrai P, Horvath G, et al: Agrin immunohistochemistry facilitates the determination of primary versus metastatic origin of liver carcinomas. Hum Pathol. 41(9):1310–1319, 2010. 850. Hughes NR, Goodman ZD, Bhathal PS: An immunohistochemical profile of the so-called bile duct adenoma: clues to pathogenesis. Am J Surg Pathol. 34(9):1312–1318, 2010. 851. Nakanuma Y, Sripa B, Vatanasapt V, et al: Intrahepatic cholangiocarcinoma. In Hamilton SR, Aaltonen LA, editors: Tumours of the Digestive System, Lyon, 2000, International Agency for Research on Cancer (IARC), pp 173–180. 852. Tihan T, Blumgart L, Klimstra DS: Clear cell papillary carcinoma of the liver: an unusual variant of peripheral cholangiocarcinoma. Hum Pathol. 29(2):196–200, 1998. 853. Jain R, Fischer S, Serra S, et al: The use of Cytokeratin 19 (CK19) immunohistochemistry in lesions of the pancreas,
gastrointestinal tract, and liver. Appl Immunohistochem Mol Morphol. 18(1):9–15, 2010. 854. Haglund C, Lindgren J, Roberts PJ, et al: Difference in tissue expression of tumour markers CA 19-9 and CA 50 in hepatocellular carcinoma and cholangiocarcinoma. Br J Cancer. 63(3):386– 389, 1991. 855. Ohshio G, Ogawa K, Kudo H, et al: Immunohistochemical studies on the localization of cancer associated antigens DU-PAN-2 and CA19-9 in carcinomas of the digestive tract. J Gastroenterol Hepatol. 5(1):25–31, 1990. 856. Loy TS, Sharp SC, Andershock CJ, et al: Distribution of CA 19-9 in adenocarcinomas and transitional cell carcinomas. An immunohistochemical study of 527 cases. Am J Clin Pathol. 99(6):726– 728, 1993. 857. Yu L, Feng M, Kim H, et al: Mesothelin as a potential therapeutic target in human cholangiocarcinoma. J Cancer. 1:141–149, 2010. 858. Wu Y, Wang T, Ye S, et al: Detection of hepatitis B virus DNA in paraffin-embedded intrahepatic and extrahepatic cholangiocarcinoma tissue in the northern Chinese population. Hum Pathol. 43(1):56–61, 2012. 859. Aishima S, Kuroda Y, Nishihara Y, et al: Gastric mucin phenotype defines tumour progression and prognosis of intrahepatic cholangiocarcinoma: gastric foveolar type is associated with aggressive tumour behaviour. Histopathology. 49(1):35–44, 2006. 860. Mall AS, Tyler MG, Ho SB, et al: The expression of MUC mucin in cholangiocarcinoma. Pathol Res Pract. 206(12):805–809, 2010. 861. Huang XY, Ke AW, Shi GM, et al: Overexpression of CD151 as an adverse marker for intrahepatic cholangiocarcinoma patients. Cancer. 116(23):5440–5451, 2010. 862. Tan FL, Ooi A, Huang D, et al: p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma and its involvement in cell motility and invasion. Int J Cancer. 126(10):2353–2361, 2010. 863. Lee D, Do IG, Choi K, et al: The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas. Mod Pathol. 25(1):131–139, 2012. 864. Xu D, Matsuo Y, Ma J, et al: Cancer cell-derived IL-1alpha promotes HGF secretion by stromal cells and enhances metastatic potential in pancreatic cancer cells. J Surg Oncol. 102(5):469–477, 2010. 865. Shafizadeh N, Grenert JP, Sahai V, et al: Epidermal growth factor receptor and HER-2/neu status by immunohistochemistry and fluorescence in situ hybridization in adenocarcinomas of the biliary tree and gallbladder. Hum Pathol. 41(4):485–492, 2010. 866. Patsenker E, Wilkens L, Banz V, et al: The alphavbeta6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma. J Hepatol. 52(3):362–369, 2010. 867. Thelen A, Scholz A, Weichert W, et al: Tumor-associated angiogenesis and lymphangiogenesis correlate with progression of intrahepatic cholangiocarcinoma. Am J Gastroenterol. 105(5):1123–1132, 2010. 868. Nakajima T, Tajima Y, Sugano I, et al: Intrahepatic cholangiocarcinoma with sarcomatous change. Clinicopathologic and immunohistochemical evaluation of seven cases. Cancer. 72(6): 1872–1877, 1993. 869. Imazu H, Ochiai M, Funabiki T: Intrahepatic sarcomatous cholangiocarcinoma. J Gastroenterol. 30(5):677–682, 1995. 870. Sumiyoshi S, Kikuyama M, Matsubayashi Y, et al: Carcinosarcoma of the liver with mesenchymal differentiation. World J Gastroenterol. 13(5):809–812, 2007. 871. Shinoda M, Shimazu M, Mukai M, et al: Spindle cell carcinoma of the intrahepatic bile duct in a patient with primary sclerosing cholangitis. J Gastroenterol. 38(11):1091–1096, 2003. 872. Nomura K, Aizawa S, Ushigome S: Carcinosarcoma of the liver. Arch Pathol Lab Med. 124(6):888–890, 2000. 873. Lim BJ, Kim KS, Lim JS, et al: Rhabdoid cholangiocarcinoma: a variant of cholangiocarcinoma with aggressive behavior. Yonsei Med J. 45(3):543–546, 2004. 874. Honda M, Enjoji M, Sakai H, et al: Case report: intrahepatic cholangiocarcinoma with rhabdoid transformation. J Gastroenterol Hepatol. 11(8):771–774, 1996.
References 875. Wagner LM, Garrett JK, Ballard ET, et al: Malignant rhabdoid tumor mimicking hepatoblastoma: a case report and literature review. Pediatr Dev Pathol. 10(5):409–415, 2007. 876. White FV, Dehner LP, Belchis DA, et al: Congenital disseminated malignant rhabdoid tumor: a distinct clinicopathologic entity demonstrating abnormalities of chromosome 22q11. Am J Surg Pathol. 23(3):249–256, 1999. 877. Stroescu C, Herlea V, Dragnea A, et al: The diagnostic value of cytokeratins and carcinoembryonic antigen immunostaining in differentiating hepatocellular carcinomas from intrahepatic cholangiocarcinomas. J Gastrointestin Liver Dis. 15(1):9–14, 2006. 878. Lodi C, Szabo E, Holczbauer A, et al: Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas. Mod Pathol. 19(3):460–469, 2006. 879. He F, Yan Q, Fan L, et al: PBK/TOPK in the differential diagnosis of cholangiocarcinoma from hepatocellular carcinoma and its involvement in prognosis of human cholangiocarcinoma. Hum Pathol. 41(3):415–424, 2010. 880. Loy TS, Quesenberry JT, Sharp SC: Distribution of CA 125 in adenocarcinomas. An immunohistochemical study of 481 cases. Am J Clin Pathol. 98(2):175–179, 1992. 881. Huang J, Zhang X, Tang Q, et al: Prognostic significance and potential therapeutic target of VEGFR2 in hepatocellular carcinoma. J Clin Pathol. 64(4):343–348, 2011. 882. Leelawat K, Thongtawee T, Narong S, et al: Strong expression of CD133 is associated with increased cholangiocarcinoma progression. World J Gastroenterol. 17(9):1192–1198, 2011. 883. Morine Y, Shimada M, Iwahashi S, et al: Role of histone deacetylase expression in intrahepatic cholangiocarcinoma. Surgery. 151(3):412–419, 2012. 884. Leger-Ravet MB, Borgonovo G, Amato A, et al: Carcinosarcoma of the liver with mesenchymal differentiation: a case report. Hepatogastroenterology. 43(7):255–259, 1996. 885. Lao XM, Chen DY, Zhang YQ, et al: Primary carcinosarcoma of the liver: clinicopathologic features of 5 cases and a review of the literature. Am J Surg Pathol. 31(6):817–826, 2007. 886. Siren J, Karkkainen P, Luukkonen P, et al: A case report of biliary cystadenoma and cystadenocarcinoma. Hepatogastroenterology. 45(19):83–89, 1998. 887. Yanase M, Ikeda H, Ogata I, et al: Primary smooth muscle tumor of the liver encasing hepatobiliary cystadenoma without mesenchymal stroma. Am J Surg Pathol. 23(7):854–859, 1999. 888. Terada T, Nakanuma Y, Ohta T, et al: Mucin-histochemical and immunohistochemical profiles of epithelial cells of several types of hepatic cysts. Virchows Arch A Pathol Anat Histopathol. 419(6):499–504, 1991. 889. Maruyama S, Hirayama C, Yamamoto S, et al: Hepatobiliary cystadenoma with mesenchymal stroma in a patient with chronic hepatitis C. J Gastroenterol. 38(6):593–597, 2003. 890. Scott FR, More L, Dhillon AP: Hepatobiliary cystadenoma with mesenchymal stroma: expression of oestrogen receptors in formalin-fixed tissue. Histopathology. 26(6):555–558, 1995. 891. Grayson W, Teare J, Myburgh JA, et al: Immunohistochemical demonstration of progesterone receptor in hepatobiliary cystadenoma with mesenchymal stroma. Histopathology. 29(5):461– 463, 1996. 892. Stocker JT, Schmidt D: Hepatoblastoma. In Hamilton SR, Aaltonen LA, editors: Tumours of the Digestive System, Lyon, 2000, International Agency for Research on Cancer (IARC), pp 184–189. 893. Ruck P, Xiao JC, Kaiserling E: Immunoreactivity of sinusoids in hepatoblastoma: an immunohistochemical study using lectin UEA-1 and antibodies against endothelium-associated antigens, including CD34. Histopathology. 26(5):451–455, 1995. 894. Al Nassan A, Sughayer M, Matalka I, et al: INI1 (BAF 47) immunohistochemistry is an essential diagnostic tool for children with hepatic tumors and low alpha fetoprotein. J Pediatr Hematol Oncol. 32(2):e79–e81, 2010. 895. Trobaugh-Lotrario AD, Finegold MJ, Feusner JH: Rhabdoid tumors of the liver: rare, aggressive, and poorly responsive to standard cytotoxic chemotherapy. Pediatr Blood Cancer. 57(3):423–428, 2011.
583.e21
896. Yuri T, Danbara N, Shikata N, et al: Malignant rhabdoid tumor of the liver: case report and literature review. Pathol Int. 54(8):623–629, 2004. 897. Purcell R, Childs M, Maibach R, et al: Potential biomarkers for hepatoblastoma: Results from the SIOPEL-3 study. Eur J Cancer 2011. 898. Purcell R, Childs M, Maibach R, et al: HGF/c-Met related activation of beta-catenin in hepatoblastoma. J Exp Clin Cancer Res. 30:96, 2011. 899. Turato C, Buendia MA, Fabre M, et al: Over-expression of SERPINB3 in hepatoblastoma: a possible insight into the genesis of this tumour? Eur J Cancer. 48(8):1219–1226, 2012. 900. Balta Z, Sauerbruch T, Hirner A, et al: [Primary neuroendocrine carcinoma of the liver: From carcinoid tumor to small-cell hepatic carcinoma: case reports and review of the literature.]. Pathologe. 29(1):53–60, 2008. 901. Cho MS, Lee SN, Sung SH, et al: Sarcomatoid hepatocellular carcinoma with hepatoblastoma-like features in an adult. Pathol Int. 54(6):446–450, 2004. 902. Wang JH, Dhillon AP, Sankey EA, et al: “Neuroendocrine” differentiation in primary neoplasms of the liver. J Pathol. 163(1):61–67, 1991. 903. Ishak KG, Anthony PP, Niederau C, et al: Mesenchymal tumours of the liver. In Hamilton SR, Aaltonen LA, editors: Tumours of the Digestive System, Lyon, 2000, International Agency for Research on Cancer (IARC), pp 191–198. 904. Zamboni G, Pea M, Martignoni G, et al: Clear cell “sugar” tumor of the pancreas. A novel member of the family of lesions characterized by the presence of perivascular epithelioid cells. Am J Surg Pathol. 20(6):722–730, 1996. 905. Shi H, Cao D, Wei L, et al: Inflammatory angiomyolipomas of the liver: a clinicopathologic and immunohistochemical analysis of 5 cases. Ann Diagn Pathol. 14(4):240–246, 2010. 906. Tryggvason G, Blondal S, Goldin RD, et al: Epithelioid angiomyolipoma of the liver: case report and review of the literature. APMIS. 112(9):612–616, 2004. 907. Lupinacci RM, Rocha Mde S, Herman P: Hepatic epithelioid hemangioendothelioma: an unusual lesion of the liver. Clin Gastroenterol Hepatol. 10(2):e15–e16, 2012. 908. Zhang Z, Chen HJ, Yang WJ, et al: Infantile hepatic hemangioendothelioma: a clinicopathologic study in a Chinese population. World J Gastroenterol. 16(36):4549–4557, 2010. 909. Emamaullee JA, Edgar R, Toso C, et al: Vascular endothelial growth factor expression in hepatic epithelioid hemangioendothelioma: Implications for treatment and surgical management. Liver Transpl. 16(2):191–197, 2010. 910. Yesim G, Gupse T, Zafer U, et al: Mesenchymal hamartoma of the liver in adulthood: immunohistochemical profiles, clinical and histopathological features in two patients. J Hepatobiliary Pancreat Surg. 12(6):502–507, 2005. 911. Shintaku M, Watanabe K: Mesenchymal hamartoma of the liver: A proliferative lesion of possible hepatic stellate cell (Ito cell) origin. Pathol Res Pract. 206(7):532–536, 2010. 912. Shehata BM, Gupta NA, Katzenstein HM, et al: Undifferentiated embryonal sarcoma of the liver is associated with mesenchymal hamartoma and multiple chromosomal abnormalities: a review of eleven cases. Pediatr Dev Pathol. 14(2):111–116, 2011. 913. Perez-Gomez RM, Soria-Cespedes D, de Leon-Bojorge B, et al: Diffuse membranous immunoreactivity of CD56 and paranuclear dot-like staining pattern of cytokeratins AE1/3, CAM5.2, and OSCAR in undifferentiated (embryonal) sarcoma of the liver. Appl Immunohistochem Mol Morphol. 18(2):195–198, 2010. 914. Li Y, Du Y, Yang HF: Primary hepatic lymphoma: a large mass surrounding multiple intrahepatic vessels. Clin Gastroenterol Hepatol. 9(5):e41–e42, 2011. 915. Zen Y, Fujii T, Nakanuma Y: Hepatic pseudolymphoma: a clinicopathological study of five cases and review of the literature. Mod Pathol. 23(2):244–250, 2010. 916. Sallah S, Smith SV, Lony LC, et al: Gamma/delta T-cell hepatosplenic lymphoma: review of the literature, diagnosis by flow cytometry and concomitant autoimmune hemolytic anemia. Ann Hematol. 74(3):139–142, 1997. 917. Nagata S, Tomoeda M, Kubo C, et al: Malignant mesothelioma of the peritoneum invading the liver and mimicking metastatic
583.e22
Immunohistology of Pancreas and Hepatobiliary Tract
carcinoma: a case report. Pathol Res Pract. 207(6):395–398, 2011. 918. Matsumoto T, Ojima H, Akishima-Fukasawa Y, et al: Solitary hepatic lymphangioma: report of a case. Surg Today. 40(9):883– 889, 2010. 919. Ross JS, Del Rosario A, Bui HX, et al: Primary hepatic leiomyosarcoma in a child with the acquired immunodeficiency syndrome. Hum Pathol. 23(1):69–72, 1992. 920. Shivathirthan N, Kita J, Iso Y, et al: Primary hepatic leiomyosarcoma: Case report and literature review. World J Gastrointest Oncol. 3(10):148–152, 2011. 921. Scheimberg I, Cullinane C, Kelsey A, et al: Primary hepatic malignant tumor with rhabdoid features. A histological, immunocytochemical, and electron microscopic study of four cases and a review of the literature. Am J Surg Pathol. 20(11):1394– 1400, 1996.
922. Shek TW, Ho FC, Ng IO, et al: Follicular dendritic cell tumor of the liver. Evidence for an Epstein-Barr virus-related clonal proliferation of follicular dendritic cells. Am J Surg Pathol. 20(3):313–324, 1996. 923. Salakos C, Nikolakopoulou NM, De Verney Y, et al: Anaplastic lymphoma kinase (ALK) positive inflammatory pseudotumor of the liver: conservative treatment and long-term follow-up. Eur J Pediatr Surg. 20(4):278–280, 2010. 924. Shek TW, Ng IO, Chan KW: Inflammatory pseudotumor of the liver. Report of four cases and review of the literature. Am J Surg Pathol. 17(3):231–238, 1993. 925. Isse K, Grama K, Abbott IM, et al: Adding value to liver (and allograft) biopsy evaluation using a combination of multiplex quantum dot immunostaining, high-resolution whole-slide digital imaging, and automated image analysis. Clin Liver Dis. 14(4):669–685, 2010.
C H A P T E R 1 6
IMMUNOHISTOLOGY OF THE PROSTATE GEORGE J. NETTO, JONATHAN I. EPSTEIN
Overview 584 Biology of Antigens and Antibodies 584 Diagnostic Immunohistochemistry of Specific Prostate Lesions 587 Beyond Immunohistochemistry: Theranostic and Genomic Applications 606 Summary 614
the pathologist correctly identify many morphologic mimics of PCa that could lead to a false-positive interpretation. The serious patient care consequences and medicolegal implications of a false-positive diagnosis of PCa are evident. This chapter discusses the utility of IHC markers, as well as the genomic applications, in accurately diagnosing and predicting the prognosis of PCa.
Biology of Antigens and Antibodies Overview Used in the proper setting, ancillary techniques can be a great adjunct to light microscopy to obtain an accurate diagnosis in urologic pathology. In the last decade, a plethora of molecular biomarkers have been evaluated for their potential role in enhancing our ability to predict disease progression, response to therapy, and survival in prostate cancer (PCa) patients.1-6 These research efforts have been greatly facilitated by the wealth of information garnered from gene-expression array studies and by sophisticated bioinformatics tools that help evaluate the overwhelming datasets generated from genomic, transcriptomic, and proteomic studies. These genomic technologies continue to yield new markers that can in turn be evaluated for clinical utility in a high-throughput manner by using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)–labeled tissue microarrays and state-of-the-art image-analysis systems.7-10 In prostate biopsies, immunomarkers that facilitate a diagnosis of carcinoma in a small focus of atypical glands are of great utility. The latter are especially valuable in organs such as the prostate, in which a repeat biopsy does not always reach the target focus for additional sampling. The layer of assurance rendered by multiple immunostains used in prostate biopsy is due in part to their amenability to be simultaneously applied in the same section when only limited tissue is available. Ancillary techniques are equally important in helping 584
Principal Antibodies Many different antibodies are used for the IHC evaluation of urologic neoplasms. Generally utilized epithelial, neuroendocrine, and mesenchymal antibodies are discussed in other chapters. In each section we will summarize antibodies of particular importance for the neoplasms covered in that section.
Prostate-Specific Antigen Prostate-specific antigen (PSA) is a serine protease member of the human glandular kallikrein family. PSA is a 34-kD glycoprotein of 237 amino acids with high sequence homology with human glandular kallikrein 2 (HK2). It is almost exclusively synthesized in the prostate ductal and acinar epithelium and is found in normal, hyperplastic, and malignant prostate tissue.11 PSA liquefies the seminal fluid coagulum through proteolysis of the gel-forming proteins, thus releasing spermatozoa. It can reach the serum by diffusion from the luminal cells through the basal cell layer, glandular basement membrane, and extracellular matrix (ECM). Measuring total serum PSA levels is currently the mainstay of PCa detection, and numerous studies have shown that patients with PCa have, in general, elevated serum PSA levels. The most commonly used cutoff for PSA is 4 ng/ mL. When serum PSA concentrations are 4 to 10 ng/ mL, the incidence of cancer detection on prostate biopsy in men with a normal digital rectal exam (DRE) is approximately 25%. With serum PSA levels higher than 10 ng/mL, the incidence of PCa on biopsy increases
Biology of Antigens and Antibodies
to approximately 67%. However, the risk of cancer is proportional to the serum PSA level even at values less than 4 ng/mL. As large screening trials have demonstrated, clinically significant cancers occur in men with serum PSA levels of 2.5 to 4.0 ng/mL, thus some experts have proposed lowering the PSA cutoff to 2.5 ng/mL to improve early detection of cancer in younger men.11 Once PSA gains access into the circulation, most remains bound to serine protease inhibitors. The three most recognizable inhibitors are α-1–antichymotrypsin (α1-AT), α-2–macroglobulin, and α-1–protein inhibitor. PSA bound to α1-AT is the most immunoreactive and clinically the most useful in diagnosing PCa. A smaller fraction (5% to 40%) of the measurable serum PSA is free (noncomplexed) PSA. Therefore the total serum PSA measured reflects both free and complexed PSA. It has been demonstrated that the percent of free PSA can improve the specificity of PSA testing for PCa. A free PSA value of less than 10% is worrisome for cancer. More recently, additional isoforms of free PSA have been discovered and were detailed in a review by Gretzer and Partin.11 PSA is first secreted in the form of a precursor termed pro-PSA. This inactive form of the enzyme constitutes the majority of free PSA in serum in men with PCa, making the relative increase of serum pro-PSA a risk marker of PCa. Benign PSA (BPSA) refers to a cleaved form of PSA from benign prostatic hyperplasia tissue. Measurement of the ratio of pro-PSA to BPSA has been proposed as a means of improving the accuracy of diagnosing cancer in men with a very low percentage of free PSA levels, who are at relatively high risk of cancer.9,12 Serum PSA tests may also be used to monitor patients after therapy to detect early recurrence. Following radical prostatectomy, the serum PSA should drop to undetectable levels. Elevated serum PSA levels following radical prostatectomy (>0.2 ng/mL) indicate recurrent or persistent disease. Following radiotherapy for PCa, serum PSA values will decrease to a nadir, although not to the same extent as those following radical prostatectomy. Three subsequent rises in serum PSA values after radiotherapy indicates treatment failure. Although PSA expression in extraprostatic tissues and tumors other than PCA have been rarely demonstrated (Box 16-1), for all practical purposes, PSA expression at the IHC level is a specific and sensitive marker of prostatic lineage of differentiation with as much as 97.4% sensitivity found in a recent study from our group.13 Urethral, periurethral, and perianal glands are among normal tissues that have been rarely reported to show PSA reactivity. Extraprostatic neoplasms that occasionally express PSA include urethral and periurethral adenocarcinoma, cloacogenic carcinoma, pleomorphic adenoma of salivary gland, salivary duct carcinoma, and rare mammary carcinomas.14-16 Although a rare report indicated PSA expression in intestinal-type urachal adenocarcinoma of bladder, we failed to reveal such expression in a recent study of villous adenoma and adenocarcinoma of bladder.17 The latter is especially important from a differential diagnosis point of view, given the topographic proximity of the two organs.
585
Box 16-1 PROSTATE-SPECIFIC ANTIGEN IMMUNOREACTIVITY IN EXTRAPROSTATIC TISSUES AND TUMORS Extraprostatic Tissues Urethra and periurethral glands (male and female) Bladder, including cystitis cystica and glandularis Anal glands (male) Urachal remnants Neutrophils Extraprostatic Tumors Urethral and periurethral gland adenocarcinoma (female) Villous adenoma and adenocarcinoma of the bladder Extramammary Paget disease of the male external genitalia Pleomorphic adenoma of the salivary glands (male) Carcinoma of the salivary glands (male) Breast carcinoma
Although weaker intensity of PSA expression can be encountered in higher Gleason grade PCa, we were recently able to demonstrate a high degree of PSA immunostain sensitivity (97.4%) in high-grade prostate carcinoma, even when transcription-mediated amplification (TMA) sampling was used.13 Likewise, PSA expression is very valuable in defining a prostatic primary site of origin during the evaluation of a poorly differentiated metastatic carcinoma.
Prostate-Specific Membrane Antigen Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein expressed in prostate tissue and, to a lesser extent, in peripheral and central nervous system, small intestinal, and salivary gland tissues. PSMA expression has also been documented in endothelial cells of the neovasculature of many solid tumors, including renal cell carcinoma (RCC).18-22 In prostate, PSMA is expressed by benign and malignant prostatic epithelial cells, with a higher extent of staining seen in the latter.23 It is also expressed by high-grade prostatic intraepithelial neoplasia (PIN).23 PSMA expression correlates with PCa stage and Gleason grade.24 The increase in both expression and enzymatic activity of PSMA in aggressive PCa points to a selective advantage imparted on cells that express PSMA, thereby contributing to the development and progression of PCa.25 Increased PSMA expression is an independent predictor of PCa recurrence,25,26 and PSMA expression is maintained in hormone-refractory PCa, thus increasing its utility in such settings.20,27 Several imaging strategies exploit PSMA specificity to PCa and are currently in use for PCa diagnostic imaging.28-31 Furthermore, PSMA is under investigation as a target of therapy in PCa and other solid tumors, given its expression by the neovasculature of extraprostatic tumors.32-34 Cytoplasmic and, to a lesser degree, membranous PSMA expression has been recently documented in 11% of analyzed urinary bladder adenocarcinomas,17 a fact worth noting when the differential diagnosis includes prostatic and bladder adenocarcinoma.
586
Immunohistology of the Prostate
Prostatic Acid Phosphatase/ Prostate-Specific Acid Phosphatase Prostate-specific acid phosphatase (PSAP) is one of the earlier prostate lineage markers to be exploited for immunolabeling of PCa before the discovery of PSA. Currently, the use of PSAP as a marker of prostatic differentiation has declined given its relative lack of specificity compared with PSA and the more variable staining of PSAP in higher grade PCa.35,36
Prostein/P501S P501S is a 553–amino acid protein localized to the Golgi complex. It is expressed in both benign and neoplastic prostate tissues. Typical P501S stain has a perinuclear cytoplasmic (Golgi) location and a speckeled pattern. Expression is retained in poorly differentiated and metastatic PCa. P501S demonstrated up to 99% sensitivity in a recent study from our group by Sheridan and colleagues.37 In rare metastatic lesions, P501S positivity may be encountered in the presence of PSA-negative expression, making it an advantageous addition to a prostate lineage immunopanel. To date, P501S expression has not been shown in extraprostatic carcinomas, which makes it of great utility in differentiating high-grade PCa from other highgrade carcinomas, including colorectal and urothelial carcinoma (URCa).13,17,37-41
α-Methylacyl–Coenzyme-A Racemase/P504S Alpha-methylacyl–CoA racemase (AMACR) is mainly localized to peroxisomal structures and plays a critical role in peroxisomal beta oxidation of branched chain fatty acid. In their original detailed IHC analysis, Luo and associates42 demonstrated that both prostate carcinomas and high-grade PINs consistently revealed a significantly higher expression than that of matched normal prostate epithelium. Both untreated and hormone-refractory PCa metastases generally maintain a strong positive reactivity for AMACR. An overall PCa sensitivity and specificity of 97% and 92%, respectively, have been shown in a multiinstitutional study by Jiang and Woda.43 Cytoplasmic AMACR staining combined with absence of basal cell markers, such as the nuclear protein p63 and high-molecular-weight cytokeratins (HMWCKs), has proved to be of greatest utility in
providing an added layer of assurance in establishing the diagnosis of PCa on small needle biopsy foci.43-46 However, AMACR expression has been repeatedly demonstrated in high-grade PIN and in some benign mimics of PCa, such as glandular and partial atrophy and adenosis; therefore AMACR is of limited utility as a single marker in resolving the differential diagnosis of PCa in such lesions. A panel of immunostains that includes AMACR, HMWCK, and p63 (positive AMACR immunostaining [Table 16-1] along with negative basal cell markers) is recommended in the interrogation of atypical prostatic glandular foci.47-54
High-Molecular-Weight Cytokeratins High-molecular-weight cytokeratins (HMWCKs) are of great utility in highlighting the presence or absence of basal cells in a focus of atypical prostate glands.55-58 34βE12 is currently the most widely used clone, both individually or as a component of a three-antibody cocktail that includes a second basal cell marker, such as p63, and AMACR. Alternatively, CK5/6 can be used as the HMWCK marker individually or in combination with p63 and AMACR. A recent study by Abrahams and colleagues59 seems to suggest a superior sensitivity for CK5/6 as an HMWCK in prostate biopsies fixed in Hollande’s fixative. Following initial examination of hematoxylin and eosin (H&E)–stained routine sections, the application of such a cocktail to previously prepared, unstained, intervening sections is recommended in biopsies where establishing the presence or absence of basal cells in a questionable focus will lead to a definitive resolution of a benign or malignant diagnosis, respectively.60
p63 The p53 homolog p63 encodes for different isotypes that can either transactivate p53 reporter genes (TAp63) or act as p53-dominant negatives (ΔNp63), and p63 is expressed in the basal or myoepithelial cells of many epithelial organs; its germline inactivation in the mouse results in agenesis of organs such as skin appendages and the breast. In the prostate, p63 expression is limited to basal cells and is absent in secretory and neuroendocrine cells,61 and ΔNp63α isotype is the most abundantly represented isotype in normal prostate basal cells. Recent experimental evidence also suggests that the P63 gene is essential for normal stem cell function in the prostate.62 Several studies have confirmed the
TABLE 16-1 Immunoreactivity of α-Methylacyl-Coenzyme-A Racemase in Benign and Neoplastic Prostate Benign
Immunoreactive Range (%)
Immunoreactive Glands (%)
Intensity (1 to 3+)
8 (0-10)
4.6 (0-24.5)
1+
AAH
14 (10-17)
15.1 (1-50)
High PIN
88 (80-100)
21.8 (2.7-5.7)
Cancer
97 (80-100)
AAH, Atypical adenomatous hyperplasia; PIN, prostatic intraepithelial neoplasia.
35 (6.2-78.2)
1+ 1+ to 2+ 2 to 3+
Diagnostic Immunohistochemistry of Specific Prostate Lesions
587
TABLE 16-2 Immunophenotype of Basal Cell Layer in the Prostate Biomarker
Function
Findings
Androgen receptors
Nuclear receptors necessary for prostatic epithelial growth
Strong immunoreactivity; also present in cancer cells
PSA
Enzyme that liquefies the seminal coagulum
Present in rare basal cells; mainly in secretory luminal cells
Keratin 34βE12
Keratins 5, 10, 11
Strong immunoreactivity; most commonly used for diagnostic purposes
TP53
A member of the TP63 gene family
Strong immunoreactivity; most commonly used for diagnostic purposes
S-100A6
Calcium-binding protein
Strong immunoreactivity
EGFR
Membrane-bound 170-kD glycoprotein which mediates the activity of EGF
Strong immunoreactivity; rare in cancer cells
GSTP1
Enzyme that activates electrophilic carcinogens
Strong immunoreactivity; rare in cancer
ECAM
Epithelial cell adhesion molecule
Strong immunoreactivity; absent in cancer
TGF-β
Growth factor that regulates cell proliferation and differentiation
Strong immunoreactivity; absent in cancer
ECAM, Epithelial cell adhesion molecule; EGFR, epidermal growth factor receptor; GSTP1, glutathione S-transferase gene; PSA, prostatespecific antigen; TGF, transforming growth factor.
clinical utility of p63 immunostain as a prostate basal cell marker, and some studies suggest a slight sensitivity advantage for p63 over HMWCK alone.63-67 Additionally, the use of basal cell HMWCK and p63 cocktails may reduce the staining variability that may be encountered in basal cells and may further decrease the falsenegative and false-positive rates of basal cell labeling by either marker alone (Table 16-2).65,68 Finally, given the fact that immunostains for basal cell markers are typically used in a “negative” diagnostic mode, to show absence of basal cells in PCa, sole reliance on such markers is not advocated, and the identification of a combination of major and minor histologic features of PCa is crucial for achieving clinical diagnostic accuracy. In this regard, consideration should be given to the fact that benign prostatic glands from the transition zone are subject to basal cell staining variability that may result in occasional negative basal cell staining in such benign glands.65 Furthermore, basal cells can be retained, albeit very rarely, in individual glands in otherwise typical acinar PCa focus, and the constellation of diagnostic features are to be relied on in such rare cases. We have recently described an intriguing p63-positive, HMWCK-negative variant of PCa, in which nuclear p63 staining is seen in secretory PCa cells in a nonbasal distribution.69
NKX3-1 NKX3-1 is a prostate-specific androgen-regulated homeobox gene required for tissue differentiation, whose loss of function leads to carcinogenesis. In normal prostate, NKX3-1 controls differentiation and protects against oxidative damage by regulating gene expression in conjunction with other transcription factors. In cancer, a relative loss of NKX3-1 expression occurs as a result of loss of heterozygosity (LOH), promoter
methylation, or alterations in NKX3-1 degradation. Downregulation of NKX3-1 protein leads to increased prostate epithelial cell proliferation, differentiation, and susceptibility to DNA damage, thereby furthering oncogenic insult. In addition to benign and malignant prostatic epithelium, NKX3-1 expression is found in normal testis, bronchial mucous glands, and infiltrating lobular carcinoma of the breast. Sensitivity of NKX3-1 for PCa has ranged from 68% to 94.7%. In poorly differentiated PCa, NKX3-1 appears to be superior to PSA, which may show a relatively focal weaker staining in that subset.13,70-72
Diagnostic Immunohistochemistry of Specific Prostate Lesions Immunohistochemistry in Small Focus of Prostate Carcinoma The use of IHC markers to help establish the diagnosis of carcinoma in a morphologically atypical small focus of prostate glands is currently a common laboratory practice. As mentioned above, used individually or with two or three markers combined in a panel, HMWCK, p63, and AMACR offer great help in ensuring absence of a basal layer combined with positive AMACR labeling in such small foci (Fig. 16-1). Such a panel is also of use in distinguishing a small focus of PCa infiltrating adjacent to a high-grade PIN lesion from the glandular outpouching of high-grade PIN where an interrupted (patchy) layer of basal cells would still be identified with the aid of immunostains (Figs. 16-2 and 16-3). Fully characterizing and delineating the group of atypical acini in question based on a combination of established H&E morphologic features of malignancy before their interrogation by immunostains is the key to a
588
Immunohistology of the Prostate
A
B
C Figure 16-1 A, The utility of immunostains in support of diagnosis of a small focus of prostate carcinoma. B, Lack of basal cell layer is illustrated by high-molecular-weight keratin (CK903). C, Positive staining in the tumor glands for α-methylacyl-coenzyme-A racemase is shown.
successful diagnostic approach in PCa. The key H&E morphologic features include small acinar architecture, single-layer lining, straight luminal borders, amphophilic cytoplasm, nuclear enlargement and atypia, presence of prominent nucleoli, wispy or blue mucin content, dense eosinophilic secretions or “cancer”
A
crystalloids, and presence of mucinous fibroplasia. The demonstration of an increasing combination of the above morphologic features in the presence of a supportive immunostaining pattern will allow for a significant increment in diagnostic confidence when faced with increasingly smaller-sized atypical foci on a needle
B
Figure 16-2 A, Cribriform high-grade prostatic intraepithelial neoplasia. B, Preservation of basal layer staining with high-molecular-weight cytokeratin is shown.
Diagnostic Immunohistochemistry of Specific Prostate Lesions
A
B
C
D
589
Figure 16-3 A and B, Atypical glands adjacent to high-grade prostatic intraepithelial neoplasia (PIN). Given the presence of an interrupted (patchy) layer of basal cells, the possibility of glandular outpouchings of PIN could not be excluded with the aid of immunostains (p63 in C and high-molecular-weight keratin in D).
biopsy.73 If such a confidence level is unobtainable despite the application of immunostains, a diagnosis of focus of atypical glands suspicious but not diagnostic of malignancy should be rendered, with a recommendation for a close repeat follow-up biopsy to rule out malignancy (Fig. 16-4). Routine initial use of immunostain cocktails as a screening tool before H&E examination, to facilitate identification of basal cell negative foci, is not advocated for obvious reasons that include cost, misallocation of resources, and potential detriment to diagnostic accuracy. On the other hand, when used judiciously, the role of HMWCK in decreasing diagnostic uncertainty expressed in prostate needle biopsies has been established in several large studies, including a College of American Pathology (CAP) Q-probes study of more than 15,000 biopsies.55-58 In a large study from our center, 34βE12 stains either helped to establish (14%), confirmed (58%), or changed (2%) our diagnoses when applied to questionable/atypical foci. In an additional 18% of cases, the diagnosis remained (or became) equivocal despite the use of HMWCK.55 False-negative staining of basal cells with HMWCK can occur for a variety of technical reasons, including
suboptimal antigen retrieval, and this should be taken into consideration. Finally, it is worthy to note that a very low but existent false-positive HMWCK immunostaining of PCa cells can be encountered (0.2% to 2.8%),65,68 characteristically in a non–basal cell distribution pattern (Fig. 16-5). Another complicating issue in interpreting basal cell immunostaining results is the p63-positive, HMWCK-negative rare variant of PCa mentioned previously (Fig. 16-6).69
KEY DIAGNOSTIC POINTS Small Focus of Prostate Carcinoma • Small acinar architecture, single-layer lining, straight luminal borders, amphophilic cytoplasm, nuclear enlargement and atypia, presence of prominent nucleoli, wispy or blue mucin content, dense eosinophilic secretions or “cancer” crystalloids, and presence of mucinous fibroplasia are apparent. • If additional immunostains do not render confidence, focal atypical glands suspicious for carcinoma should prompt follow-up biopsy.
590
Immunohistology of the Prostate
A
B
C
D
Figure 16-4 Prostate adenocarcinoma diagnosis supported by absence of basal layer on immunostains in two groups of atypical glands (A) and infiltration among benign glands (B). Use of immunostains for high-molecular-weight cytokeratin (C) and p63 (D).
Immunohistochemistry in Benign Mimics of Prostate Adenocarcinoma PROSTATIC ATROPHY
Figure 16-5 A focus of prostate adenocarcinoma shows scattered tumor cells with positive expression of high-molecular-weight cytokeratin in a nonbasal distribution. Such findings alone should not deter the pathologist from making a diagnosis of carcinoma if all other features of malignancy are satisfied.
Partial atrophy (PTAT) and postatrophic hyperplasia (PAH) are the most problematic morphologic variants of atrophy that may mimic PCa. In fact, PTAT is the most common mimic of PCa on needle biopsy, mainly because of the presence of disorganized acini lined by pale cytoplasm with occasional acini that retain a full height of cytoplasm and contain slightly enlarged nuclei with notable nucleoli. PTAT foci can morphologically mimic “atrophic” PCa. In difficult PTAT lesions, immunostains for basal cell markers help highlight the presence of basal cells. HMWCKs (34βE12, CK5/6) and p63 show patchy positivity in basal cells in at least some of the glands (Figs. 16-7). Lack of positivity in some glands should not be misinterpreted as PCa as long as the negative and positive glands share similar cytologic features.74 It is also important to remember that AMACR can be expressed by some PTAT acini.75 Only rarely does the clinician need to resort to immunostains to recognize simple atrophy and PAH
Diagnostic Immunohistochemistry of Specific Prostate Lesions
A
B
C
D
591
Figure 16-6 A, Rare variant of prostate adenocarcinoma shows aberrant expression of p63 but not high-molecular-weight cytokeratin (HMWCK). B, Note nuclear positivity for p63 in tumor glands (combo p63-34βE12). C, Note infiltrating tumor glands showing tumor cells staining for p63. D, The tumor is negative for HMWCK.
lesions. The latter two variants of atrophy demonstrate a continuous basal layer on immunostains, and they are usually negative for AMACR (Fig. 16-8). ADENOSIS
Adenosis is a common mimic of PCa both on needle biopsy and on transurethral resection of prostate (TURP).76-78 Given its preferential occurrence in the transition zone, adenosis is more frequently seen in TURP (1.6%) compared with needle biopsy (0.8%). Adenosis is characterized by a nodular proliferation of small glands. Within such nodules, larger elongated glands with papillary infolding and branching lumina share identical nuclear and cytoplasmic features with the admixed smaller, more suspicious glands. In contrast, small PCa glands usually stand out cytologically from adjacent benign larger glands. To avoid misinterpretation of adenosis, the constellation of histologic features in a given lesion should outweigh the significance of any one diagnostic feature79 given the fact that several features are shared between adenosis and PCa. Therefore in difficult cases, IHC for HMWCK can be of great utility to demonstrate the
patchy positivity of basal cells in adenosis (Fig. 16-9).80 Lack of positivity for HMWCK in some of the glands should not be misinterpreted as evidence of PCa as long as the negative and positive glands share similar cytologic features. Of note, AMACR can be focally or diffusely expressed in adenosis in as many as 10% of cases.75 SCLEROSING ADENOSIS
Sclerosing adenosis is a rare lesion mainly encountered in TURP specimens performed for urinary obstruction. Very rarely, it may also be sampled on needle biopsy, leading to potential overdiagnosis as PCa. Sclerosing adenosis is composed of a relatively well-circumscribed proliferation of well-formed glands admixed with single epithelial cells set in a background of dense spindle cell proliferation. The glandular structures are similar to those seen in adenosis. Some glands are surrounded by a distinct eosinophilic hyaline sheath–like collarette. The lining epithelial cells usually lack atypia, and a basal cell layer can be appreciated on H&E. Establishing the diagnosis of sclerosing adenosis in examples that demonstrate atypical features such as presence of crystalloids, mitotic figures, and prominent nucleoli requires
592
Immunohistology of the Prostate
A
B
C
D
E
KEY DIAGNOSTIC POINTS Atrophy, Adenosis, and Sclerosing Adenosis • Atrophy, adenosis, and sclerosing adenosis may mimic prostate cancer, and basal marker immunostains p63 and 34βE12 may aid in the diagnosis. • Adenosis and sclerosing adenosis are most commonly seen in TURP specimens.
Figure 16-7 Foci of partial atrophy (A and B) show patchy basal cell layer positivity with high-molecular-weight keratin (C) and p63 (D). Partial atrophy glands may display positive α-methylacyl–CoA racemase staining (D and E).
the aid of immunostains. Basal cells and spindle cells are unique in their true myoepethelial differentiation as indicated by coexpression of keratin and muscle-specific actin (MSA; Fig. 16-10). The latter is not expressed by basal cells in normal prostate glands.81-84 XANTHOMA
Prostatic xanthomas are rare but could be potentially misleading lesions in small and distorted needle-tissue fragments. Typical low microscopic appearance is that of a small well-circumscribed solid nodule; examples
Diagnostic Immunohistochemistry of Specific Prostate Lesions
A
593
B
Figure 16-8 A, Focus of prostatic postatrophic hyperplasia. B, Continuous basal cell layer on high-molecular-weight cytokeratin immunostain.
A
B
Figure 16-9 A, Prostate gland with a focus of adenosis. B, Patchy staining of basal cells by using high-molecular-weight cytokeratin.
A
B Figure 16-10 A, Sclerosing adenosis of the prostate gland. B, Positive smooth muscle actin staining in myoepithelial cells.
594
Immunohistology of the Prostate
B
A
Figure 16-11 A, Focus of xanthoma involving prostate. B, CD68 positivity.
architecturally set as infiltrative cords and individual cells are more troubling. Xanthoma cells have a uniform appearance and contain abundant foamy cytoplasm and bland nuclei without prominent nucleoli (Fig. 16-11). Mitotic activity is usually lacking.85 However, mitotic figures are also rare in PCa. Immunostains should be obtained if the possibility of xanthoma is suspected based on morphology. Expression of histiocytic markers such as CD68 and lack of cytokeratin (CAM5.2) staining support the diagnosis.86,87
Posttherapy Changes in Prostate Adenocarcinoma ANTIANDROGEN THERAPY
Currently, luteinizing hormone–releasing hormone (LHRH) agonist (leuprolide), typically in association with the antiandrogen flutamide, is the most commonly used form of hormonal therapy in PCa. Both benign and neoplastic prostatic tissue can be significantly altered
A
with hormonal therapy.88-93 Under hormone deprivation, neoplastic acini usually acquire an atrophic appearance and can mimic benign atrophic glands because of the relative lack of nuclear atypia and absence of prominent nucleoli. At times, treated PCa glands develop pyknotic nuclei with abundant xanthomatous cytoplasm, and when present as scattered cells, they will closely resemble foamy histiocytes (Fig. 16-12). IHC for PSA or pancytokeratin can aid in the diagnosis of carcinoma. As with their untreated counterparts, PCa cells after hormonal therapy demonstrate a lack of HMWCK staining. Following hormonal therapy, a decrease may be noted in immunoreactivity with prostate lineage markers such as PSA, P501S, PSMA, and PSAP. However, with the exception of tumors that develop focal squamous differentiation, resulting in adenosquamous carcinomas, most tumors will maintain at least focal labeling with these antibodies.94-98 In our laboratory, we find that utilizing a panel of three of the above markers— PSA, PSMA, and P501S—will increase the sensitivity for prostatic differentiation. Finally, it is worth
B
Figure 16-12 A, Prostate adenocarcinoma showing changes prior hormonal therapy effect. Staining with α-methylacyl–CoA racemase (AMACR) highlights the scattered residual tumor cells that lack a basal cell layer. (B; Combination AMACR and CK 34βE12).
Diagnostic Immunohistochemistry of Specific Prostate Lesions
remembering that squamous components of recurrent or metastatic hormone-independent prostatic adenosquamous carcinoma will only rarely and very focally be positive for prostate lineage markers such as PSA, PSMA, P501S, and PSAP and will diffusely express HMWCK in these areas.94-98 KEY DIAGNOSTIC POINTS Posthormonal Therapy Histology • Following hormonal therapy, prostate cancer cells demonstrate a lack of high-molecular-weight cytokeratin staining. Following hormonal therapy, a decrease in immunoreactivity may be seen with prostate lineage markers such as PSA, P501S, PSMA, and PSAP.
RADIATION THERAPY
Besides surgery, external beam radiation and or interstitial radiotherapy (brachytherapy) are two additional standard treatment options for localized PCa with a curative intent.99 Radiated nonneoplastic prostatic glands undergo glandular atrophy, squamous metaplasia, and cytologic atypia.100 The marked epithelial atypia,
A
Figure 16-13 A, Prostate adenocarcinoma showing changes of prior radiation therapy effect. B, Lack of a basal cell layer is demonstrated on high-molecular-weight cytokeratin immunostain. C, Positivity for prostate-specific antigen is shown.
595
especially following brachytherapy, tends to persist for several years.101 The distinction between irradiated nonneoplastic prostatic glands and prostate carcinoma can be difficult, especially if the history of prior treatment is not provided and is not considered by the pathologist. On low magnification, radiated benign prostate glands maintain their normal architectural lobular configuration. On higher magnification, piling up of the nuclei occurs within irradiated benign glands with a recognizable basal cell layer (Fig. 16-13). The finding of scattered, markedly atypical nuclei with a degenerative, hyperchromatic, and smudgy appearance within wellformed acini is typical of radiated benign glands. In contrast, radiated glands of PCa are lined by a single cell layer with typical pyknotic nuclei and foamy cytoplasm. PCa that is sufficiently differentiated to form glands rarely manifests the degree of atypia seen with radiation. In difficult cases, HMWCK can aid in the diagnosis of irradiated prostate by identifying basal cells within benign radiated glands to prevent a false-positive interpretation of carcinoma.102-104 Another scenario in which radiation treatment can introduce diagnostic difficulty when recurrent or residual PCa displays marked and extensive radiation effect in the form of glands or individual cells with
B
C
596
Immunohistology of the Prostate
abundant vacuolated cytoplasm that takes on a histiocytic appearance. The nuclei lack apparent nucleoli and are pyknotic with smudged chromatin.105 Pancytokeratin (AE1/AE3 and CAM5.2) and CD68 markers can be used to illustrate the epithelial nature of treated PCa. In most cases, treated PCa will retain its PSA and PSAP positivity105 as well as its expression of AMACR (P504S).103,104 However, as mentioned above, recurrent or metastatic radiated PCa that displays a sarcomatoid, squamous, or adenosquamous phenotype may only focally be positive for prostate lineage markers while expressing HMWCK.94-98 When postradiation clinical response is evaluated, prostate biopsies are obtained 1 year after conclusion of treatment. Negative biopsies and the presence of residual PCa displaying severe radiation effect portend good response. Residual tumor without demonstrable radiation effect is considered a strong predictor of clinical failure. The expression of proliferation markers (MIB-1 or PCNA) in postradiated cancer has also been shown to correlate with clinical failure.105,106
Prostatic Duct Carcinoma Less than 1% (0.4% to 0.8%) of PCa shows distinctive tall columnar cells in papillary or cribriform structures
and is classified as prostatic duct adenocarcinoma,107-109 which can be encountered as a single pattern of tumor differentiation or, more frequently, is found admixed with “usual” acinar differentiation. Prostatic duct adenocarcinomas show a variety of architectural patterns that include a papillary exophytic architecture, seen in a periurethral location, lined by tall pseudostratified epithelial cells and a cribriform pattern, more commonly seen deeper within the tissue, formed by back-to-back large glands with slitlike lumina. It is not uncommon to find areas of papillary formation admixed with cribriform patterns. An important point is that ductal adenocarcinomas, because they arise in ducts, may show residual staining for HMWCK and p63 (Fig. 16-14). Prostatic duct adenocarcinomas can invade as single glands lined by tall columnar cells, unlike the cuboidal cells that characterize acinar prostatic carcinoma. The single infiltrating glands of prostatic duct adenocarcinoma may resemble infiltrating colonic adenocarcinoma. The differentiation between prostatic duct adenocarcinoma and secondary involvement of the prostate by colonic adenocarcinoma is usually made by finding more typical prostatic duct adenocarcinoma elsewhere in the biopsy. Rarely in such settings, IHC demonstration of PSA or other prostate lineage markers, such as P501S, is needed to identify a prostatic duct
A B
Figure 16-14 A and B, Prostate ductal adenocarcinoma. C, Lack of a basal cell layer on high-molecular-weight cytokeratin and p63 stains is shown.
C
Diagnostic Immunohistochemistry of Specific Prostate Lesions
adenocarcinoma. Adding β-catenin, CDX-2, and villin, all of which are positive in colon cancer, to the IHC panel can be of further utility in such a differential, although uncommonly prostatic ductal adenocarcinomas are CDX2 positive.110,111 Prostatic duct adenocarcinoma on TURP specimens can also mimic papillary URCa. Nuclear features can be helpful in such a differential, because nuclei in URCa tend to be more pleomorphic and angulated.112 PSA and PSAP positivity and negative reactivity for GATA3 thrombomodulin and uroplakin in prostatic duct adenocarcinoma can also be useful.110,113,114
Neuroendocrine Prostatic Neoplasms It is somewhat controversial whether neuroendocrine differentiation in typical adenocarcinomas worsens prognosis.115-117 Three studies evaluated the prognostic role of neuroendocrine differentiation in organ-confined prostate adenocarcinoma. No prognostic role was found in the first study, whereas the two subsequent larger studies, including ours, found only a marginal prognostic role insufficient to be useful clinically.118-120 In the single study that analyzed neuroendocrine differentiation in PCa on needle biopsy, no relationship with prognosis could be established.121 According to the 1999 CAP Consensus Statement,122 neuroendocrine differentiation is still considered a category III prognostic factor not sufficiently studied to demonstrate its prognostic value. As in other organs, the spectrum of neuroendocrine neoplasms in the prostate include carcinoid tumors, small cell carcinoma, and large cell neuroendocrine carcinoma as defined in the lung by Travis and colleagues.123 True carcinoid tumors of the prostate are extremely rare. Recently, a total of three such cases have been reported with documented negative immunoreactivity for PSA and PSAP and otherwise typical carcinoid tumor morphology and immunoprofile.124-126 All three patients came to medical attention with normal serum PSA levels; they lacked clinical features of carcinoid syndrome. Several additional cases have been reported in which at least a focal “carcinoid like” appearance has been present.127,128 None of these patients had carcinoid syndrome, and all such cases have been positive with antibodies for PSA and PSAP. They have clinically behaved like ordinary prostate carcinomas. A more appropriate designation for these lesions is prostatic adenocarcinomas with neuroendocrine differentiation. The basis for a diagnosis of small cell carcinoma of the prostate is the presence of morphologic features similar to those found in small cell carcinomas of the lung as defined in the 1999 World Health Organization (WHO) classification criteria.129-131 In approximately 50% of the cases, the tumors are mixed small cell carcinoma and adenocarcinoma of the prostate. As with other unusual subtypes of PCa, we do not assign a Gleason score to small cell carcinoma, only to the conventional adenocarcinoma component. Immunohistochemically, the small cell component is positive for one or more neuroendocrine markers—neuron-specific
597
enolase (NSE), synaptophysin, chromogranin, or CD56—and is negative for markers of prostatic differentiation such as PSA, PSMA, P501S, and PSAP. A minority of small cell carcinomas is positive for prostatic markers to varying degrees and may be negative for neuroendocrine markers. In a recent study by Yao and colleagues,131 strong chromogranin and synaptophysin positivity was present in 61% and 89%, respectively, of prostatic small cell carcinoma studied. PSA and PSAP were positive in 17% and 24% of cases, respectively. In 24% and 35% of cases, positivity was noted for p63 and HMWCK, markers typically negative in prostatic carcinoma yet expressed in normal basal cells of the prostate. In our recent and largest study on small cell carcinoma of the prostate,40 we found most small cell carcinomas (88%) to be positive for at least one neuroendocrine marker. We also found P501S and PSMA to be better in identifying the prostatic origin of small cell carcinoma than PSA, although the majority (60%) of prostatic small cell carcinomas were still negative for all three markers (Fig. 16-15). The latter, together with the above-cited heterogeneity of the prostatic small cell carcinoma immunophenotype, is consistent with an origin from multipotential transient amplifying cells closely related to stem cells.131-133 Ordonez and colleagues134 originally reported that thyroid transcription factor 1 (TTF-1) was positive in 96% of small cell carcinomas of the lung and was negative in all three prostate small cell cancers. Subsequent studies, including our own, have demonstrated TTF-1 expression in the majority of small cell carcinomas of the prostate, limiting its utility in distinguishing primary small cell carcinoma of the prostate from a metastasis from the lung.40,131,135 Small cell carcinoma of the prostate continues to have a dismal outcome, with average survival of less than 1 year. No difference in prognosis is observed among patients with pure small cell carcinomas or mixed glandular and small cell carcinomas. Tumor immunoprofile does not affect survival. A review by Mackey and colleagues136 concluded that hormonal manipulation and systemic chemotherapy had little effect on the natural history of prostate small cell carcinoma. Others suggest treatment of small cell carcinoma of the prostate with the same combination chemotherapy used to treat small cell carcinomas in other sites.137,138 It remains to be seen whether new targeted therapy strategies currently under investigation in small cell carcinoma of lung may be applicable to small cell carcinoma of prostate, expressing targets such as c-kit, Bcl-2, and CD56. Large cell neuroendocrine carcinoma (LCNEC) of the prostate is an extremely rare occurrence. In the largest series on the topic by Evans and associates,139 only one out of seven cases was a de novo LCNEC; the remaining six cases represented progression from prior acinar adenocarcinoma following long-standing hormonal therapy. LCNEC is composed of sheets and ribbons of amphophilic cells with large nuclei, coarse chromatin, and prominent nucleoli. Mitotic activity is brisk, and foci of necrosis are common. A minor (<10%) component of conventional prostate adenocarcinoma
598
Immunohistology of the Prostate
A
B
C
D
E
showing hormonal deprivation effect was identified in all but the single de novo case. The LCNEC component was strongly positive for CD56, CD57, chromogranin A, synaptophysin, and P504S. PSA and PSAP expression was present in the conventional component but was focal or absent in the LCNEC areas. All six patients with available follow-up died, and mean survival was 7 months.
Figure 16-15 A and C, Small cell carcinoma of prostate in association with acinar prostate carcinoma. D, Focal weak positivity for prostate-specific antigen. E, Positive staining for P501S.
Urothelial Carcinoma that Involves Prostate and Prostatic Urethra Prostatic involvement by URCa can result from direct invasion of an infiltrating bladder cancer into prostate stroma and through extension of urothelial tumor through an intraductal route with or without
Diagnostic Immunohistochemistry of Specific Prostate Lesions
subsequent stromal invasion of the prostate.140,141 In the first scenario, direct bladder wall to prostate invasion, the prognosis of the URCa of the bladder is equivalent in survival to cases of bladder carcinoma with regional lymph node metastases. A common diagnostic problem in this setting is differentiating between a poorly differentiated URCa of the bladder and a poorly differentiated prostatic adenocarcinoma in a TURP specimen. Because therapy differs significantly, the distinction between these two entities is crucial. Even in poorly differentiated prostatic carcinomas, relatively little pleomorphism or mitotic activity is typical compared with poorly differentiated URCa. A more subtle finding is that the cytoplasm of prostatic adenocarcinoma is often very foamy and pale, imparting a “soft” appearance. In contrast, URCas may demonstrate hard, glassy eosinophilic cytoplasm or more prominent squamous differentiation. The findings of infiltrating cords of cells or focal cribriform glandular differentiation are other features more typical of prostatic adenocarcinoma. Although the above distinction between URCa and prostatic adenocarcinoma on H&E-stained sections is valid for almost all cases, we have seen rare cases in which prostate adenocarcinoma has had marked pleomorphism identical to URCa. Consequently, in a poorly differentiated tumor that involves the bladder and prostate without any glandular differentiation typical of prostate adenocarcinoma, the case should be worked up immunohistochemically, given the high stakes of a misdiagnosis. Approximately 95% of poorly differentiated prostatic adenocarcinomas show PSA and PSAP staining, although it may be focal.142-144 Whereas some authors have demonstrated a superiority of PSA over PSAP in staining prostatic carcinoma, others have found poorly differentiated prostatic carcinomas that lacked PSA staining but still maintained immunoreactivity to PSAP.144-146 In our lab, PSA has in general been more sensitive. Monoclonal antibodies to PSAP have lower sensitivities than their polyclonal counterparts but are more specific.147 We have compared PSA staining in a group of poorly differentiated prostatic adenocarcinomas with “poor” PSA staining to newer prostate-specific markers, including PSMA, P501S (prostein), and NKX3-1 (Fig. 16-16).13 Completely negative staining was seen in 15% (PSA), 12% (PSMA), 17% (P501S), and 5% (NKX3-1) of the cases, and 5% were negative for all four markers. A similar 5% rate of false negativity is found when combining PSA and PSAP stains.148 Therefore the lack of immunoreactivity to prostatespecific markers in a poorly differentiated tumor within the prostate, especially in small samples, does not exclude the diagnosis of a poorly differentiated prostatic adenocarcinoma. With only a few exceptions, immunoperoxidase staining for PSA and PSAP is very specific for prostatic tissue. Situations that can cause diagnostic difficulty include PSA and PSAP within periurethral glands and cystitis cystica and cystitis glandularis in both men and women.149-151 Other examples of cross-reactive staining include anal glands in men (PSA, PSAP) and urachal remnants (PSA).152,153 Some intestinal carcinoids and pancreatic islet cell tumors are strongly reactive with antibodies to PSAP yet are negative with
599
antibodies to PSA.154 Periurethral gland carcinomas in women and various salivary gland tumors may also be PSA and PSAP positive.155,156 Although adenocarcinomas of the bladder, whether as a pure tumor or with mixed URCa, have also been reported to be positive for PSA or PSAP, there has yet to be a case reported positive for both.157,158 In a poorly differentiated tumor occurring in the bladder and the prostate, where the differential diagnosis is between high-grade prostatic adenocarcinoma and URCa, focal strong staining for either marker can be used reliably to make the diagnosis of prostatic adenocarcinoma, because PSAP and PSA false positivity have not been convincingly described in URCas.159,160 In general, various cytokeratins (CK7, CK20, HMWCK) show strong positivity in cases of URCa involving the prostate. Although CK7 and CK20 are more frequently seen in URCa, as compared with adenocarcinoma of the prostate, they may also be positive in adenocarcinoma of the prostate, such that in our experience, they are not that helpful in this differential diagnosis.148,161 We and others have found HMWCK to be positive in more than 90% of URCas.13,162 In contrast, HMWCK is only rarely (8%) expressed, and usually in a very small percentage of cells, in adenocarcinoma of the prostate.13 Another useful marker in differentiating high-grade URCa from prostatic adenocarcinoma is p63. Using tissue microarrays, we found p63 to have a greater specificity, albeit with lower sensitivity, for URCa compared with HMWCK (100% specificity and 83% sensitivity; Fig. 16-17).13 Other markers that also appear highly specific but are only of modest sensitivity for URCa include uroplakin and thrombomodulin (49% to 69% sensitivity). More recently, GATA3 has become the favored marker with the highest sensitivity and specificity for urothelial carcinoma versus prostate cancer. If intraductal URCa is identified on TURP or transurethral biopsy, patients usually will be recommended for radical cystoprostatectomy. The finding of intraductal URCa also has been demonstrated to increase the risk of urethral recurrence following cystoprostatectomy, such that its identification may also result in prophylactic total urethrectomy. IHC stains for basal cells (HMWCK, p63) may in some cases only outline the prostatic basal cells and in other cases may label the intraductal URCa. The diagnosis of URCa on prostate needle biopsy is especially difficult. Clinically, urothelial carcinoma that involves the prostate can mimic prostatic adenocarcinoma in terms of findings on DRE and ultrasound, along with the potential for an elevated serum PSA level.163 Histologic features and IHC studies are therefore essential to establish the correct diagnosis. URCa that involves the prostate differs from adenocarcinoma of the prostate both architecturally and cytologically, and it typically forms nests of tumor, whereas poorly differentiated PCa tends to form sheets, individual cells, or cords. In our study, urothelial carcinoma that involved the prostate contained areas of necrosis in 43% of cases. Necrosis is an unusual finding even in high-grade adenocarcinoma of the prostate. The presence of an intraductal growth in which preexisting benign prostate glands are
600
Immunohistology of the Prostate
A
B
C
D
E
filled with solid nests of tumor also differs from highgrade PIN, which is composed of flat, tufting, papillary, or cribriform patterns. The presence of squamous differentiation seen in 14% of our cases would also be unusual for adenocarcinoma of the prostate. Cytologically, URCas that involve the prostate tend to show greater nuclear pleomorphism, variably prominent nucleoli, and increased mitotic activity compared with even poorly differentiated prostate adenocarcinoma. In
Figure 16-16 High-grade prostate adenocarcinoma (A) showing weak focal prostate-specific antigen positivity (B) but strong diffuse staining for P501S (C) and prostate-specific membrane antigen (D). Nuclear staining for NKX3-1 is also encountered (E).
high-grade adenocarcinomas of the prostate, nuclei tend to be more uniform from one to another with centrally located prominent eosinophilic nucleoli. Mitotic figures in high-grade PCa are typically not as frequent compared with what is seen in URCa on biopsy. Finally, the presence of stromal inflammation, seen in 76% of our cases of URCa on biopsy, differs from the typical lack of associated inflammation seen with ordinary adenocarcinoma of the prostate (Fig. 16-18).
Diagnostic Immunohistochemistry of Specific Prostate Lesions
A
B
C
D
Figure 16-17 A, High-grade urothelial carcinoma. B, Focal highmolecular-weight cytokeratin positivity. Strong diffuse nuclear staining for p63 (C) and S-100A protein (E) and diffuse cytoplasmic staining for thrombomodulin (D).
E
601
602
Immunohistology of the Prostate
A
B
C
D
Figure 16-18 A and B, Intraductal spread of urothelial carcinoma (URCa) involving prostate gland. C, URCa undermines the secretory prostate epithelial layer, as shown on prostate-specific antigen stains. D, URCa cells react positively with high-molecular-weight cytokeratin.
KEY DIAGNOSTIC POINTS Prostate Carcinoma vs. Urothelial Carcinoma in Prostate • Distinction is crucial for appropriate therapy. • Cellular/nuclear pleomorphism, necrosis, squamous differentiation, and intraductal sheet pattern of growth along with high-molecular-weight cytokeratin/p63 immunostaining all strongly support urothelial carcinoma.
Secondary Involvement of Prostate by Colorectal Adenocarcinoma Another source of secondary tumor extension into prostate is the topographically adjacent colorectal tract. Here again, attention to some characteristic morphologic features should raise the possibility of a secondary spread. The presence of goblet/columnar cell differentiation, pseudostratified basally located nuclei, and characteristic “dirty necrosis” are more likely encountered in colorectal carcinoma (CRCa).111,164,165 One should be cautioned that single infiltrating glands of prostatic duct
adenocarcinoma can resemble infiltrating colonic adenocarcinoma. The differentiation between prostatic duct adenocarcinoma and secondary involvement of the prostate by CRCa can be facilitated by finding more typical prostatic duct adenocarcinoma elsewhere within the biopsy. An IHC profile of positive nuclear CDX-2 staining, positive nuclear staining for β-catenin (cytoplasmic staining can occur in PCa) and positive staining for CK20 in the face of negative reactivity for PSA, NKX3.1, and P501S can be used to confirm the diagnosis of CRCa spread.111,164,165
Prostatic Mesenchymal Tumors As in any other organ, immunostains can be of great utility in resolving a variety of spindle cell mesenchymal lesions that occur in the prostate, including benign and malignant smooth muscle neoplasms, peripheral nerve sheath tumors, and rhabdomyosarcoma (Table 16-3).166,167 Here we will focus our discussion on four lesions that pose a unique differential challenge when encountered in a prostatic biopsy: 1) stromal tumors of uncertain malignant potential (STUMPs) and stromal sarcomas, 2) smooth muscle neoplasms (leiomyomas/
Diagnostic Immunohistochemistry of Specific Prostate Lesions
603
TABLE 16-3 Immunohistochemistry of Prostatic Mesenchymal Lesions STUMP
SS
Leiomyosarcoma
Rhabdomyosarcoma
IMT
SFT
GIST
CD34
+
+
−
−
−
+
+
SMA
S
−
+
+
+
−
S
Desmin
S
−
+
+
+
−
S
Myogenin
−
−
−
+
−
−
−
c-kit
−
−
−
−
S
−
+
ALK-1
−
−
−
−
+
−
−
PR
+
+
S
−
−
S
−
Reactivity: +, almost always positive; S, sometimes positive; –, negative. GIST, Gastrointestinal stromal tumor; IMT, inflammatory myofibroblastic tumor; PR, progesterone receptor; SFT, solitary fibrous tumor; SMA, smooth muscle actin; SS, stromal sarcoma; STUMPs, stromal tumors of uncertain malignant potential.
leimyosarcomas), 3) solitary fibrous tumors (SFTs), and 4) gastrointestinal stromal tumors (GISTs). STROMAL TUMORS OF UNCERTAIN MALIGNANT POTENTIAL AND STROMAL SARCOMAS
Stromal tumors of uncertain malignant potential and stromal sarcomas (STUMPs) are rare but distinct tumors of the specialized prostatic stroma as currently recognized in the 2004 WHO Classification of Tumors of the Urinary System and Male Genital Organs.168 STUMPs present most commonly with lower urinary tract obstruction, abnormal digital rectal examination, hematuria, hematospermia, palpable rectal mass, or elevated serum PSA levels.169-171 On gross examination, a STUMP appears as a white-tan solid or solid cystic nodule that may range in size; lesions may be microscopic or large cystic lesions up to 15 cm in size. Microscopically, STUMPs present with diverse histologic patterns. Four histologic patterns of STUMP have been described: 1) hypercellular stroma that contain scattered atypical degenerative appearing cells; 2) hypercellular stroma that consist of bland fusiform stromal cells with eosinophilic cytoplasm; 3) leaflike hypocellular fibrous stroma covered by benign-appearing prostatic epithelium, similar in morphology to a benign phyllodes tumor of the breast; and 4) myxoid stroma– containing bland stromal cells that often lack admixed glands. Some cases exhibit a mixture of the above patterns. Immunostains demonstrate that STUMPs are positive for CD34 and vimentin and variably positive for smooth muscle actin (SMA) and desmin (see Table 16-3). Not surprisingly, given their presumed derivation from the prostatic stroma, progesterone receptor is frequently found on immunostaining, although estrogen receptor is less commonly positive. C-kit and S-100 have been negative in all cases examined, a feature of value in distinguishing STUMPs from other spindle cell tumors, such as SFT and schwannoma. Although STUMPs are generally considered to represent a benign neoplastic stromal process, a subset of STUMPs has been associated with stromal sarcoma on a synchronous or metasynchronous biopsy, suggesting a malignant progression in at least some cases.169 There
appears to be no correlation between the pattern of STUMP and association with stromal sarcoma. Stromal sarcoma may arise de novo, or it may exist in association with either a preexisting or concurrent STUMP. Stromal sarcomas demonstrate either a solid growth with storiform, epithelioid, fibrosarcomatous, or patternless patterns or may infiltrate between benign prostatic glands. Less commonly, stromal sarcomas may demonstrate leaflike glands with underlying hypercellular stroma, which are also termed malignant phyllodes tumors. Stromal sarcomas have one or more of the following four features within the spindle cell component: 1) hypercellularity, 2) cytologic atypia, 3) mitotic figures, and 4) necrosis. Stromal sarcomas can be further subclassified into low- and high-grade lesions; high-grade tumors show moderate to marked pleomorphism and hypercellularity, often with increased mitotic activity and occasional necrosis. IHC findings in stromal sarcomas are similar to those of STUMPs, with strong vimentin reactivity and positivity for CD34 and progesterone receptor. In a subset of cases studied, pancytokeratin and CAM5.2 stains were negative. Stromal sarcomas can extend out of the prostate and metastasize to distant sites, such as bone, lung, abdomen, and retroperitoneum. The variability of STUMPs clinical behavior and their occasional association or progression to stromal sarcomas make for a challenging patient management plan. STUMPs warrant close follow-up, and definitive resection should be considered in younger individuals. Factors to consider in deciding whether to proceed with definitive resection for STUMPs diagnosed on biopsy include patient age and treatment preference, presence and size of the lesion on rectal exam or imaging studies, and extent of the lesion on tissue sampling. Expectant management with close clinical follow-up could be considered in an older individual with a limited lesion on biopsy, when there is no lesion identified on digital rectal exam or on imaging studies. SMOOTH MUSCLE NEOPLASMS (LEIOMYOMA/LEIOMYOSARCOMA)
Leiomyoma contains well-organized fascicles and may demonstrate degenerative features, such as hyalinization and calcification, that are not commonly seen in stromal
604
Immunohistology of the Prostate
nodules, their main differential diagnosis. Large solitary leiomyomas that are symptomatic are rare.172,173 Leiomyomas should demonstrate virtually no mitotic activity and minimal if any nuclear atypia, with the exception of occasional scattered degenerative nuclei. Sarcomas of the prostate account for 0.1% to 0.2% of all malignant prostatic tumors. Leiomyosarcoma is the most common sarcoma of prostate, and lesions range in size from 3 to 21 cm. Microscopically, leiomyosarcomas are hypercellular and composed of intersecting bundles of spindle cells with moderate to severe atypia. The vast majority of leiomyosarcomas have been high grade with frequent mitoses and necrosis, although we have encountered a rare low-grade prostatic leiomyosarcoma.174 Low-grade leiomyosarcomas are distinguished from leiomyomas by a moderate amount of atypia, focal areas of increased cellularity, scattered mitotic figures, and/or a focally infiltrative growth pattern around benign prostate glands at the perimeter. Unlike some stromal sarcomas, leiomyosarcomas lack admixed normal glands, except entrapped glands at the periphery. Immunohistochemically, leiomyosarcomas commonly express vimentin, actin, and desmin. Cytokeratin expression is observed in approximately one quarter of cases. In addition, some leiomyosarcomas have been reported to express progesterone receptor, similar to STUMPs and stromal sarcomas (see Table 16-3).175,176
A
Figure 16-19 A and B, Solitary fibrous tumor involving prostate. C, Strong CD34 positivity.
Leiomyosarcomas have a poor clinical outcome characterized by multiple recurrences, and 50% to 75% of patients die of their disease within 2 to 5 years. In the study by Sexton and colleagues,177 variables predictive of a favorable prognosis included presentation with metastasis and complete surgical resection. Optimal treatment requires a multimodal approach rather than surgery alone. SOLITARY FIBROUS TUMOR
Fewer than 20 cases of SFT involving the prostate have been reported.178 Some older reported cases of hemangiopericytoma of the prostate may also be today classified as SFT. Microscopically, prostatic SFTs appear similar to those identified in extraprostatic sites. Uniform spindled cells with bland nuclei are arranged in a “patternless” pattern in a background of variable ropy collagen and a hemangiopericytomatous appearance.178 None of the reported prostatic SFTs have behaved in an aggressive fashion. However, based on the behavior of SFTs in other sites and the finding in some prostatic SFTs of hypercellularity, pleomorphism, necrosis, and infiltrative margins, careful long-term clinical follow-up is warranted. IHC generally reveals diffuse reactivity for CD34, vimentin, and Bcl-2, although rare SFTs may lack some of these markers (Fig. 16-19). Staining for
B
C
Diagnostic Immunohistochemistry of Specific Prostate Lesions
CD99, β-catenin, p53, SMA, and muscle-specific actin (MSA) has also been reported. These tumors are typically negative for pancytokeratin, S-100, and CD117 (c-kit). GASTROINTESTINAL STROMAL TUMOR
Although gastrointestinal stromal tumors (GISTs) may occur as a primary prostatic process on imaging and clinical studies, such cases are typically large masses that arise from the rectum or perirectal space that only compress the prostate. Very rarely, GIST may invade the prostate. There is not yet a fully documented example of a GIST arising within the prostate.179-181 Typically, GIST is not considered in the differential diagnosis of spindle cell lesions of the prostate, although the unique management of these tumors underscores the importance of recognizing them. Unfortunately, several patients have undergone pelvic exenteration, irradiation, and chemotherapy for a misdiagnosis of a GIST as a “pelvic sarcoma.”180 So-called prostatic GISTs present with urinary obstructive symptoms, rectal fullness, and abnormal digital rectal examination.179-181
605
Microscopically, they show identical features to lesions within the GI tract. GIST is composed of spindle cells with a fascicular growth pattern, occasional epithelioid features, and focal dense collagenous stroma (Fig. 16-20). When present, a fascicular or palisading growth pattern and perinuclear vacuoles along with a lack of collagen deposition aid in the discrimination of GISTs from SFTs and STUMPs. Tumors with malignant potential show elevated mitotic rates of more than 5 per 50 hpf, cytologically malignant features (high cellularity and overlapping nuclei), and/or necrosis. Immunohisochemically, CD117/c-kit is uniformly expressed in all cases, and CD34 is positive in almost all cases studied. S100, desmin, and SMA are negative. On prostate needle biopsy, before rendering a diagnosis of SFT, schwannoma, leiomyosarcoma, or stromal sarcoma, GIST should be considered in the differential diagnosis. Furthermore, immunostains for CD117 should be performed to verify the diagnosis. CD34 is not discriminatory, because it is positive in GISTs, SFTs, and specialized prostatic stromal tumors, and it is variably positive in schwannomas. It is, however, typically negative in smooth muscle tumors. Strong positive staining for
A
B
C
Figure 16-20 A and B, Gastrointestinal stromal tumor involving prostate biopsy. C, Strong CD117 staining.
606
Immunohistology of the Prostate
desmin can help discriminate smooth muscle tumors from other lesions. Similarly, positive immunoreactivity to S-100 may aid in diagnosing neural tumors. SMA is typically expressed in smooth muscle tumors and is variably positive in STUMPs and GISTs and typically negative in SFTs and schwannomas. A subset of patients treated with the c-kit tyrosine kinase inhibitor imatinib (Gleevec) following the diagnosis of “prostatic” GIST demonstrated a subsequent reduction in tumor size.181
Beyond Immunohistochemistry: Theranostic and Genomic Applications The continuous debate on whether current serum PSAbased screening strategies potentially lead to overtreatment of a subset of PCa patients has further fueled the interest in pursuing clinicopathologic and molecular parameters that may help identify patients with biologically “significant” PCas.182,183 Also gaining momentum is the parallel pursuit of clinicopathologic algorithms and criteria that can accurately predict “insignificant” PCa, generally defined as tumors that lack the biologic potential to affect disease-specific mortality and morbidity within a given patient life expectancy. As alternative PCa management approaches such as proactive surveillance are increasingly offered, accurate identification of insignificant PCa becomes more pressing. Meanwhile, prostate needle biopsy remains the gold standard for establishing the diagnosis of PCa in patients with elevated serum PSA and or positive digital rectal exam. At this time, firmly established parameters such as clinical stage, pathologic stage, histologic Gleason grade, and serum PSA levels are routinely used for prognostication and guidance of disease management.184-186 Given the existing need to improve upon the prognostic and predictive power of the above established parameters, an extensive list of molecular biomarkers have been evaluated in the last decade for their potential role in enhancing our ability to predict disease progression, response to therapy, and survival based on the discovery of the key genetic alterations involved in the progression of PCa (Fig. 16-21).187-191 The perceived need to identify objective markers to supplement, or conceivably supplant, the more subjective established histologic parameters has been a major driving force behind biomarker discovery efforts. It is crucial to recognize and account for the potential variability that can exist even with the new molecular parameters. Sources of variability include differences in molecular technique methodologies, tissue fixation and processing, interobserver and intraobserver variability (in IHC based biomarkers), and differences in cut-off points.192 Furthermore, illustration of statistical significance for a particular biomarker does not alone assure its utility in a given patient, therefore a promising prognostic or therapeutic target biomarker should endure a rigorous evidence-based analysis and be validated in large prospective clinical trials before transition into use in
Normal prostate epithelium
TMPRSS2-ERG rearrangement
Proliferative inflammatory atrophy
GSTP1 promoter hypermethylation
PIN
Critical telomeres shortening C-myc alteration
p27 loss NKX3.1 loss Localized PCa PTEN loss AR mutation AR amplification
Metastatic PCa
Telomerase activation: immortalization
Castration resistant PCa Figure 16-21 Somatic genetic alterations involved in the pathogenetic steps of prostate cancer progression. AR, Androgen receptor; PCa, prostatic carcinoma; PIN, prostatic intraepithelial neoplasia.
standard practice.193 Table 16-4 lists salient genetic and epigenetic alterations in PCa.
Emerging Prognostic Factors According to the 1999 CAP Consensus Statement, prognostic and predictive factors in prostate carcinoma are stratified into three categories. Category I factors are considered proven to be useful in clinical practice and include preoperative PSA, tumor-node-metastasis (TNM) stage, Gleason grade, and surgical margins.194 Category II factors that have been extensively studied but await statistically robust trials include tumor volume, histologic type, and DNA ploidy analysis. Category III factors are those that still need additional studies to assure their prognostic utility before undergoing clinical trials. Currently pursued prognostic molecular biologic markers in PCa are categorized as level III factors. The wide array of molecular-based PCa markers include proliferation index (Ki-67), microvessel density, nuclear morphometry, tumor suppression genes (TP53, p21, p27, NKX3-1, PTEN, Rb), oncogenes (BCL2, MYC, EZH2, ERBB2), adhesion molecules (CD44, E-cadherin), PIK3CA/AKT1/MTOR (mechanistic target of rapamycin) pathway,195 apoptosis regulators (survivin, transforming growth factor β-1), androgen receptor status, neuroendocrine differentiation markers, and prostate tissue lineage–specific markers expression (PSA, PSAP, PSMA).196-198 PROLIFERATION INDEX
A single study has demonstrated that proliferation of cancer on biopsy as measured by Ki-67 and percentage of cells in S-phase and G2M better correlated with PSA failure after radical prostatectomy than did biopsy grade.199 Two additional studies have shown a similar role for Ki-67 index measurement as an independent
Beyond Immunohistochemistry: Theranostic and Genomic Applications
607
TABLE 16-4 Genetic and Epigenetic Alterations in Prostate Cancer Gene and Gene Type
Location
Notes
CDKN1B
12p13.1-p12
Encodes cyclin-dependent kinase inhibitor p27. One allele is frequently deleted in primary PCa.
NKX3-1
8p21.2
Encodes prostate-restricted homeobox protein that can suppress the growth of prostate epithelial cells. One allele is frequently deleted in primary PCa.
PTEN
10q23.31
Encodes phosphatase and tensin homolog, suppresses cell proliferation and increases apoptosis. One allele is frequently lost in primary PCa tumors. Mutations are found more frequently in metastatic PCa.
TP53
17p13.1
Mutations are uncommon early but occur in about 50% of advanced or castration-resistant PCa.
MYC
8q24
This transcription factor regulates genes involved in cell proliferation, senescence, apoptosis, and cell metabolism; mRNA levels are increased in all stages. Low-level amplification of the MYC locus is common in advanced PCa.
ERG
21q22.3
Fusion transcripts with the 5′ portion of androgen-regulated gene (TMPRSS2) arise from deletion or chromosomal rearrangements commonly found in PCa.
ETV1-ETV4
7p21.3, 19q13.12, 1q21-q23, 17q21.31
Encodes ETS-like transcription factors 1 through 4, which are proposed to be new oncogenes for prostate cancer. Fusion transcripts with the 5′ portion of androgen-regulated gene (TMPRSS2) arise from chromosomal rearrangements commonly found in all disease stages.
AR
Xq11-12
Encodes the androgen receptor. Protein is expressed in most PCa. Locus is amplified or mutated in advanced and castration-resistant PCa.
Activation of the enzyme telomerase
Maintains telomere function and contributes to cell immortalization. Activated in most PCa, mechanism of activation may be through MYC activation.
GSTP1
11q13
Encodes the enzyme that catalyzes the conjugation of reduced glutathione to electrophilic substrates; functions to detoxify carcinogens. Inactivated in more than 90% of PCa by somatic hypermethylation of the CpG island within the upstream regulatory region.
Telomere dysfunction
Chromosome termini
Contributes to chromosomal instability. Shortened telomeres are found in more than 90% of PIN lesions and PCa lesions.
Centrosome abnormalities
NA
Contributes to chromosomal instability. Centrosomes are structurally and numerically abnormal in most PCa.
Various
The hypermethylation of CpG islands within upstream regulatory regions occurs in most primary tumors and metastatic lesions. The functional significance of these changes is not yet known.
Tumor Suppressor Genes
Oncogenes
Caretaker Genes
Other Somatic Changes PTGS2, APC, MDR1, EDNRB, RASSF1A, rarB2
ETS, Erythroblast transformation specific; NA, not applicable; PCa, prostate cancer; PIN, prostatic intraepithelial neoplasia.
prognosticator in prostatectomy specimens.200,201 Conflicting reports have been furthered by others.202-205 ANGIOGENESIS
The mean number of microscopic blood vessels in tissue is higher in PCa and in PIN than in normal prostate
tissue. In a study to evaluate microvessel density (MVD) on needle biopsy, the authors found that MVD, when combined with Gleason score and preoperative PSA, provided improved ability to predict extraprostatic extension at radical prostatectomy.206 Although microvessel density was significant in the multivariate analysis, Gleason score and serum PSA were much
608
Immunohistology of the Prostate
more powerful predictors of extraprostatic disease. Three additional studies revealed a prognostic role for MVD in prostatectomy specimens.207-209 Others, however, failed to confirm such a role.210-212 Differences in vascular antibodies used and topography of vessel measurements could account for the variable results. It appears that MVD will have a marginal adjunctive role, if any, next to current established parameters.
epigenetic hypermethylation.230 Yegnasubramanian and associates231,232 found hypermethylation of GSTP1, APC, RASSF1, COX2, and MDR1 both in localized and metastatic PCa, whereas hypermethylation of other genes—such as ERα, hMLH1, and p14/CDKN2A (formerly INK4a)—were more likely to be found in later stages of PCa progression, suggesting two “waves” of epigenetic alterations.
NEUROENDOCRINE DIFFERENTIATION
ERG–ETS GENE FUSIONS
Two studies have shown neuroendocrine differentiation to be predictive of prognosis in organ-confined prostate carcinoma.213,214 However, several more recent studies have shown no prognostic role for neuroendocrine differentiation on needle biopsy or prostatectomy.215-218
The discovery by Tomlins and colleagues233,234 of a recurrent chromosomal rearrangement in more than half of their analyzed PCa cases is ranked as one of the most notable in solid tumor biology given the sheer prevalence of PCa. The recurrent chromosomal rearrangements lead to a fusion of the androgen-responsive promoter elements of the TMPRSS2 gene (21q22) to one of three members of the E26 transformation– specific (ETS) transcription factors family members— ERG, ETV1, and ETV4—located at chromosomes 21q22, 7p21, and 17q21 respectively (see Fig. 16-21). Although the prognostic role of assessing TMPRSS2ETS rearrangements in PCa tissue samples has been called into question by recent well-designed large cohort studies,235,236 the discovery will no doubt have great implications in terms of furthering our understanding of the steps involved in the development and pathogenesis of PCa, and it will provide a new marker for molecular diagnosis and potential targets of therapy in PCa.237-246 The potential diagnostic and prognostic role of detecting TMPRSS2-ERG in post prostate massage urine samples requires further investigation.247-249 Figure 16-22 depicts a commonly used FISH split-apart–based approach for the evaluation of ERG gene fusion. Recently, commercial anti-ERG monoclonal antibodies became available that make it possible to use IHC for evaluating ERG protein expression as a surrogate approach to detecting TMPRSS2-ERG fusion by FISH. We and others have demonstrated a strong correlation between ERG overexpression by IHC and ERG fusion status with more than 86% sensitivity and specificity rates. ERG IHC may offer an accurate, simpler, and less costly alternative for evaluation of ERG fusion status in PCa on needle biopsy and radical prostatectomy samples (Fig. 16-23).250,251
MORPHOMETRY/KARYOMETRY
Nuclear morphometric measurements on needle biopsy have also been demonstrated in limited studies to correlate with PCa prognosis. The same group, however, found lack of correlation in nuclear measurements between needle and corresponding prostatectomy tissue.219,220 Several studies from our institution have looked at the prognostic role of the integration of multiple nuclear morphometry parameters and variables221,222 and at the potentials of integration of nuclear morphometry parameters with other established factors, such as grade and stage. The latter appear to enhance the prediction accuracy of established parameters.223,224 PROSTATIC LINEAGE–SPECIFIC MARKERS
The expression of prostatic markers such as PSAP and PSMA225,226 have been linked to prognosis in an occasional study. The degree of PSA expression does not appear to correlate with progression.226 EPIGENETIC CHANGES IN PROSTATE CANCER
Changes in DNA methylation marks, accompanied by epigenetic gene silencing, appear to be the earliest somatic genome changes in PCa.227 A new generation of assay strategies for detection of specific DNA sequences that carry 5-meC offer promising opportunities for clinical tests for potential PCa screening, detection, diagnosis, staging, and risk stratification. Hypermethylation of glutathione S-transferase 1 (GSTP1) transcriptional regulatory sequences has been consistently detected in more than 90% of PCa. GSTP1 encodes an enzyme responsible for detoxifying electrophiles and oxidants, thus shielding cells from genome damage. Loss of GSTP1 expression appears to be an early event in the initiation of prostatic carcinogenesis as evidenced by the presence of GSTP1 methylation in 5% to 10% of proliferative inflammatory atrophy (PIA) lesions, thought by some to be the earliest PCa precursors, and in more than 70% of high-grade PIN lesions.228,229 In addition to GSTP1, more than 40 other genes have been shown to be altered by
PIK3CA/MTOR PATHWAY
The PIK3CA/MTOR pathway plays an important role in cell growth, proliferation, and oncogenesis in PCa.252-258 PTEN is a negative regulator of this pathway. Several recent well-designed retrospective studies have revealed that loss of PTEN tumor suppressor gene activity and the ensuing MTOR pathway activation is associated with poor prognosis in PCa. In a recent, large, nested, case-control tissue microarray–based study from our institution, we were able to show loss of immunoexpression of PTEN to be a predictor of biochemical recurrence following radical prostatectomy independent of Gleason grade, cancer stage, and other
Beyond Immunohistochemistry: Theranostic and Genomic Applications
TMPRSS2 5
609
ERG 3
5
3
5
3
A
TMPRSS2 5
ERG 3
B Figure 16-22 Fluorescence in situ hybridization analysis by using ERG split-apart probes. A, The presence of juxtaposed red and green signals that occasionally form a yellow signal indicates lack of TMPRSS2-ERG fusion in the benign glands. B, Loss of green signal in one allele indicates the presence of TMPRSS2-ERG fusion by deletion involving the 5′ ERG region, as shown in the malignant glands.
clinicopathologic parameters.259 In a second study from our group by Lotan and colleagues,260 the prognostic role of PTEN alteration was further linked to adverse pathologic features and decreased time to metastatic disease in a surgical cohort of high-risk PCa patients. The correlation of PTEN immuostains with genomic loss of the PTEN gene was also established in the later study. The MTOR pathway is also a potential target for PCa treatment, and several rapamycin analogs are currently being tested as potential therapeutic agents for PCa.257,261 We recently reported the results of a pilot study to evaluate the pharmacodynamic efficacy of neoadjuvant rapamycin therapy in PCa.261 Using IHC analysis, we found a significant decrease in Phos-S6 protein, the main downstream effector of the MTOR pathway, in patients receiving a neoadjuvant mTOR inhibitor.261
OTHER TUMOR SUPPRESSOR GENES AND ONCOGENES
Among tumor suppressor genes, the role of P53 expression in predicting prognosis in prostate carcinoma has been extensively studied. Brewster and colleagues found P53 expression and Gleason score in needle biopsy to be independent predictors of biochemical relapse after radical prostatectomy.262 Another study found P53 status on prostatectomy, but not needle biopsies, to be predictive, which raises the issue of sampling.263 Many studies to evaluate prostatectomy specimens found P53 to be of prognostic significance independent of grade, stage, or margin status.204,212,264-269 More recent genomewide studies seem to support the prognostic role of P53 alterations (see the section below on integrated
610
Immunohistology of the Prostate
A
B
C
D
E
F
Figure 16-23 ERG overexpression, as demonstrated by immunohistochemistry, is a simple surrogate method for evaluating TMPRSS2-ERG fusion in prostate adenocarcinoma. A to D, ERG-positive expression in cases graded as Gleason 6 and 8, respectively, that were also positive for TMPRSS2-ERG fusion by fluorescence in situ hybridization (FISH). E to F, Lack of ERG expression in a Gleason grade 6 tumor that lacked TMPRSS2-ERG fusion by FISH. (ERG immunostains; A, C, and E, ×100; B, D, and F, ×200).
genomics).270 The majority of studies of another tumor suppressor gene, p27, a cell-cycle inhibitor, have also supported a correlation with progression after prostatectomy. Although less robust evidence exists for the prognostic role of p21,271 a downstream mediator of P53, and transcription factors such as NKX3-1,272,273 a preponderance of evidence supports a prognostic role
for Bcl2200,262,264,266,268 and myc oncogenes274,275 as potential adjuncts to histologic prognostic parameters. INTEGRATED GENOMICS
Gene-expression profiling studies using complementary DNA (cDNA) microarrays that contain 26,000 genes
Beyond Immunohistochemistry: Theranostic and Genomic Applications
identified three subclasses of prostate tumors based on distinct patterns of gene expression.8 High-grade and advanced-stage tumors and those associated with recurrence were disproportionately represented among two of the three subtypes, one of which also included most lymph node metastases. Furthermore, two surrogate genes were differentially expressed among tumor subgroups by IHC. These included MUC1, a gene highly expressed in the subgroups with aggressive clinicopathologic features, and AZGP1, a gene highly expressed in the favorable subgroup. The surrogate genes were strong predictors of tumor recurrence independent of tumor grade, stage, and preoperative PSA levels. Such study suggests that prostate tumors can be usefully classified according to their gene-expression patterns, and these tumor subtypes may provide a basis for improved prognostication and treatment stratification. In another study, Tomlins and colleagues276 used laser-capture microdissection to isolate 101 cell populations to illustrate gene-expression profiles of PCa progression from benign epithelium to metastatic disease. By analyzing expression signatures in the context of more than 14,000 molecular concepts, or sets of biologically connected genes, the authors generated an integrative model of progression. Molecular critical transitions in progression included protein biosynthesis, ETS family transcriptional targets, androgen signaling, and cell proliferation. Known prognostic markers such as grade could be ascribed to the noted attenuated androgensignaling signature seen in high-grade cancer (Gleason pattern 4), similar to metastatic PCa, which may reflect dedifferentiation and may explain the clinical association of grade and prognosis. Taken together, these data show that analyzing gene-expression signatures in the context of a compendium of molecular concepts is useful in understanding cancer biology. Lapointe and colleagues276 complemented their above-mentioned gene-expression findings by looking for associated copy-number alterations using arraybased comparative genomic hybridization (array CGH). They were able to identify recurrent copy-number aberrations that correspond to three prognostically distinct groups of PCa: 1) deletions at the 5q21 and 6q15 deletion group, associated with favorable outcome; 2) an 8p21 (NKX3-1) and 21q22 deletion group, resulting in TMPRSS2-ERG fusion and 8q24 (MYC) and 16p13 gains; and 3) loss at 10q23 (PTEN) and 16q23 groups that correlated with metastatic disease and aggressive outcome. Finally, in a recent genome-wide analysis of PCa, Taylor and associates277 elegantly illustrated how detailed annotation of PCa genomes can impact our understanding of the disease and its treatment strategy. Assessing DNA copy number, messenger RNA (mRNA) expression, and focused exon resequencing in 218 PCa tumors, the authors identified the role of nuclear receptor coactivator NCOA2 as a novel oncogene in 11% of PCa cases. TMPRSS2-ERG fusion was associated with novel prostate-specific deletion at chromosome 3p14 that may implicate FOXP1, RYBP, and SHQ1 as potential cooperative tumor suppressors. Most intriguing was their ability to define clusters of low-risk and high-risk
611
disease beyond that achieved by Gleason score using DNA copy-number data. As shown in Figure 16-24, six clusters of PCa tumors are identified by unsupervised hierarchical clustering with distinct risk for biochemical recurrence. Markert and colleagues270 also illustrated the potential utility of molecular signatures as a prognosticator in PCa. The authors assessed microarray datasets that characterized 281 PCa patients from a Swedish watchfulwaiting cohort; mRNA microarray signature profiles for gene signatures that reflected embryonic stem cell (ESC), induced pleuripotent stem cell (iPSC), and polycomb repressive complex 2 phenotypes (PRC2) were assessed in addition to inactivation of the tumor suppressor P53 and PTEN loss and the TMPRSS2-ERG fusion. Unsupervised clustering identified a PCa subset with “stemlike signatures” combined with P53 and PTEN inactivation to be associated with very poor survival outcome. PCa tumors characterized by TMPRSS2ERG fusion had intermediate survival outcome, whereas remaining groups demonstrated more favorable outcome (Fig. 16-25). These exciting findings were further validated in an independent clinical cohort at Memorial Sloan-Kettering Cancer Center. This classification was independent of Gleason score and therefore can provide added value in prognostication in patients with lower Gleason grade PCa. In summary, genomic studies suggest that PCa develops via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating PCa biology and explain in part the clinical heterogeneity of the disease.
Emerging Early Detection Markers and Targets of Therapy Markers of PCa detection that can be applied to blood, urine, or prostatic secretion fluid (ejaculate or prostate massage fluids) have been the focus of active recent research. Markers that have been investigated in the urine or prostatic secretions include gene promoter hypermethylation profile assays278-281 and DD3 (differential display code 3), now known as PCA3, a noncoding RNA initially identified by Bussemakers and associates282 as one of the most specific markers of PCa. The PCA3 gene is located on chromosome 9q21.2 (Fig. 16-26). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay to detect PCA3 can be applied to blood, urine, or prostatic fluid.283 Evaluation of PCA3 in postattentive prostate massage urine samples by using transcription mediated amplification (TMA) technology has been shown to be superior to serum PSA in predicting biopsy outcome; sensitivity and specificity are approximately 70% and 80%, respectively, with a negative predictive value of 90%.284-287 The PCA3 PROGENSA assay is the first such assay to be approved by the U.S. Food and Drug Administration (FDA). Encouraging data from the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial support a role for evaluation of PCA3
612
Immunohistology of the Prostate
METs
6q 7 8p 8q 13q 16q 18q 1
2
3
4
5
6
A 1.0
Cluster 2
Probability of freedom from biochemical recurrence
0.8
Cluster 6 Cluster 3
0.6
** **
* *
Cluster 4 Cluster 1
0.4
** * ** *
Cluster 5 0.2 **P < .005 0
*P < .05 0
15
30
45 60 Time (months)
75
90
105
B Figure 16-24 Clinically distinct groups of prostate cancer (PCa) are identified based on genomic alterations. A, Unsupervised hierarchical clustering of copy-number alterations identified six groups (clusters) of PCa. B, Statistically significant differences in freedom from biochemical recurrence are found among the six groups. Modified from Aslan G, Irer B, Tuna B, et al: Analysis of NKX3.1 expression in prostate cancer tissues and correlation with clinicopathologic features. Pathol Res Pract 2006;202:93-98.
in postattentive prostate massage urine sample in predicting positive prostate needle biopsy in immediately subsequent, as well as future, biopsies following initial negative biopsy. PCA3 may also have a role in predicting the risk for higher Gleason score and larger tumor volume on radical retropubic prostatectomy (RRP). If confirmed, the latter could be of great value in a treatment options algorithm and in delineation of candidates
for active surveillance.288-291 Multiplex urine assays to include PCA3, TMPRSS2-ERG, SPINK1, and Golph2 are also under evaluation, and recent data suggest improved performance of such assays compared with PCA3 alone.292 Finally, several markers are being investigated as potential targets of therapy for PCa. The list includes tyrosine kinase receptors (EGFR), angiogenesis targets
Cluster 1 Cluster 2
1
Cluster 3 Cluster 4
0.8
Cluster 5
0.6 0.4 0.2 0
0
50
100 150 200 Time (m)
250
A
1.2 Stem-like (Cluster 1)
1
Other Censored
0.8
P = 4.4109e -08
0.6 0.4 0.2 0
0
50
100 150 200 Time (m)
250
B Size (%)
613
1.2
Estimated overall survival functions
1.2
Estimated overall survival functions
Estimated overall survival functions
Beyond Immunohistochemistry: Theranostic and Genomic Applications
Gleason low (6, 7)
1
Gleason high (8, 9, 10) Censored
0.8
P 1.0e 13
0.6 0.4 0.2 0
0
50
100 150 200 Time (m)
250
C
Major Gleason Gleason
Death from PC (%)
Follow- Followup time up (m) censored
DSS time (m)
Fusion (FISH)
Age
P value (KM)
0.81** 57.34****
0.13
42.88***
0.13
74.84
4.41E008
3.61
0.76**
93.04
0.16
73.62
0.58****
74.71
0.005
7.25
3.44
0.54
103.44
0.38
72.00
0.11
72.46
0.39
0.27
7.03
3.41
0.51
108.05
0.28
69.16
0.07**
74.89
0.17
0.27
6.72**
3.27****
0.48* 120.61***
0.32
80.97
0.04***
73.2
1
ESC | P53 | PTEN
0.11
8.22****
3.91***
TMPRSS2-ERG fusion
0.18
7.49
Cytokine | RAS | Mesenchyme
0.17
Transitional PRC2 | differentiated
*P .05, **P .01, ***P .001, ****P .0001
D
9p24.1 9p24.2 9p24.3
9p21.1 9p321.2 9p21.3 9p22.1 9p22.2 9p22.3 9p23
9p13.3
9q11 9p11.1 9p11.2 9p12 9p13.1 9p13.2
9q12
9q21.31 9q21.2 9q21.13 9q21.12 9q21.11 9q13
9q21.32
9q21.33
9q22.33 9q22.32 9q22.31 9q22.2 9q22.1
9q31.3 9q31.2 9q31.1
9q32
9q34.2 9q34.13 9q34.12 9q34.11 9q33.3 9q33.2 9q33.1
9q34.3
Figure 16-25 Clinical outcome data for the Swedish watchful-waiting cohort in distinct molecular profile subgroups found by signature profiling. A to C, Kaplan-Meier estimates for survival functions for the different subgroups include a side-by-side comparison of survival analysis based on signature profiling (A and B) and Gleason score (C). D, Clinical variables for the subgroups show a highly significant prognostic value for the stemlike subtype. Significance of assignments is indicated by asterisks. DSS, Disease-specific survival; FISH, fluorescence in situ hybridization; KM, Kaplan-Meier; PC, prostatic carcinoma. Modified from Markert EK, Mizuno H, Vazquez A, Levine AJ: Molecular classification of prostate cancer using curated expression signatures. Proc Natl Acad Sci U S A 2011;108:21276-21281.
9q21.2 1
2
3
4
14 kb 2 kb
1 3
4a
4b
1 3
4a
4b
1 3
4a
4c
0.5 kb
Figure 16-26 Structure of the PCA3 gene (formerly DD3). The gene is located at the chromosome 9q21-22 locus and consists of four exons. Alternative polyadenylation at three different positions in exon 4—4a, 4b, and 4c—gives rise to three different-sized transcripts. The most frequently found transcript contains exons 1, 3, 4a, and 4b.
614
Immunohistology of the Prostate
(VEGF),293 fatty acid synthase (FAS),294 PIK3CA/ AKT1/MTOR,257,261,295 endothelin receptors,296,297 and PSMA298-301 to name a few.
Summary A wide array of molecular markers discussed in this chapter may be utilized in the near future as adjuncts to currently established prognostic parameters and early
detection markers. Loss of PTEN and PCA3 are two such markers that are most likely to soon gain widespread utilization. Current research efforts are also focused on biologic markers that can serve as targets of therapy. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Srigley JR, Amin M, Boccon-Gibod L, et al: Prognostic and predictive factors in prostate cancer: Historical perspectives and recent international consensus initiatives. Scand J Urol Nephrol Suppl. 216:8–19, 2005. 2. Schalken JA, Bergh A, Bono A, et al: Molecular prostate cancer pathology: Current issues and achievements. Scand J Urol Nephrol Suppl. 216:82–93, 2005. 3. Epstein JI, Amin M, Boccon-Gibod L, et al: Prognostic factors and reporting of prostate carcinoma in radical prostatectomy and pelvic lymphadenectomy specimens. Scand J Urol Nephrol Suppl. 216:34–63, 2005. 4. DeMarzo AM, Nelson WG, Isaacs WB, et al: Pathological and molecular aspects of prostate cancer. Lancet. 361:955–964, 2003. 5. Amin M, Boccon-Gibod L, Egevad L, et al: Prognostic and predictive factors and reporting of prostate carcinoma in prostate needle biopsy specimens. Scand J Urol Nephrol Suppl. 216:20– 33, 2005. 6. Nelson WG, De Marzo AM, Isaacs WB: Prostate cancer. N Engl J Med. 349:366–381, 2003. 7. Prowatke I, Devens F, Benner A, et al: Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays. Br J Cancer. 96:82–88, 2007. 8. Lapointe J, Li C, Higgins JP, et al: Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci U S A. 101:811–816, 2004. 9. Khan MA, Sokoll LJ, Chan DW, et al: Clinical utility of proPSA and “benign” PSA when percent free PSA is less than 15%. Urology. 64:1160–1164, 2004. 10. Tomlins SA, Mehra R, Rhodes DR, et al: Integrative molecular concept modeling of prostate cancer progression. Nat Genet. 39:41–51, 2007. 11. Gretzer MB, Partin AW: PSA markers in prostate cancer detection. Urol Clin North Am. 30:677–686, 2003. 12. Shen S, Lepor H, Yaffee R, et al: Ultrasensitive serum prostate specific antigen nadir accurately predicts the risk of early relapse after radical prostatectomy. J Urol. 173:777–780, 2005. 13. Chuang AY, DeMarzo AM, Veltri RW, et al: Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma. Am J Surg Pathol. 31:1246–1255, 2007. 14. Kamoshida S, Tsutsumi Y: Extraprostatic localization of prostatic acid phosphatase and prostate-specific antigen: Distribution in cloacogenic glandular epithelium and sex-dependent expression in human anal gland. Hum Pathol. 21:1108–1111, 1990. 15. James GK, Pudek M, Berean KW, et al: Salivary duct carcinoma secreting prostate-specific antigen. Am J Clin Pathol. 106:242– 247, 1996. 16. Diamandis EP: Prostate specific antigen—new applications in breast and other cancers. Anticancer Res. 16:3983–3984, 1996. 17. Lane Z, Hansel DE, Epstein JI: Immunohistochemical expression of prostatic antigens in adenocarcinoma and villous adenoma of the urinary bladder. Am J Surg Pathol. 32:1322–1326, 2008. 18. Chang SS, O’Keefe DS, Bacich DJ, et al: Prostate-specific membrane antigen is produced in tumor-associated neovasculature. Clin Cancer Res. 5:2674–2681, 1999. 19. Chang SS, Reuter VE, Heston WD, et al: Five different antiprostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res. 59:3192–3198, 1999. 20. Chang SS, Reuter VE, Heston WD, et al: Comparison of anti-prostate-specific membrane antigen antibodies and other immunomarkers in metastatic prostate carcinoma. Urology. 57:1179–1183, 2001. 21. Silver DA, Pellicer I, Fair WR, et al: Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 3:81–85, 1997. 22. Murphy GP, Elgamal AA, Su SL, et al: Current evaluation of the tissue localization and diagnostic utility of prostate specific membrane antigen. Cancer. 83:2259–2269, 1998. 23. Marchal C, Redondo M, Padilla M, et al: Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia. Histol Histopathol. 19:715–718, 2004.
614.e1
24. Bostwick DG, Pacelli A, Blute M, et al: Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: A study of 184 cases. Cancer. 82:2256– 2261, 1998. 25. Yao V, Parwani A, Maier C, et al: Moderate expression of prostate-specific membrane antigen, a tissue differentiation antigen and folate hydrolase, facilitates prostate carcinogenesis. Cancer Res. 68:9070–9077, 2008. 26. Ross JS, Sheehan CE, Fisher HA, et al: Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res. 9:6357– 6362, 2003. 27. Chang SS, Reuter VE, Heston WD, et al: Short term neoadjuvant androgen deprivation therapy does not affect prostate specific membrane antigen expression in prostate tissues. Cancer. 88:407–415, 2000. 28. Bander NH: Technology insight: Monoclonal antibody imaging of prostate cancer. Nat Clin Pract Urol. 3:216–225, 2006. 29. Bander NH, Milowsky MI, Nanus DM, et al: Phase I trial of 177lutetium-labeled J591, a monoclonal antibody to prostatespecific membrane antigen, in patients with androgenindependent prostate cancer. J Clin Oncol. 23:4591–4601, 2005. 30. Elsasser-Beile U, Wolf P, Gierschner D, et al: A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer. Prostate. 66:1359–1370, 2006. 31. Sodee DB, Sodee AE, Bakale G: Synergistic value of singlephoton emission computed tomography/computed tomography fusion to radioimmunoscintigraphic imaging of prostate cancer. Semin Nucl Med. 37:17–28, 2007. 32. Mannweiler S, Amersdorfer P, Trajanoski S, et al: Heterogeneity of prostate-specific membrane antigen (PSMA) expression in prostate carcinoma with distant metastasis. Pathol Oncol Res. 2008. 33. Milowsky MI, Nanus DM, Kostakoglu L, et al: Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced solid tumors. J Clin Oncol. 25:540–547, 2007. 34. Olson WC, Heston WD, Rajasekaran AK: Clinical trials of cancer therapies targeting prostate-specific membrane antigen. Rev Recent Clin Trials. 2:182–190, 2007. 35. van Dieijen-Visser MP, Delaere KP, Gijzen AH, et al: A comparative study on the diagnostic value of prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) in patients with carcinoma of the prostate gland. Clin Chim Acta. 174:131–140, 1988. 36. Ersev A, Ersev D, Turkeri L, et al: The relation of prostatic acid phosphatase and prostate specific antigen with tumour grade in prostatic adenocarcinoma: An immunohistochemical study. Prog Clin Biol Res. 357:129–134, 1990. 37. Sheridan T, Herawi M, Epstein JI, et al: The role of P501S and PSA in the diagnosis of metastatic adenocarcinoma of the prostate. Am J Surg Pathol. 31:1351–1355, 2007. 38. Kusumi T, Koie T, Tanaka M, et al: Immunohistochemical detection of carcinoma in radical prostatectomy specimens following hormone therapy. Pathol Int. 58:687–694, 2008. 39. Osunkoya AO, Netto GJ, Epstein JI: Colorectal adenocarcinoma involving the prostate: Report of 9 cases. Hum Pathol. 38:1836– 1841, 2007. 40. Wang W, Epstein JI: Small cell carcinoma of the prostate. A morphologic and immunohistochemical study of 95 cases. Am J Surg Pathol. 32:65–71, 2008. 41. Yin M, Dhir R, Parwani AV: Diagnostic utility of p501s (prostein) in comparison to prostate specific antigen (PSA) for the detection of metastatic prostatic adenocarcinoma. Diagn Pathol. 2:41, 2007. 42. Luo J, Zha S, Gage WR, et al: Alpha-methylacyl-CoA racemase: A new molecular marker for prostate cancer. Cancer Res. 62:2220–2226, 2002. 43. Jiang Z, Woda BA: Diagnostic utility of alpha-methylacyl CoA racemase (P504S) on prostate needle biopsy. Adv Anat Pathol. 11:316–321, 2004. 44. Jiang Z, Wu CL, Woda BA, et al: P504S/alpha-methylacyl-CoA racemase: A useful marker for diagnosis of small foci of prostatic
614.e2
Immunohistology of the Prostate
carcinoma on needle biopsy. Am J Surg Pathol. 26:1169–1174, 2002. 45. Jiang Z, Li C, Fischer A, et al: Using an AMACR (P504S)/34betaE12/p63 cocktail for the detection of small focal prostate carcinoma in needle biopsy specimens. Am J Clin Pathol. 123:231–236, 2005. 46. Magi-Galluzzi C, Luo J, Isaacs WB, et al: Alpha-methylacyl-CoA racemase: A variably sensitive immunohistochemical marker for the diagnosis of small prostate cancer foci on needle biopsy. Am J Surg Pathol. 27:1128–1133, 2003. 47. Kunju LP, Rubin MA, Chinnaiyan AM, et al: Diagnostic usefulness of monoclonal antibody P504S in the workup of atypical prostatic glandular proliferations. Am J Clin Pathol. 120:737– 745, 2003. 48. Molinie V, Fromont G, Sibony M, et al: Diagnostic utility of a p63/alpha-methyl-CoA-racemase (p504s) cocktail in atypical foci in the prostate. Mod Pathol. 17:1180–1190, 2004. 49. Zhou M, Aydin H, Kanane H, et al: How often does alphamethylacyl-CoA-racemase contribute to resolving an atypical diagnosis on prostate needle biopsy beyond that provided by basal cell markers? Am J Surg Pathol. 28:239–243, 2004. 50. Zhou M, Jiang Z, Epstein JI: Expression and diagnostic utility of alpha-methylacyl-CoA-racemase (P504S) in foamy gland and pseudohyperplastic prostate cancer. Am J Surg Pathol. 27:772– 778, 2003. 51. Farinola MA, Epstein JI: Utility of immunohistochemistry for alpha-methylacyl-CoA racemase in distinguishing atrophic prostate cancer from benign atrophy. Hum Pathol. 35:1272–1278, 2004. 52. Dorer R, Odze RD: AMACR immunostaining is useful in detecting dysplastic epithelium in barrett’s esophagus, ulcerative colitis, and crohn’s disease. Am J Surg Pathol. 30:871–877, 2006. 53. Lin F, Brown RE, Shen T, et al: Immunohistochemical detection of P504S in primary and metastatic renal cell carcinomas. Appl Immunohistochem Mol Morphol. 12:153–159, 2004. 54. Sanderson SO, Sebo TJ, Murphy LM, et al: An analysis of the p63/alpha-methylacyl coenzyme A racemase immunohistochemical cocktail stain in prostate needle biopsy specimens and tissue microarrays. Am J Clin Pathol. 121:220–225, 2004. 55. Wojno KJ, Epstein JI: The utility of basal cell-specific anticytokeratin antibody (34 beta E12) in the diagnosis of prostate cancer. A review of 228 cases. Am J Surg Pathol. 19:251–260, 1995. 56. Kahane H, Sharp JW, Shuman GB, et al: Utilization of high molecular weight cytokeratin on prostate needle biopsies in an independent laboratory. Urology. 45:981–986, 1995. 57. Oliai BR, Kahane H, Epstein JI: Can basal cells be seen in adenocarcinoma of the prostate?: An immunohistochemical study using high molecular weight cytokeratin (clone 34betaE12) antibody. Am J Surg Pathol. 26:1151–1160, 2002. 58. Novis DA, Zarbo RJ, Valenstein PA: Diagnostic uncertainty expressed in prostate needle biopsies. A college of american pathologists Q-probes study of 15,753 prostate needle biopsies in 332 institutions. Arch Pathol Lab Med. 123:687–692, 1999. 59. Abrahams NA, Ormsby AH, Brainard J: Validation of cytokeratin 5/6 as an effective substitute for keratin 903 in the differentiation of benign from malignant glands in prostate needle biopsies. Histopathology. 41:35–41, 2002. 60. Humphrey PA: Diagnosis of adenocarcinoma in prostate needle biopsy tissue. J Clin Pathol. 60:35–42, 2007. 61. Signoretti S, Waltregny D, Dilks J, et al: P63 is a prostate basal cell marker and is required for prostate development. Am J Pathol. 157:1769–1775, 2000. 62. Grisanzio C, Signoretti S: P63 in prostate biology and pathology. J Cell Biochem. 103:1354–1368, 2008. 63. Shah RB, Zhou M, LeBlanc M, et al: Comparison of the basal cell-specific markers, 34betaE12 and p63, in the diagnosis of prostate cancer. Am J Surg Pathol. 26:1161–1168, 2002. 64. Shah RB, Kunju LP, Shen R, et al: Usefulness of basal cell cocktail (34betaE12 + p63) in the diagnosis of atypical prostate glandular proliferations. Am J Clin Pathol. 122:517–523, 2004. 65. Zhou M, Shah R, Shen R, et al: Basal cell cocktail (34betaE12 + p63) improves the detection of prostate basal cells. Am J Surg Pathol. 27:365–371, 2003. 66. Signoretti S, Loda M: Defining cell lineages in the prostate epithelium. Cell Cycle. 5:138–141, 2006.
67. Weinstein MH, Signoretti S, Loda M: Diagnostic utility of immunohistochemical staining for p63, a sensitive marker of prostatic basal cells. Mod Pathol. 15:1302–1308, 2002. 68. Ali TZ, Epstein JI: False positive labeling of prostate cancer with high molecular weight cytokeratin: P63 a more specific immunomarker for basal cells. Am J Surg Pathol. 2008. 69. Osunkoya AO, Hansel DE, Sun X, et al: Aberrant diffuse expression of p63 in adenocarcinoma of the prostate on needle biopsy and radical prostatectomy: Report of 21 cases. Am J Surg Pathol. 32:461–467, 2008. 70. Aslan G, Irer B, Tuna B, et al: Analysis of NKX3.1 expression in prostate cancer tissues and correlation with clinicopathologic features. Pathol Res Pract. 202:93–98, 2006. 71. Bowen C, Bubendorf L, Voeller HJ, et al: Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res. 60:6111–6115, 2000. 72. Abate-Shen C, Shen MM, Gelmann E: Integrating differentiation and cancer: The Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis. Differentiation. 76:717–727, 2008. 73. Magi-Galluzzi C, Epstein JI: Threshold for diagnosing prostate cancer over time. Hum Pathol. 34:1116–1118, 2003. 74. Farinola MA, Epstein JI: Utility of immunohistochemistry for alpha-methylacyl-CoA racemase in distinguishing atrophic prostate cancer from benign atrophy. Hum Pathol. 35:1272–1278, 2004. 75. Yang XJ, Wu CL, Woda BA, et al: Expression of alpha-methylacylCoA racemase (P504S) in atypical adenomatous hyperplasia of the prostate. Am J Surg Pathol. 26:921–925, 2002. 76. Gaudin PB, Epstein JI: Adenosis of the prostate. histologic features in needle biopsy specimens. Am J Surg Pathol. 19:737–747, 1995. 77. Gaudin PB, Epstein JI: Adenosis of the prostate. histologic features in transurethral resection specimens. Am J Surg Pathol. 18:863–870, 1994. 78. Bostwick DG, Amin MB, Dundore P, et al: Architectural patterns of high-grade prostatic intraepithelial neoplasia. Hum Pathol. 24:298–310, 1993. 79. Kramer CE, Epstein JI: Nucleoli in low-grade prostate adenocarcinoma and adenosis. Hum Pathol. 24:618–623, 1993. 80. Hedrick L, Epstein JI: Use of keratin 903 as an adjunct in the diagnosis of prostate carcinoma. Am J Surg Pathol. 13:389–396, 1989. 81. Luque RJ, Lopez-Beltran A, Perez-Seoane C, et al: Sclerosing adenosis of the prostate. histologic features in needle biopsy specimens. Arch Pathol Lab Med. 127:e14–e16, 2003. 82. Sakamoto N, Tsuneyoshi M, Enjoji M: Sclerosing adenosis of the prostate. histopathologic and immunohistochemical analysis. Am J Surg Pathol. 15:660–667, 1991. 83. Grignon DJ, Ro JY, Srigley JR, et al: Sclerosing adenosis of the prostate gland. A lesion showing myoepithelial differentiation. Am J Surg Pathol. 16:383–391, 1992. 84. Jones EC, Clement PB, Young RH: Sclerosing adenosis of the prostate gland. A clinicopathological and immunohistochemical study of 11 cases. Am J Surg Pathol. 15:1171–1180, 1991. 85. Nelson RS, Epstein JI: Prostatic carcinoma with abundant xanthomatous cytoplasm. Foamy gland carcinoma. Am J Surg Pathol. 20:419–426, 1996. 86. Chuang AY, Epstein JI: Xanthoma of the prostate: A mimicker of high-grade prostate adenocarcinoma. Am J Surg Pathol. 31:1225–1230, 2007. 87. Sebo TJ, Bostwick DG, Farrow GM, et al: Prostatic xanthoma: A mimic of prostatic adenocarcinoma. Hum Pathol. 25:386–389, 1994. 88. Balaji KC, Rabbani F, Tsai H, et al: Effect of neoadjuvant hormonal therapy on prostatic intraepithelial neoplasia and its prognostic significance. J Urol. 162:753–757, 1999. 89. Tetu B, Srigley JR, Boivin JC, et al: Effect of combination endocrine therapy (LHRH agonist and flutamide) on normal prostate and prostatic adenocarcinoma. A histopathologic and immunohistochemical study. Am J Surg Pathol. 15:111–120, 1991. 90. Murphy WM, Soloway MS, Barrows GH: Pathologic changes associated with androgen deprivation therapy for prostate cancer. Cancer. 68:821–828, 1991. 91. Smith DM, Murphy WM: Histologic changes in prostate carcinomas treated with leuprolide (luteinizing hormone-releasing
References hormone effect). distinction from poor tumor differentiation. Cancer. 73:1472–1477, 1994. 92. Armas OA, Aprikian AG, Melamed J, et al: Clinical and pathobiological effects of neoadjuvant total androgen ablation therapy on clinically localized prostatic adenocarcinoma. Am J Surg Pathol. 18:979–991, 1994. 93. Vailancourt L, Ttu B, Fradet Y, et al: Effect of neoadjuvant endocrine therapy (combined androgen blockade) on normal prostate and prostatic carcinoma. A randomized study. Am J Surg Pathol. 20:86–93, 1996. 94. Accetta PA, Gardner WA: Squamous metastases from prostatic adenocarcinoma. Prostate. 3:515–521, 1982. 95. Accetta PA, Gardner WA, Jr: Adenosquamous carcinoma of prostate. Urology. 22:73–75, 1983. 96. Parwani AV, Kronz JD, Genega EM, et al: Prostate carcinoma with squamous differentiation: An analysis of 33 cases. Am J Surg Pathol. 28:651–657, 2004. 97. Vernon SE, Williams WD: Pre-treatment and post-treatment evaluation of prostatic adenocarcinoma for prostatic specific acid phosphatase and prostatic specific antigen by immunohistochemistry. J Urol. 130:95–98, 1983. 98. Grignon D, Troster M: Changes in immunohistochemical staining in prostatic adenocarcinoma following diethylstilbestrol therapy. Prostate. 7:195–202, 1985. 99. Pisansky TM: External-beam radiotherapy for localized prostate cancer. N Engl J Med. 355:1583–1591, 2006. 100. Bostwick DG, Egbert BM, Fajardo LF: Radiation injury of the normal and neoplastic prostate. Am J Surg Pathol. 6:541–551, 1982. 101. Magi-Galluzzi C, Sanderson H, Epstein JI: Atypia in nonneoplastic prostate glands after radiotherapy for prostate cancer: Duration of atypia and relation to type of radiotherapy. Am J Surg Pathol. 27:206–212, 2003. 102. Brawer MK, Nagle RB, Pitts W, et al: Keratin immunoreactivity as an aid to the diagnosis of persistent adenocarcinoma in irradiated human prostates. Cancer. 63:454–460, 1989. 103. Yang XJ, Laven B, Tretiakova M, et al: Detection of alphamethylacyl-coenzyme A racemase in postradiation prostatic adenocarcinoma. Urology. 62:282–286, 2003. 104. Martens MB, Keller JH: Routine immunohistochemical staining for high-molecular weight cytokeratin 34-beta and alphamethylacyl CoA racemase (P504S) in postirradiation prostate biopsies. Mod Pathol. 19:287–290, 2006. 105. Crook JM, Bahadur YA, Robertson SJ, et al: Evaluation of radiation effect, tumor differentiation, and prostate specific antigen staining in sequential prostate biopsies after external beam radiotherapy for patients with prostate carcinoma. Cancer. 79:81–89, 1997. 106. Scalzo DA, Kallakury BV, Gaddipati RV, et al: Cell proliferation rate by MIB-1 immunohistochemistry predicts postradiation recurrence in prostatic adenocarcinomas. Am J Clin Pathol. 109:163–168, 1998. 107. Bostwick DG, Kindrachuk RW, Rouse RV: Prostatic adenocarcinoma with endometrioid features. clinical, pathologic, and ultrastructural findings. Am J Surg Pathol. 9:595–609, 1985. 108. Greene LF, Farrow GM, Ravits JM, et al: Prostatic adenocarcinoma of ductal origin. J Urol. 121:303–305, 1979. 109. Epstein JI, Woodruff JM: Adenocarcinoma of the prostate with endometrioid features. A light microscopic and immunohistochemical study of ten cases. Cancer. 57:111–119, 1986. 110. Hameed O, Humphrey PA: Immunohistochemistry in diagnostic surgical pathology of the prostate. Semin Diagn Pathol. 22:88– 104, 2005. 111. Owens CL, Epstein JI, Netto GJ: Distinguishing prostatic from colorectal adenocarcinoma on biopsy samples: Th role of morphology and immunohistochemistry. Arch Pathol Lab Med. 131:599–603, 2007. 112. Grignon DJ: Unusual subtypes of prostate cancer. Mod Pathol. 17:316–327, 2004. 113. Mai KT, Collins JP, Veinot JP: Prostatic adenocarcinoma with urothelial (transitional cell) carcinoma features. Appl Immunohistochem Mol Morphol. 10:231–236, 2002. 114. Oxley J, Abbott C: Thrombomodulin immunostaining and ductal carcinoma of the prostate. Histopathology. 33:391–392, 1998.
614.e3
115. Bostwick DG, Qian J, Pacelli A, et al: Neuroendocrine expression in node positive prostate cancer: Correlation with systemic progression and patient survival. J Urol. 168:1204–1211, 2002. 116. Abrahamsson PA: Neuroendocrine cells in tumour growth of the prostate. Endocr Relat Cancer. 6:503–519, 1999. 117. Abrahamsson PA, Falkmer S, Falt K, et al: The course of neuroendocrine differentiation in prostatic carcinomas. an immunohistochemical study testing chromogranin A as an “endocrine marker”. Pathol Res Pract. 185:373–380, 1989. 118. Cohen RJ, Glezerson G, Haffejee Z: Neuro-endocrine cells—a new prognostic parameter in prostate cancer. Br J Urol. 68:258– 262, 1991. 119. Weinstein MH, Partin AW, Veltri RW, et al: Neuroendocrine differentiation in prostate cancer: Enhanced prediction of progression after radical prostatectomy. Hum Pathol. 27:683–687, 1996. 120. Theodorescu D, Broder SR, Boyd JC, et al: Cathepsin D and chromogranin A as predictors of long term disease specific survival after radical prostatectomy for localized carcinoma of the prostate. Cancer. 80:2109–2119, 1997. 121. Casella R, Bubendorf L, Sauter G, et al: Focal neuroendocrine differentiation lacks prognostic significance in prostate core needle biopsies. J Urol. 160:406–410, 1998. 122. Bostwick DG, Grignon DJ, Hammond ME, et al: Prognostic factors in prostate cancer. college of american pathologists consensus statement 1999. Arch Pathol Lab Med. 124:995–1000, 2000. 123. Travis WD, Linnoila RI, Tsokos MG, et al: Neuroendocrine tumors of the lung with proposed criteria for large-cell neuroendocrine carcinoma. an ultrastructural, immunohistochemical, and flow cytometric study of 35 cases. Am J Surg Pathol. 15:529– 553, 1991. 124. Murali R, Kneale K, Lalak N, et al: Carcinoid tumors of the urinary tract and prostate. Arch Pathol Lab Med. 130:1693– 1706, 2006. 125. Freschi M, Colombo R, Naspro R, et al: Primary and pure neuroendocrine tumor of the prostate. Eur Urol. 45:166,69; discussion 169–170, 2004. 126. Tash JA, Reuter V, Russo P: Metastatic carcinoid tumor of the prostate. J Urol. 167:2526–2527, 2002. 127. Zarkovic A, Masters J, Carpenter L: Primary carcinoid tumour of the prostate. Pathology. 37:184–186, 2005. 128. Ghannoum JE, DeLellis RA, Shin SJ: Primary carcinoid tumor of the prostate with concurrent adenocarcinoma: A case report. Int J Surg Pathol. 12:167–170, 2004. 129. Ro JY, Tetu B, Ayala AG, et al: Small cell carcinoma of the prostate. II. immunohistochemical and electron microscopic studies of 18 cases. Cancer. 59:977–982, 1987. 130. Tetu B, Ro JY, Ayala AG, et al: Small cell carcinoma of the prostate. part I. A clinicopathologic study of 20 cases. Cancer. 59:1803–1809, 1987. 131. Yao JL, Madeb R, Bourne P, et al: Small cell carcinoma of the prostate: An immunohistochemical study. Am J Surg Pathol. 30:705–712, 2006. 132. Foster CS, Dodson A, Karavana V, et al: Prostatic stem cells. J Pathol. 197:551–565, 2002. 133. Uzgare AR, Xu Y, Isaacs JT: In vitro culturing and characteristics of transit amplifying epithelial cells from human prostate tissue. J Cell Biochem. 91:196–205, 2004. 134. Ordonez NG: Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol. 24:1217–1223, 2000. 135. Agoff SN, Lamps LW, Philip AT, et al: Thyroid transcription factor-1 is expressed in extrapulmonary small cell carcinomas but not in other extrapulmonary neuroendocrine tumors. Mod Pathol. 13:238–242, 2000. 136. Mackey JR, Au HJ, Hugh J, et al: Genitourinary small cell carcinoma: Determination of clinical and therapeutic factors associated with survival. J Urol. 159:1624–1629, 1998. 137. Rubenstein JH, Katin MJ, Mangano MM, et al: Small cell anaplastic carcinoma of the prostate: Seven new cases, review of the literature, and discussion of a therapeutic strategy. Am J Clin Oncol. 20:376–380, 1997. 138. Amato RJ, Logothetis CJ, Hallinan R, et al: Chemotherapy for small cell carcinoma of prostatic origin. J Urol. 147:935–937, 1992.
614.e4
Immunohistology of the Prostate
139. Evans AJ, Humphrey PA, Belani J, et al: Large cell neuroendocrine carcinoma of prostate: A clinicopathologic summary of 7 cases of a rare manifestation of advanced prostate cancer. Am J Surg Pathol. 30:684–693, 2006. 140. Chibber PJ, McIntyre MA, Hindmarsh JR, et al: Transitional cell carcinoma involving the prostate. Br J Urol. 53:605–609, 1981. 141. Schellhammer PF, Bean MA, Whitmore WF, Jr: Prostatic involvement by transitional cell carcinoma: Pathogenesis, patterns and prognosis. J Urol. 118:399–403, 1977. 142. Svanholm H: Evaluation of commercial immunoperoxidase kits for prostatic specific antigen and prostatic specific acid phosphatase. Acta Pathol Microbiol Immunol Scand [A]. 94:7–12, 1986. 143. Ellis DW, Leffers S, Davies JS, et al: Multiple immunoperoxidase markers in benign hyperplasia and adenocarcinoma of the prostate. Am J Clin Pathol. 81:279–284, 1984. 144. Ford TF, Butcher DN, Masters JR, et al: Immunocytochemical localisation of prostate-specific antigen: Specificity and application to clinical practice. Br J Urol. 57:50–55, 1985. 145. Feiner HD, Gonzalez R: Carcinoma of the prostate with atypical immunohistological features. clinical and histologic correlates. Am J Surg Pathol. 10:765–770, 1986. 146. Keillor JS, Aterman K: The response of poorly differentiated prostatic tumors to staining for prostate specific antigen and prostatic acid phosphatase: A comparative study. J Urol. 137:894–896, 1987. 147. Epstein JI: PSAP and PSA as immunohistochemical markers. Urol Clin North Am. 20:757–770, 1993. 148. Mhawech P, Uchida T, Pelte MF: Immunohistochemical profile of high-grade urothelial bladder carcinoma and prostate adenocarcinoma. Hum Pathol. 33:1136–1140, 2002. 149. Nowels K, Kent E, Rinsho K, et al: Prostate specific antigen and acid phosphatase-reactive cells in cystitis cystica and glandularis. Arch Pathol Lab Med. 112:734–737, 1988. 150. Pollen JJ, Dreilinger A: Immunohistochemical identification of prostatic acid phosphatase and prostate specific antigen in female periurethral glands. Urology. 23:303–304, 1984. 151. Tepper SL, Jagirdar J, Heath D, et al: Homology between the female paraurethral (skene’s) glands and the prostate. immunohistochemical demonstration. Arch Pathol Lab Med. 108:423– 425, 1984. 152. Kamoshida S, Tsutsumi Y: Extraprostatic localization of prostatic acid phosphatase and prostate-specific antigen: Distribution in cloacogenic glandular epithelium and sex-dependent expression in human anal gland. Hum Pathol. 21:1108–1111, 1990. 153. Golz R, Schubert GE: Prostatic specific antigen: Immunoreactivity in urachal remnants. J Urol. 141:1480–1482, 1989. 154. Sobin LH, Hjermstad BM, Sesterhenn IA, et al: Prostatic acid phosphatase activity in carcinoid tumors. Cancer. 58:136–138, 1986. 155. van Krieken JH: Prostate marker immunoreactivity in salivary gland neoplasms. A rare pitfall in immunohistochemistry. Am J Surg Pathol. 17:410–414, 1993. 156. Spencer JR, Brodin AG, Ignatoff JM: Clear cell adenocarcinoma of the urethra: Evidence for origin within paraurethral ducts. J Urol. 143:122–125, 1990. 157. Grignon DJ, Ro JY, Ayala AG, et al: Primary adenocarcinoma of the urinary bladder. A clinicopathologic analysis of 72 cases. Cancer. 67:2165–2172, 1991. 158. Epstein JI, Kuhajda FP, Lieberman PH: Prostate-specific acid phosphatase immunoreactivity in adenocarcinomas of the urinary bladder. Hum Pathol. 17:939–942, 1986. 159. Heyderman E, Brown BM, Richardson TC: Epithelial markers in prostatic, bladder, and colorectal cancer: An immunoperoxidase study of epithelial membrane antigen, carcinoembryonic antigen, and prostatic acid phosphatase. J Clin Pathol. 37:1363– 1369, 1984. 160. Nadji M, Tabei SZ, Castro A, et al: Prostatic-specific antigen: An immunohistologic marker for prostatic neoplasms. Cancer. 48:1229–1232, 1981. 161. Genega EM, Hutchinson B, Reuter VE, et al: Immunophenotype of high-grade prostatic adenocarcinoma and urothelial carcinoma. Mod Pathol. 13:1186–1191, 2000. 162. Varma M, Morgan M, Amin MB, et al: High molecular weight cytokeratin antibody (clone 34betaE12): A sensitive marker for differentiation of high-grade invasive urothelial carcinoma from prostate cancer. Histopathology. 42:167–172, 2003.
163. Oliai BR, Kahane H, Epstein JI: A clinicopathologic analysis of urothelial carcinomas diagnosed on prostate needle biopsy. Am J Surg Pathol. 25:794–801, 2001. 164. Hameed O, Humphrey PA: Immunohistochemistry in diagnostic surgical pathology of the prostate. Semin Diagn Pathol. 22:88– 104, 2005. 165. Lane Z, Epstein JI, Ayub S, Netto GJ: Prostatic adenocarcinoma in colorectal biopsy: Clinical and pathologic features. Hum Pathol. 39:543–549, 2008. 166. Rames RA, Smith MT: Malignant peripheral nerve sheath tumor of the prostate: A rare manifestion of neurofibromatosis type 1. J Urol. 162:165–166, 1999. 167. Raney RB, Anderson JR, Barr FG, et al: Rhabdomyosarcoma and undifferentiated sarcoma in the first two decades of life: A selective review of intergroup rhabdomyosarcoma study group experience and rationale for intergroup rhabdomyosarcoma study V. J Pediatr Hematol Oncol. 23:215–220, 2001. 168. Eble JN, Sauter G, Epstein JI, et al: The world health organization classification of tumours of the urinary system and male genital system. Lyon, France, 2004, IARC Press, pp 209–211. 169. Gaudin PB, Rosai J, Epstein JI: Sarcomas and related proliferative lesions of specialized prostatic stroma: A clinicopathologic study of 22 cases. Am J Surg Pathol. 22:148–162, 1998. 170. Bostwick DG, Hossain D, Qian J, et al: Phyllodes tumor of the prostate: Long-term followup study of 23 cases. J Urol. 172:894– 899, 2004. 171. Herawi M, Epstein JI: Specialized stromal tumors of the prostate: A clinicopathologic study of 50 cases. Am J Surg Pathol. 30:694–704, 2006. 172. Michaels MM, Brown HE, Favino CJ: Leiomyoma of prostate. Urology. 3:617–620, 1974. 173. Regan JB, Barrett DM, Wold LE: Giant leiomyoma of the prostate. Arch Pathol Lab Med. 111:381–382, 1987. 174. Stenram U, Holby LE: A case of circumscribed myosarcoma of the prostate. Cancer. 24:803–806, 1969. 175. Kelley TW, Borden EC, Goldblum JR: Estrogen and progesterone receptor expression in uterine and extrauterine leiomyosarcomas: An immunohistochemical study. Appl Immunohistochem Mol Morphol. 12:338–341, 2004. 176. Cheville JC, Dundore PA, Nascimento AG, et al: Leiomyosarcoma of the prostate. report of 23 cases. Cancer. 76:1422–1427, 1995. 177. Sexton WJ, Lance RE, Reyes AO, et al: Adult prostate sarcoma: The M. D. anderson cancer center experience. J Urol. 166:521– 525, 2001. 178. Herawi M, Epstein JI: Solitary fibrous tumor on needle biopsy and transurethral resection of the prostate: A clinicopathologic study of 13 cases. Am J Surg Pathol. 31:870–876, 2007. 179. Van der Aa F, Sciot R, Blyweert W, et al: Gastrointestinal stromal tumor of the prostate. Urology. 65:388, 2005. 180. Madden JF, Burchette JL, Raj GV, et al: Anterior rectal wall gastrointestinal stromal tumor presenting clinically as prostatic mass. Urol Oncol. 23:268–272, 2005. 181. Herawi M, Montgomery EA, Epstein JI: Gastrointestinal stromal tumors (GISTs) on prostate needle biopsy: A clinicopathologic study of 8 cases. Am J Surg Pathol. 30:1389–1395, 2006. 182. Andriole GL, Crawford ED, Grubb RL, 3rd, et al, PLCO Project Team: Mortality results from a randomized prostate-cancer screening trial. N Engl J Med. 360:1310–1319, 2009. 183. Schroder FH, Hugosson J, Roobol MJ, et al, ERSPC Investigators: Screening and prostate-cancer mortality in a randomized european study. N Engl J Med. 360:1320–1328, 2009. 184. Stephenson AJ, Scardino PT, Eastham JA, et al: Preoperative nomogram predicting the 10-year probability of prostate cancer recurrence after radical prostatectomy. J Natl Cancer Inst. 98:715–717, 2006. 185. Stephenson AJ, Scardino PT, Eastham JA, et al: Postoperative nomogram predicting the 10-year probability of prostate cancer recurrence after radical prostatectomy. J Clin Oncol. 23:7005– 7012, 2005. 186. Partin AW, Kattan MW, Subong EN, et al: Combination of prostate-specific antigen, clinical stage, and gleason score to predict pathological stage of localized prostate cancer. A multiinstitutional update. JAMA. 277:1445–1451, 1997. 187. Amin M, Boccon-Gibod L, Egevad L, et al: Prognostic and predictive factors and reporting of prostate carcinoma in prostate
References needle biopsy specimens. Scand J Urol Nephrol Suppl. 216:20– 33, 2005. 188. DeMarzo AM, Nelson WG, Isaacs WB, et al: Pathological and molecular aspects of prostate cancer. Lancet. 361:955–964, 2003. 189. De Marzo AM, DeWeese TL, Platz EA, et al: Pathological and molecular mechanisms of prostate carcinogenesis: Implications for diagnosis, detection, prevention, and treatment. J Cell Biochem. 91:459–477, 2004. 190. Nelson WG, De Marzo AM, Isaacs WB: Prostate cancer. N Engl J Med. 349:366–381, 2003. 191. Srigley JR, Amin M, Boccon-Gibod L, et al: Prognostic and predictive factors in prostate cancer: Historical perspectives and recent international consensus initiatives. Scand J Urol Nephrol Suppl. 216:8–19, 2005. 192. Epstein JI, Amin M, Boccon-Gibod L, et al: Prognostic factors and reporting of prostate carcinoma in radical prostatectomy and pelvic lymphadenectomy specimens. Scand J Urol Nephrol Suppl. 216:34–63, 2005. 193. Hammond ME, Fitzgibbons PL, Compton CC, et al: College of american pathologists conference XXXV: Solid tumor prognostic factors-which, how and so what? summary document and recommendations for implementation. cancer committee and conference participants. Arch Pathol Lab Med. 124:958–965, 2000. 194. Bostwick DG, Grignon DJ, Hammond ME, et al: Prognostic factors in prostate cancer. college of american pathologists consensus statement 1999. Arch Pathol Lab Med. 124:995–1000, 2000. 195. Kremer CL, Klein RR, Mendelson J, et al: Expression of mTOR signaling pathway markers in prostate cancer progression. Prostate. 66:1203–1212, 2006. 196. Sanchez D, Rosell D, Honorato B, et al: Androgen receptor mutations are associated with gleason score in localized prostate cancer. BJU Int. 98:1320–1325, 2006. 197. Wikstrom P, Bergh A, Damber JE: Transforming growth factorbeta1 and prostate cancer. Scand J Urol Nephrol. 34:85–94, 2000. 198. Wikstrom P, Damber J, Bergh A: Role of transforming growth factor-beta1 in prostate cancer. Microsc Res Tech. 52:411–419, 2001. 199. Diaz JI, Mora LB, Austin PF, et al: Predictability of PSA failure in prostate cancer by computerized cytometric assessment of tumoral cell proliferation. Urology. 53:931–938, 1999. 200. Keshgegian AA, Johnston E, Cnaan A: Bcl-2 oncoprotein positivity and high MIB-1 (ki-67) proliferative rate are independent predictive markers for recurrence in prostate carcinoma. Am J Clin Pathol. 110:443–449, 1998. 201. Bubendorf L, Tapia C, Gasser TC, et al: Ki67 labeling index in core needle biopsies independently predicts tumor-specific survival in prostate cancer. Hum Pathol. 29:949–954, 1998. 202. Bettencourt MC, Bauer JJ, Sesterhenn IA, et al: Ki-67 expression is a prognostic marker of prostate cancer recurrence after radical prostatectomy. J Urol. 156:1064–1068, 1996. 203. Cheng L, Pisansky TM, Sebo TJ, et al: Cell proliferation in prostate cancer patients with lymph node metastasis: A marker for progression. Clin Cancer Res. 5:2820–2823, 1999. 204. Stapleton AM, Zbell P, Kattan MW, et al: Assessment of the biologic markers p53, ki-67, and apoptotic index as predictive indicators of prostate carcinoma recurrence after surgery. Cancer. 82:168–175, 1998. 205. Vis AN, van Rhijn BW, Noordzij MA, et al: Value of tissue markers p27(kip1), MIB-1, and CD44s for the pre-operative prediction of tumour features in screen-detected prostate cancer. J Pathol. 197:148–154, 2002. 206. Bostwick DG, Wheeler TM, Blute M, et al: Optimized microvessel density analysis improves prediction of cancer stage from prostate needle biopsies. Urology. 48:47–57, 1996. 207. Silberman MA, Partin AW, Veltri RW, et al: Tumor angiogenesis correlates with progression after radical prostatectomy but not with pathologic stage in gleason sum 5 to 7 adenocarcinoma of the prostate. Cancer. 79:772–779, 1997. 208. Strohmeyer D, Rossing C, Strauss F, et al: Tumor angiogenesis is associated with progression after radical prostatectomy in pT2/ pT3 prostate cancer. Prostate. 42:26–33, 2000.
614.e5
209. Strohmeyer D, Strauss F, Rossing C, et al: Expression of bFGF, VEGF and c-met and their correlation with microvessel density and progression in prostate carcinoma. Anticancer Res. 24:1797– 1804, 2004. 210. Gettman MT, Bergstralh EJ, Blute M, et al: Prediction of patient outcome in pathologic stage T2 adenocarcinoma of the prostate: Lack of significance for microvessel density analysis. Urology. 51:79–85, 1998. 211. Gettman MT, Pacelli A, Slezak J, et al: Role of microvessel density in predicting recurrence in pathologic stage T3 prostatic adenocarcinoma. Urology. 54:479–485, 1999. 212. Krupski T, Petroni GR, Frierson HF, Jr, et al: Microvessel density, p53, retinoblastoma, and chromogranin A immunohistochemistry as predictors of disease-specific survival following radical prostatectomy for carcinoma of the prostate. Urology. 55:743– 749, 2000. 213. Theodorescu D, Broder SR, Boyd JC, et al: Cathepsin D and chromogranin A as predictors of long term disease specific survival after radical prostatectomy for localized carcinoma of the prostate. Cancer. 80:2109–2119, 1997. 214 Weinstein MH, Partin AW, Veltri RW, et al: Neuroendocrine differentiation in prostate cancer: Enhanced prediction of progression after radical prostatectomy. Hum Pathol. 27:683–687, 1996. 215. McWilliam LJ, Manson C, George NJ: Neuroendocrine differentiation and prognosis in prostatic adenocarcinoma. Br J Urol. 80:287–290, 1997. 216. Cohen RJ, Glezerson G, Haffejee Z: Neuro-endocrine cells—a new prognostic parameter in prostate cancer. Br J Urol. 68:258– 262, 1991. 217. Casella R, Bubendorf L, Sauter G, et al: Focal neuroendocrine differentiation lacks prognostic significance in prostate core needle biopsies. J Urol. 160:406–410, 1998. 218. Shariff AH, Ather MH: Neuroendocrine differentiation in prostate cancer. Urology. 68:2–8, 2006. 219. Zhang YH, Kanamaru H, Oyama N, et al: Prognostic value of nuclear morphometry on needle biopsy from patients with prostate cancer: Is volume-weighted mean nuclear volume superior to other morphometric parameters? Urology. 55:377–381, 2000. 220. Zhang YH, Kanamaru H, Oyama N, et al: Comparison of nuclear morphometric results between needle biopsy and surgical specimens from patients with prostate cancer. Urology. 54:763–766, 1999. 221. Veltri RW, Miller MC, Partin AW, et al: Ability to predict biochemical progression using gleason score and a computergenerated quantitative nuclear grade derived from cancer cell nuclei. Urology. 48:685–691, 1996. 222. Khan MA, Walsh PC, Miller MC, et al: Quantitative alterations in nuclear structure predict prostate carcinoma distant metastasis and death in men with biochemical recurrence after radical prostatectomy. Cancer. 98:2583–2591, 2003. 223. Partin AW, Steinberg GD, Pitcock RV, et al: Use of nuclear morphometry, gleason histologic scoring, clinical stage, and age to predict disease-free survival among patients with prostate cancer. Cancer. 70:161–168, 1992. 224. Potter SR, Miller MC, Mangold LA, et al: Genetically engineered neural networks for predicting prostate cancer progression after radical prostatectomy. Urology. 54:791–795, 1999. 225. Ross JS, Sheehan CE, Fisher HA, et al: Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res. 9:6357– 6362, 2003. 226. Hammond ME, Sause WT, Martz KL, et al: Correlation of prostate-specific acid phosphatase and prostate-specific antigen immunocytochemistry with survival in prostate carcinoma. Cancer. 63:461–466, 1989. 227. Nelson WG, De Marzo AM, Yegnasubramanian S: Epigenetic alterations in human prostate cancers. Endocrinology. 150:3991– 4002, 2009. 228. Nakayama M, Bennett CJ, Hicks JL, et al: Hypermethylation of the human glutathione S-transferase-pi gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophy lesions but not in normal or hyperplastic epithelium of the prostate: A detailed study using laser-capture microdissection. Am J Pathol. 163:923–933, 2003.
614.e6
Immunohistology of the Prostate
229. Brooks JD, Weinstein M, Lin X, et al: CG island methylation changes near the GSTP1 gene in prostatic intraepithelial neoplasia. Cancer Epidemiol Biomarkers Prev. 7:531–536, 1998. 230. Bastian PJ, Yegnasubramanian S, Palapattu GS, et al: Molecular biomarker in prostate cancer: The role of CpG island hypermethylation. Eur Urol. 46:698–708, 2004. 231. Yegnasubramanian S, Kowalski J, Gonzalgo ML, et al: Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res. 64:1975–1986, 2004. 232. Yegnasubramanian S, Haffner MC, Zhang Y, et al: DNA hypomethylation arises later in prostate cancer progression than CpG island hypermethylation and contributes to metastatic tumor heterogeneity. Cancer Res. 68:8954–8967, 2008. 233. Tomlins SA, Rhodes DR, Perner S, et al: Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science. 310:644–648, 2005. 234. Tomlins SA, Mehra R, Rhodes DR, et al: TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer. Cancer Res. 66:3396–3400, 2006. 235. Toubaji A, Albadine R, Meeker AK, et al: Increased gene copy number of ERG on chromosome 21 but not TMPRSS2-ERG fusion predicts outcome in prostatic adenocarcinomas. Mod Pathol. 2011. 236. Gopalan A, Leversha MA, Satagopan JM, et al: TMPRSS2-ERG gene fusion is not associated with outcome in patients treated by prostatectomy. Cancer Res. 69:1400–1406, 2009. 237. Demichelis F, Fall K, Perner S, et al: TMPRSS2:ERG gene fusion associated with lethal prostate cancer in a watchful waiting cohort. Oncogene. 26:4596–4599, 2007. 238. Lotan TL, Toubaji A, Albadine R, et al: TMPRSS2-ERG gene fusions are infrequent in prostatic ductal adenocarcinomas. Mod Pathol. 2009. 239. Yoshimoto M, Joshua AM, Cunha IW, et al: Absence of TMPRSS2:ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome. Mod Pathol. 21:1451–1460, 2008. 240. FitzGerald LM, Agalliu I, Johnson K, et al: Association of TMPRSS2-ERG gene fusion with clinical characteristics and outcomes: Results from a population-based study of prostate cancer. BMC Cancer. 8:230, 2008. 241. Mao X, Shaw G, James SY, et al: Detection of TMPRSS2:ERG fusion gene in circulating prostate cancer cells. Asian J Androl. 10:467–473, 2008. 242. Perner S, Mosquera JM, Demichelis F, et al: TMPRSS2-ERG fusion prostate cancer: An early molecular event associated with invasion. Am J Surg Pathol. 31:882–888, 2007. 243. Saramaki OR, Harjula AE, Martikainen PM, et al: TMPRSS2:ERG fusion identifies a subgroup of prostate cancers with a favorable prognosis. Clin Cancer Res. 14:3395–3400, 2008. 244. Falzarano SM, Navas M, Simmerman K, et al: ERG rearrangement is present in a subset of transition zone prostatic tumors. Mod Pathol. 23:1499–1506, 2010. 245. Netto GJ: TMPRSS2-ERG fusion as a marker of prostatic lineage in small-cell carcinoma. Histopathology. 57:633; author reply 633–634, 2010. 246. Albadine R, Latour M, Toubaji A, et al: TMPRSS2-ERG gene fusion status in minute (minimal) prostatic adenocarcinoma. Mod Pathol. 22:1415–1422, 2009. 247. Rostad K, Hellwinkel OJ, Haukaas SA, et al: TMPRSS2:ERG fusion transcripts in urine from prostate cancer patients correlate with a less favorable prognosis. APMIS. 117:575–582, 2009. 248. Rice KR, Chen Y, Ali A, et al: Evaluation of the ETS-related gene mRNA in urine for the detection of prostate cancer. Clin Cancer Res. 16:1572–1576, 2010. 249. Nguyen PN, Violette P, Chan S, et al: A panel of TMPRSS2:ERG fusion transcript markers for urine-based prostate cancer detection with high specificity and sensitivity. Eur Urol. 59:407–414, 2011. 250. Park K, Tomlins SA, Mudaliar KM, et al: Antibody-based detection of ERG rearrangement-positive prostate cancer. Neoplasia. 12:590–598, 2010. 251. Chaux A, Albadine R, Toubaji A, et al: Immunohistochemistry for ERG expression as a surrogate for TMPRSS2-ERG fusion detection in prostatic adenocarcinomas. Am J Surg Pathol. 35:1014–1020, 2011.
252. Wu Y, Chhipa RR, Cheng J, et al: Androgen receptor-mTOR crosstalk is regulated by testosterone availability: Implication for prostate cancer cell survival. Anticancer Res. 30:3895–3901, 2010. 253. Bismar TA, Yoshimoto M, Vollmer RT, et al: PTEN genomic deletion is an early event associated with ERG gene rearrangements in prostate cancer. BJU Int. 2010. 254. Bubendorf L: Words of wisdom. re: Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate. Eur Urol. 56:882–883, 2009. 255. Han B, Mehra R, Lonigro RJ, et al: Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression. Mod Pathol. 22:1083–1093, 2009. 256. King JC, Xu J, Wongvipat J, et al: Cooperativity of TMPRSS2ERG with PI3-kinase pathway activation in prostate oncogenesis. Nat Genet. 41:524–526, 2009. 257. Sarker D, Reid AH, Yap TA, et al: Targeting the PI3K/AKT pathway for the treatment of prostate cancer. Clin Cancer Res. 15:4799–4805, 2009. 258. Squire JA: TMPRSS2-ERG and PTEN loss in prostate cancer. Nat Genet. 41:509–510, 2009. 259. Chaux A, Peskoe SB, Gonzalez-Roibon N, et al: Loss of PTEN expression is associated with increased risk of recurrence after prostatectomy for clinically localized prostate cancer. Mod Pathol. 25:1543–1549, 2012. 260. Lotan TL, Gurel B, Sutcliffe S, et al: PTEN protein loss by immunostaining: Analytic validation and prognostic indicator for a high risk surgical cohort of prostate cancer patients. Clin Cancer Res. 17:6563–6573, 2011. 261. Armstrong AJ, Netto GJ, Rudek MA, et al: A pharmacodynamic study of rapamycin in men with intermediate- to high-risk localized prostate cancer. Clin Cancer Res. 16:3057–3066, 2010. 262. Brewster SF, Oxley JD, Trivella M, et al: Preoperative p53, bcl-2, CD44 and E-cadherin immunohistochemistry as predictors of biochemical relapse after radical prostatectomy. J Urol. 161:1238–1243, 1999. 263. Stackhouse GB, Sesterhenn IA, Bauer JJ, et al: p53 and bcl-2 immunohistochemistry in pretreatment prostate needle biopsies to predict recurrence of prostate cancer after radical prostatectomy. J Urol. 162:2040–2045, 1999. 264 Bauer JJ, Sesterhenn IA, Mostofi FK, et al: Elevated levels of apoptosis regulator proteins p53 and bcl-2 are independent prognostic biomarkers in surgically treated clinically localized prostate cancer. J Urol. 156:1511–1516, 1996. 265. Bauer JJ, Sesterhenn IA, Mostofi KF, et al: p53 nuclear protein expression is an independent prognostic marker in clinically localized prostate cancer patients undergoing radical prostatectomy. Clin Cancer Res. 1:1295–1300, 1995. 266. Moul JW, Bettencourt MC, Sesterhenn IA, et al: Protein expression of p53, bcl-2, and KI-67 (MIB-1) as prognostic biomarkers in patients with surgically treated, clinically localized prostate cancer. Surgery. 120:159, 66; discussion 166–167, 1996. 267. Osman I, Drobnjak M, Fazzari M, et al: Inactivation of the p53 pathway in prostate cancer: Impact on tumor progression. Clin Cancer Res. 5:2082–2088, 1999. 268. Theodorescu D, Broder SR, Boyd JC, et al: p53, bcl-2 and retinoblastoma proteins as long-term prognostic markers in localized carcinoma of the prostate. J Urol. 158:131–137, 1997. 269. Kuczyk MA, Serth J, Bokemeyer C, et al: The prognostic value of p53 for long-term and recurrence-free survival following radical prostatectomy. Eur J Cancer. 34:679–686, 1998. 270. Markert EK, Mizuno H, Vazquez A, et al: Molecular classification of prostate cancer using curated expression signatures. Proc Natl Acad Sci U S A. 108:21276–21281, 2011. 271. Lacombe L, Maillette A, Meyer F, et al: Expression of p21 predicts PSA failure in locally advanced prostate cancer treated by prostatectomy. Int J Cancer. 95:135–139, 2001. 272. Aslan G, Irer B, Tuna B, et al: Analysis of NKX3.1 expression in prostate cancer tissues and correlation with clinicopathologic features. Pathol Res Pract. 202:93–98, 2006. 273. Bethel CR, Faith D, Li X, et al: Decreased NKX3.1 protein expression in focal prostatic atrophy, prostatic intraepithelial neoplasia, and adenocarcinoma: Association with gleason score and chromosome 8p deletion. Cancer Res. 66:10683–10690, 2006.
References 274. Gurel B, Iwata T, Koh CM, et al: Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis. Mod Pathol. 21:1156–1167, 2008. 275. Gurel B, Iwata T, Koh CM, et al: Molecular alterations in prostate cancer as diagnostic, prognostic, and therapeutic targets. Adv Anat Pathol. 15:319–331, 2008. 276. Lapointe J, Li C, Giacomini CP, et al: Genomic profiling reveals alternative genetic pathways of prostate tumorigenesis. Cancer Res. 67:8504–8510, 2007. 277. Taylor BS, Schultz N, Hieronymus H, et al: Integrative genomic profiling of human prostate cancer. Cancer Cell. 18:11–22, 2010. 278. Bastian PJ, Ellinger J, Wellmann A, et al: Diagnostic and prognostic information in prostate cancer with the help of a small set of hypermethylated gene loci. Clin Cancer Res. 11:4097– 4106, 2005. 279. Bastian PJ, Nakayama M, De Marzo AM, et al: GSTP1 CpG island hypermethylation as a molecular marker of prostate cancer. Urologe A. 43:573–579, 2004. 280. Bastian PJ, Palapattu GS, Lin X, et al: Preoperative serum DNA GSTP1 CpG island hypermethylation and the risk of early prostate-specific antigen recurrence following radical prostatectomy. Clin Cancer Res. 11:4037–4043, 2005. 281. Bastian PJ, Yegnasubramanian S, Palapattu GS, et al: Molecular biomarker in prostate cancer: The role of CpG island hypermethylation. Eur Urol. 46:698–708, 2004. 282. Bussemakers MJ, van Bokhoven A, Verhaegh GW, et al: DD3: A new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res. 59:5975–5979, 1999. 283. de Kok JB, Verhaegh GW, Roelofs RW, et al: DD3(PCA3), a very sensitive and specific marker to detect prostate tumors. Cancer Res. 62:2695–2698, 2002. 284. Groskopf J, Aubin SM, Deras IL, et al: APTIMA PCA3 molecular urine test: Development of a method to aid in the diagnosis of prostate cancer. Clin Chem. 52:1089–1095, 2006. 285. Deras IL, Aubin SM, Blase A, et al: PCA3: A molecular urine assay for predicting prostate biopsy outcome. J Urol. 179:1587– 1592, 2008. 286. Haese A, de la Taille A, van Poppel H, et al: Clinical utility of the PCA3 urine assay in european men scheduled for repeat biopsy. Eur Urol. 54:1081–1088, 2008. 287. Sokoll LJ, Ellis W, Lange P, et al: A multicenter evaluation of the PCA3 molecular urine test: Pre-analytical effects, analytical performance, and diagnostic accuracy. Clin Chim Acta. 389:1–6, 2008. 288. Aubin SM, Reid J, Sarno MJ, et al: PCA3 molecular urine test for predicting repeat prostate biopsy outcome in populations at
614.e7
risk: Validation in the placebo arm of the dutasteride REDUCE trial. J Urol. 184:1947–1952, 2010. 289. Nakanishi H, Groskopf J, Fritsche HA, et al: PCA3 molecular urine assay correlates with prostate cancer tumor volume: Implication in selecting candidates for active surveillance. J Urol. 179:1804, 9; discussion 1809–1810, 2008. 290. van Poppel H, Haese A, Graefen M, et al: The relationship between prostate CAncer gene 3 (PCA3) and prostate cancer significance. BJU Int. 2011. 291. Aubin SM, Reid J, Sarno MJ, et al: Prostate cancer gene 3 score predicts prostate biopsy outcome in men receiving dutasteride for prevention of prostate cancer: Results from the REDUCE trial. Urology. 78:380–385, 2011. 292. Laxman B, Morris DS, Yu J, et al: A first-generation multiplex biomarker analysis of urine for the early detection of prostate cancer. Cancer Res. 68:645–649, 2008. 293. Kantoff P: Recent progress in management of advanced prostate cancer. Oncology (Williston Park). 19:631–636, 2005. 294. Pizer ES, Pflug BR, Bova GS, et al: Increased fatty acid synthase as a therapeutic target in androgen-independent prostate cancer progression. Prostate. 47:102–110, 2001. 295. Wu L, Birle DC, Tannock IF: Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts. Cancer Res. 65:2825–2831, 2005. 296. Jimeno A, Carducci M: Atrasentan: A rationally designed targeted therapy for cancer. Drugs Today (Barc). 42:299–312, 2006. 297. Jimeno A, Carducci M: Atrasentan: A novel and rationally designed therapeutic alternative in the management of cancer. Expert Rev Anticancer Ther. 5:419–427, 2005. 298. Aggarwal S, Singh P, Topaloglu O, et al: A dimeric peptide that binds selectively to prostate-specific membrane antigen and inhibits its enzymatic activity. Cancer Res. 66:9171–9177, 2006. 299. Elsasser-Beile U, Wolf P, Gierschner D, et al: A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and thera peutic targeting of prostate cancer. Prostate. 66:1359–1370, 2006. 300. Ikegami S, Yamakami K, Ono T, et al: Targeting gene therapy for prostate cancer cells by liposomes complexed with anti-prostatespecific membrane antigen monoclonal antibody. Hum Gene Ther. 17:997–1005, 2006. 301. Jayaprakash S, Wang X, Heston WD, et al: Design and synthesis of a PSMA inhibitor-doxorubicin conjugate for targeted prostate cancer therapy. Chem Med Chem. 1:299–302, 2006.
C H A P T E R 1 7
IMMUNOHISTOLOGY OF THE BLADDER, KIDNEY, AND TESTIS GEORGE J. NETTO, JONATHAN I. EPSTEIN
Overview 615 Immunohistology of the Urinary Bladder 615 Diagnostic Immunohistochemistry of Specific Bladder Neoplasms 619 Immunohistology of Renal Neoplasms 631 Immunohistology of Testicular Tumors 644 Summary 652
Overview The unprecedented advances in cancer genetics and genomics are rapidly affecting the clinical management of solid tumors. Molecular diagnostics are now an integral part of routine clinical management for patients with lung, colon, and breast cancer. In sharp contrast, molecular biomarkers continue to be noticeably absent from current management algorithms for urologic malignancies, including bladder and renal cancers. The need for new treatment options that can improve upon the modest outcomes currently associated with muscle invasive bladder cancer (MI-BC) is evident, but validated prognostic molecular biomarkers that can help clinicians identify patients in need of early aggressive management are lacking. Robust predictive biomarkers that can forecast and stratify responses to emerging targeted therapies are also needed. In recent years, the detection and treatment of renal tumors has shifted toward radiologic detection of smaller lesions with an increasing tendency toward a laparoscopic approach for partial nephrectomy, ablative cryotherapy, or radiofrequency ablation. When ablative therapy is contemplated, a proper classification of renal tumors based on a needle biopsy obtained before such therapy is even more crucial given the lack of additional sampling. The increasing number of differentially expressed renal tumor markers has been valuable in such a setting. The tremendous progress in targeted therapy for advanced renal cancer will continue to drive
parallel progress in developing predictive markers of response to the various exciting new agents. The following is a discussion of the utility of immunohistochemical (IHC) markers, genomic applications, and prognostic aspects of bladder, renal, and testicular tumors.
Immunohistology of the Urinary Bladder In 2012 more than 73,510 new cases of urothelial carcinoma (URCa) were diagnosed in the United States and led to more than 14,880 deaths.1 The estimated annual incidence worldwide is a staggering 336,000 cases. Bladder cancer is the fourth most common tumor in males and the eleventh most common in females. Because of the high rate of tumor recurrence and the need for frequent cystoscopy, URCa is the cancer with the highest cost per patient, with an annual burden of more than $3 billion to our health care system. Nevertheless, URCa presents us with unique challenges and also with opportunities, given urine samples amenable to the application of noninvasive molecular detection methods and the relative ease of delivery of molecular targeted therapy to a topographically accessible tumor.2 Clinically, URCa presents two distinct phenotypes. The first is superficial, non–muscle invasive (NMI) URCa, which represents three fourths of cases. Half of these superficial tumors will recur as NMI tumors, and only 5% to 10% will progress to muscle-invasive disease. The mainstay of therapy in superficial tumors is transurethral resection biopsy (TURB), with or without intravesical chemotherapy, and immune therapy with bacillus Calmette-Guérin (BCG). The second phenotype is the muscle-invasive URCa, which represents 20% to 30% of all URCa. Only 15% of muscle invasive URCa have a prior history of superficial URCa and represent a progression from the superficial phenotype, whereas the majority (80% to 90%) are primary de novo muscle-invasive URCa. Currently, patients who suffer from muscle-invasive high-grade tumors are destined to a disappointing 50% to 60% overall survival rate despite 615
616
Immunohistology of the Bladder, Kidney, and Testis
aggressive combined treatment modalities that include cystectomy and chemotherapy.
Biology of Principal Antigens/Antibodies CYTOKERATIN 7 AND CYTOKERATIN 20
Cytokeratins (CKs) are a family of intracytoplasmic intermediate filament proteins present in almost all epithelia. Expression of each CK molecule depends on cell type and differentiation status, therefore specific CKs can be used as markers to identify particular types of epithelial tumors (Table 17-1). CK7 is found in a wide variety of epithelia, including the columnar and glandular epithelium of the lung, cervix, and breast as well as in the bile duct, collecting ducts of the kidney, urothelium, and mesothelium, but it is not in most gastrointestinal (GI) epithelium, hepatocytes, proximal and distal tubules of the kidney, and squamous epithelium. In contrast, CK20 shows relatively restricted expression and is present in GI epithelium, Merkel cells of the epidermis, and urothelium. CK7 expression is observed in the majority of URCa, whereas CK20 expression in URCa has been reported to vary from 15% to 97% in different studies.3 Bassily and colleagues4 showed that CK20 is more frequently positive in low-grade tumors (83%) than in high-grade tumors (52%). However, Desai and associates5 showed higher expression in high-grade tumors, thus most cases of URCa are positive for both CK7 and CK20. This immunoprofile (CK7+/CK20+) is helpful, particularly in the differential diagnosis of metastatic neoplasm of uncertain primary, although other examples of CK7-positive, CK20-positive tumor include carcinomas of extrahepatic bile duct and gallbladder, pancreatic adenocarcinoma, endocervical adenocarcinoma, mucinous tumors of the ovary and upper GI tract, and mucinous bronchioloalveolar adenocarcinoma.6-9 More than half of all cases of primary adenocarcinoma of the bladder are also positive for both CK7 and CK20.10 However, given that intestinal-type primary adenocarcinomas of bladder are likely to be CK7 negative and CK20 positive, such a panel has only a limited role in the differential diagnosis with secondary bladder involvement by adenocarcinoma of colorectal origin. The utility of the combination of CK7 and CK20 IHC can be further enhanced by the addition of
tissue-specific markers such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), P501S, thyroid transcription factor 1 (TTF-1), and so on. For example, prostatic adenocarcinomas that are occasionally CK7 and CK20 positive could be distinguished from URCa by their positivity for PSA. Unequivocal strong or extensive PSA positivity should not be encountered in URCa.4,11 Several studies have suggested a diagnostic role for CK20 expression pattern in distinguishing flat urothelial carcinoma in situ (CIS) from reactive urothelial atypia. In reactive nonneoplastic lesions, CK20 expression is usually restricted to surface “umbrella” cells. In contrast, the majority of urothelial dysplasia or CIS will show at least focal positive transmucosal CK20 expression in all layers of urothelium.12-14 CK20 staining in conjunction with Ki-67 proliferation index and p53 and p16 expression have also been suggested to be of value in distinguishing reactive atypia from CIS.13-15 We do not routinely resort to immunostaining in establishing the diagnosis of CIS. Finally, a role for CK20 expression pattern as a predictor of recurrence in low-grade urothelial neoplasms has also been proposed.16-18 UROPLAKIN
Uroplakins (UPs) are urothelium-specific transmembrane proteins present in terminally differentiated superficial urothelial cells, therefore expression of UPs is expected to diminish during urothelial tumorigenesis. The majority of noninvasive and up to two thirds of advanced invasive and metastatic URCas have been shown to retain UP expression as assessed by UPIII IHC.19-23 Interestingly, in some of these studies, loss of UPIII expression was associated with significantly worse prognosis even in patients with advanced disease.20,22,24 The latter was true on multivariate analysis when established prognostic parameters, such as stage and presence of lymph node metastasis, were included.20 Although highly specific for urothelial differentiation, IHC expression of UPIII is only of moderate sensitivity (as low as 40%)25 given that UPIII messenger RNA (mRNA) can often be detected in the absence of UPIII protein.26 Attesting to their suggested urothelial histogenesis, benign Brenner tumors of the ovary also stain for UPIII.19,25,27 Interestingly, only a slim minority of malignant Brenner tumors and primary ovarian URCa (6%)
TABLE 17-1 Utility of CK7/20 in the Differential Diagnosis of Urothelial Carcinoma CK7+/CK20+
CK7−/CK20−
CK7+/CK20−
CK7−/CK20+
Urothelial Ca Pancreatic Ca Ovarian mucinous Ca
Hepatocellular Ca Renal cell Ca Prostatic Ca Squamous cell Ca Neuroendocrine Ca
Urothelial Ca Breast cancer Lung non–small cell Ca Primary seminal vesicle Ca Ovarian serous Ca Mesothelioma (all forms) Endometrial adenocarcinoma
Colorectal Ca
Ca, Carcinoma; CK, cytokeratin.
Immunohistology of the Urinary Bladder
stained positive for UPIII in the study by Logani and associates.25 THROMBOMODULIN
Thrombomodulin (TM), also designated CD141, is an endothelial cell–associated cofactor for the thrombinmediated activator of protein C. TM expression, predominantly as membranous staining, has been found in 69% to 100% of URCa.10,11,21,28 TM expression is particularly useful in differentiating URCa from high-grade prostatic adenocarcinoma, renal cell carcinoma (RCC), and adenocarcinoma of the colon and endometrium, in which TM is rarely positive.11,28 However, it should be kept in mind that TM is also expressed by nonurothelial tumors such as vascular tumors, mesothelioma, and squamous cell carcinoma (SCC).28 Compared with UPIII, TM has a higher degree of sensitivity but lower specificity as a marker for URCa. TP63
As mentioned in the section on prostate carcinoma (PCa), TP63, a homolog of the TP53 tumor suppressor gene, encodes at least six different proteins with wide range of biologic functions, including a role in urothelial differentiation. Immunostaining for p63 is normally present in more than 90% of urothelial nuclei. The majority of URCa retains a normal expression pattern of p63, but expression may be partially lost in higher grade invasive URCa.11,29 However, in a recent study, we found p63 to be superior to TM as a urothelial marker of differentiation in high-grade tumors. In combination with prostate lineage markers, p63 is a valuable marker in differentiating URCa from high-grade prostatic adenocarcinoma secondarily invading the bladder.11 HIGH-MOLECULAR-WEIGHT CYTOKERATIN
As already mentioned in the discussion on PCa, the monoclonal antibody 34βE12 reacts specifically with high-molecular-weight cytokeratins (HMWCKs) CK1, CK5, CK10, and CK14. In addition to its aforementioned utility in labeling the basal cell layer of prostatic glands, HMWCK is a highly sensitive marker of urothelial differentiation, matching the sensitivity of p63 and surpassing that of TM and UPIII.11,21 HMWCK is useful in differentiating URCa, in which it is normally positive, from PCa, which is usually negative for HMWCK. However, a cautionary note is warranted, given that HMWCK labels squamous epithelia, including areas of squamous differentiation, in posttherapy recurrent PCa lesions. HMWCK positivity restricted to areas of squamous differentiation does not exclude the diagnosis of PCa.30 Finally, IHC for HMWCKs has been cited to be helpful in distinguishing urothelial dysplasia, which shows only basal staining, from flat urothelial CIS, in which a transmucosal staining of urothelial layers is expected.31 Diffuse expression of HMWCK in lowgrade papillary URCa has been reported to be a strong predictor of tumor recurrence.17
617
GATA3
GATA3 (GATA binding protein 3 to DNA sequence [A/T]GATA[A/G]) is a member of a zinc finger transcription factor family that plays an important role in promoting and directing cell proliferation, development, and differentiation. Several recent studies have pointed to its utility as a marker for the diagnosis of URCa.32-34 In the most recent and largest study by Liu and colleagues,34 applying a commercial antibody in tissue microarray analysis (TMA) sections constructed from 1110 carcinomas and 310 normal tissues of various organs, 62 of 72 URCas (86%) were positive for GATA3. The nuclear staining is usually diffuse in more than 50% of cells. It is important to remember that in addition to URCa, 91% of ductal mammary carcinomas and 100% of lobular carcinomas also tested positive for GATA3 in the same study. Rare staining (<2% of cases) was found in endometrioid-type endometrium, whereas SCC of lung origin, prostatic carcinoma, and clear cell and papillary RCC have been shown to be negative. ANAPLASTIC LYMPHOMA KINASE
Anaplastic lymphoma receptor tyrosine kinase (ALK) is a cytoplasmic membrane tyrosine kinase receptor expressed in anaplastic large cell lymphoma. ALK expression has been detected in approximately two thirds of inflammatory myofibroblastic tumors (IMTs) of the urinary tract, also termed postoperative spindle cell nodule, inflammatory pseudotumor, and pseudosarcomatous fibromyxoid tumor.35-38 The ALK gene is located on chromosome 2p23, and rearrangements of this gene through translocations with various gene partners have been identified in IMT from a number of anatomic sites, including the urinary bladder. Two recent studies by Montgomery and associates35 and Sukov and colleagues38 showed 72% and 67% respective rates of ALK gene rearrangement in bladder IMT using a split-apart fluorescence in situ hybridization (FISH) probes strategy. Sukov and colleagues saw a tighter correlation between positive expression and rearrangement in their study. Given the close morphologic and immunophenotypic similarities between IMT and malignant spindle cell urinary bladder tumors, demonstration of ALK immunostaining or gene rearrangement by FISH can prevent unnecessary radical surgery. TP53
The tumor suppressor gene TP53 orchestrates the transcriptional regulation of cell-cycle control elements, and TP53 mutations represent the most common genetic alterations in human malignancies.39 A number of studies have revealed TP53 mutations in 40% to 60% of invasive bladder cancers and their association with a worse prognosis.40-44 The altered protein product of the mutant TP53 gene has an extended half-life, leading to its accumulation and detection by IHC techniques.45 Staining results may vary because of
618
Immunohistology of the Bladder, Kidney, and Testis
differences in specimen processing and fixation,46 and only modest correlation between TP53 mutations and protein overexpression has been shown.47,48 In addition, TP53 alterations in URCa have been shown to be predictive of increased sensitivity to chemotherapeutic agents that damage DNA.49,50 As mentioned previously in the discussion of CK20 expression patterns in urothelial CIS, one potential use of TP53 immunostaining is differentiating urothelial CIS from reactive urothelial atypia. Strong, extensive p53 positivity in more than 50% of cells is encountered in CIS, whereas reactive urothelium is usually negative or demonstrates only weak, patchy TP53 nuclear staining (Fig. 17-1, A-D).13 CDKN2A
The tumor suppressor gene CDKN2A (formerly p16) inhibits cyclin D-dependent protein kinases and thereby plays a vital role in the regulation of G1-S transition. In fact, CDKN2A gene (9p21) deletions and mutations are frequent in bladder cancer and appear to be more frequent in low-grade superficial tumors compared with higher grade invasive tumors.51,52 Several recent studies revealed a significant correlation between loss of p16 expression and progression in noninvasive (pTa) and superficially invasive (pT1) URCa.45,53
Yin and colleagues15 recently showed increased expression of p16 protein in flat urothelial CIS compared with its uniform and weak expression in normal and reactive urothelium suggesting a potential diagnostic role for p16 immunostaining in such settings. p16 immunohistochemistry cannot be used to distinguish secondary involvement of the bladder with uterine cervical carcinoma from urothelial carcinoma. RETINOBLASTOMA PROTEIN
The retinoblastoma gene (RB1) product was the first to be identified in human cancer. It encodes a nuclear protein, pRb, which plays a crucial role in cell-cycle progression by regulating cell-cycle arrest at the G1-S phase. Alterations in pRb may occur either as a result of RB1 gene mutations or as a result of loss of p16, which normally phosphorylates pRb. Loss of heterozygosity (LOH) of one RB1 gene allele in combination with mutation of the remaining allele is found more frequently in high-grade muscle-invasive URCa tumors.5457 Interestingly, both overexpression and loss of pRb expression have been associated with increased risk for bladder cancer progression.58 It appears that alterations in pRb and p53 act in a synergistic manner to promote bladder cancer progression.56,58 Thus pRb immunostaining could be a useful prognostic marker in URCa.55,56,58,59
A
B
C
D
Figure 17-1 Cytokeratin 20 (CK20) and p53 staining as an adjunct to differentiate reactive urothelial atypia (A and C) from urothelial carcinoma in situ (B and D). A and B, CK20 immunostain. C and D, p53 immunostain.
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms Urothelial Carcinoma and Variants The distinctive morphologic features of noninvasive papillary urothelial neoplasms—papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), low-grade and high-grade noninvasive papillary URCa—make their diagnosis easily achieved on hematoxylin and eosin (H&E) sections. Therefore the diagnostic role of IHC in URCa is practically limited to 1) distinguishing high-grade invasive URCa from tumors that secondarily involve the bladder from adjacent organs or, more rarely, from distant primary sites; 2) assigning a primary urothelial origin for a metastatic carcinoma of unknown primary; 3) potential utility in distinguishing CIS from reactive urothelial atypia; and 4) establishing the diagnosis in rare variants, such as plasmacytoid and sarcomatoid URCa. Urinary bladder involvement by a secondary tumor, either as a metastasis or by direct extension, occurs most commonly from colorectal (33%), prostatic (12%), and cervical (11%) sites.2,60 Less common sources include breast, stomach, lung, and melanoma primaries. Spread from a colonic or rectal primary could present a diagnostic challenge in bladder transurethral resection (TUR) samples. In fact, such secondary involvement is a more common occurrence than primary adenocarcinoma of the bladder. Differentiating a colorectal carcinoma (CRCa) spread from intestinal-type primary adenocarcinoma of bladder cannot usually be made with certainty. The presence of a background of urothelial intestinal metaplasia with associated glandular dysplasia may favor a primary origin, however, the pathologist should consider the possibility of secondary colonization of bladder urothelial mucosa by a welldifferentiated CRCa mimicking intestinal metaplasia/ dysplasia. In general, a recommendation to clinically rule out spread from a colorectal primary by imaging techniques should be forwarded in order to avoid a potentially unjustifiable radical cystectomy procedure. Immunostains that include CDX-2, β-catenin, villin, and CK7/CK20 have been shown to be helpful by some authors.10,61-63 However, some degree of overlap in staining patterns among primary enteric-type bladder adenocarcinomas and secondary colorectal adenocarcinomas still exist, which limits the utility of these markers on an individual case basis (Fig. 17-2, A-F). The second most common source of secondary tumor involvement of the bladder is PCa. Even in cases in which a prior known history of PCa is given, superimposed morphologic changes, such as squamous differentiation as a result of prior hormonal or radiation treatment, lead to additional difficulty in distinguishing PCa recurrence from a second primary URCa on a transurethral resection of prostate (TURP) or needle biopsy. As mentioned in the discussion of prostate tumors, poorly differentiated PCa may have enlarged nuclei and prominent nucleoli, yet little variability is found in nuclear shape and size in PCa. High-grade
619
URCa often reveals a more pronounced nuclear pleomorphism. URCa tends to grow in nests, even when poorly differentiated, and usually lacks the cribriform and cordlike architecture of PCa. However, in the absence of a noninvasive flat or papillary URCa component, it is difficult on limited material to distinguish high-grade PCa that involves the bladder from primary high-grade infiltrating URCa on routine H&E stained sections. Given the crucial difference in management and prognosis, resorting to immunostains is a must if the distinction could not be made with absolute certainty on morphologic grounds. As mentioned in the section on prostate, PSA and prostate-specific acid phosphatase (PSAP) have proved to be useful in identifying prostate lineage. However, the sensitivities of PSA and PSAP decrease in poorly differentiated PCa, and newer markers such as prostein (P501S), PSMA, proPSA (pPSA), and NKX3-1may be of added utility. Combining the above markers with urothelial lineage markers, such as thrombomodulin and uroplakin, will further facilitate resolving a urothelial versus a prostatic differential (Table 17-2).64 It should also be kept in mind that both UPIII and TM are of only moderate sensitivity, compared with HMWCK and p63, in labeling URCa. Recent studies have documented HMWCK positivity in more than 90% of URCa.64,65 HMWCK is only rarely and focally expressed in PCa (8%)64; and p63 has a greater specificity for URCa, albeit lower sensitivity, compared with HMWCK (100% specificity, 83% sensitivity).64 Finally, our experience shows that CK7 and CK20 are of limited utility in this differential, given that they may both be positive in a subset of PCa.66,67 More recently, GATA3 has achieved better sensitivity and specificity in labeling urothelial carcinoma and ruling out prostate adenocarcinoma. Among other rare sources of tumors that metastasize to bladder, mammary carcinoma deserves a cautionary note. The possibility of a breast metastasis should be raised when epithelial infiltration is seen in the form of cords or individual plasmacytoid to signet-ring–shaped cells that involve lamina propria without associated overlying papillary urothelial proliferation or CIS. In such cases, the differential should also include a rare variant of URCa, plasmacytoid variant (Fig. 17-3, A-C).68-72 Obtaining a proper clinical history and the use of IHC that includes estrogen and progesterone receptors, gross cystic disease fluid protein (GCDFP), UPIII, and TM will help reach a proper diagnosis. Finally, positive reactivity for CD138 in the plasmacytoid variant of URCa can lead to a misdiagnosis of plasma cell dyscrasia if a proper battery of immunostains was not utilized. In the workup of metastatic carcinoma of unknown primary origin, inclusion of GATA3, CK7, CK20, HMWCK, TM, and UPIII is needed to rule out a urothelial primary. Using a panel of four of the latter markers, excluding CK7, in a wide range of 112 urothelial tumors, Parker and colleagues21 revealed that expression pattern varied with tumor grade and stage (Table 17-3). Variant morphologic subtypes showed staining similar to that of conventional URCas. In the same study, TMA showed no UPIII immunoreactivity in tissue cores of
620
Immunohistology of the Bladder, Kidney, and Testis
A
B
C
D
E
F
Figure 17-2 A and B, Rectal adenocarcinoma secondarily involving urinary bladder. The tumor is negative for cytokeratin 7 (CK7) and is positive for CK20, CDX-2, and β-catenin as shown in C through F, respectively.
nonurothelial tumors, rendering the expression of UPIII in a tumor almost diagnostic of urothelial origin. Although coexpression of TM, HMWCK, and CK20 strongly suggests urothelial origin, none of these markers is as specific as UPIII and GATA3, given that TM is expressed in nonurothelial tumors, such as non–small cell lung carcinomas (27%) and rare lymphomas, and given that HMWCK is expressed by 43% of non–small cell lung carcinomas and mesotheliomas among others.21
Among URCa variants, sarcomatoid carcinoma deserves special attention because of its likelihood to be confused with “true” mesenchymal neoplasms such as leiomyosarcoma, osteosarcoma, and rhabdomyosarcoma. This is especially likely when heterologous elements are displayed and noninvasive papillary or in situ urothelial components are not evident. Reactivity for one or more of the markers AE1/AE3, CAM5.2, epithelial membrane antigen (EMA), HMWCK, p63, CK7, and
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms
621
TABLE 17-2 Urothelial and Prostatic Markers in the Differential Diagnosis of Prostate Cancer vs. Urothelial Carcinoma Carcinoma Prostate Urothelial
HMWCK (%)
p63 (%)
Thrombomodulin (%)
GATA3 (%)
PSA (%)
P501S (%)
0
7.9
0
5.3
94.7
97.4
86
91.4
82.9
68.6
0
0
PSMA (%)
NKX3-1 (%)
pPSA (%)
100
92.1
94.7
0
0
5.7
Data from Chuang AY, DeMarzo AM, Veltri RW, et al: Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma. Am J Surg Pathol 2007;31:1246-1255; and Liu H, Shi J, Wilkerson ML, Lin F: Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol 2012;138:57-64. HMWCK, High-molecular-weight cytokeratin; pPSA, pro-PSA; PSA, prostate-specific antigen; PSMA; prostate-specific membrane antigen.
TABLE 17-3 Immunohistochemical Results in Varying Grades and Stages of Urothelial Neoplasms Grade/Stage
UPIII n (%)
TM n (%)
LMP (n = 14)
12 (86)
12 (86)
13 (93)*
6 (43)
LG (n = 16)
12 (75)
16 (100)
10 (63)
8 (50)
HG (n = 16) INV (n = 36) MET (n = 25)
13 (81) 14 (39) 13 (52)
12 (75) 22 (61) 15 (60)
HMWCK n (%)
CK20 n (%)
11 (69)
†
12 (75)
30/34 (88)
†
17/34 (50)
24 (96)
†
10 (40)
From Parker DC, Folpe AL, Bell J, et al: Potential utility of uroplakin III, thrombomodulin, high molecular weight cytokeratin, and cytokeratin 20 in noninvasive, invasive, and metastatic urothelial (transitional cell) carcinomas. Am J Surg Pathol 2003;27:1-10. *Predominantly in basal cells. † Staining throughout the tumor. CK, Cytokeratin; HG, high grade; HMWCK, high-molecular-weight cytokeratin; INV, invasive; LG, low grade; LMP, low malignant potential; MET, metastatic; TM, thrombomodulin; UPIII, uroplakin III.
A
Figure 17-3 A and B, Plasmacytoid variant of urothelial carcinoma. C, High-molecular-weight cytokeratin highlights the tumor cells.
B
C
622
Immunohistology of the Bladder, Kidney, and Testis
CK20 supports the diagnosis of sarcomatoid carcinoma, although caution should be used with positivity for CAM5.2 and p63 because they can be seen in sarcomas. Positive reactivity for actin can be encountered in sarcomatoid carcinoma and should not mislead the observer to a diagnosis of leiomyosarcoma. As discussed below attention to differentiating sarcomatoid carcinoma from IMT is also crucial. URINARY BLADDER ADENOCARCINOMA
Primary adenocarcinomas of the bladder are relatively rare, therefore establishing their diagnosis requires the exclusion of secondary involvement by direct extension or metastatic spread. Bladder adenocarcinoma variants include signet-ring cell carcinomas and urachal, mucinous, and enteric adenocarcinomas. Distinguishing a PCa that extends into the bladder from a primary bladder adenocarcinoma is important and has both clinical and management implications; IHC markers of prostate lineage are of great utility in this regard. Although the specificity of newer prostate lineage markers have been tested against bladder URCa, the same cannot be said about their pattern of reactivity in bladder adenocarcinoma. In a recent IHC study to evaluate 37 adenocarcinomas of bladder,73 we demonstrated that a minority of bladder adenocarcinomas are positive for prostate antigens P501S and PSMA. P501S showed moderate diffuse cytoplasmic staining in 11% of cases, including enterictype and rare mucinous adenocarcinomas. The granular perinuclear staining pattern of P501S typically seen in prostatic adenocarcinoma was absent in all cases of bladder adenocarcinoma. In addition, PSMA showed diffuse cytoplasmic or membranous staining in 21% of bladder adenocarcinomas, including signet-ring, urachal, mucinous, and enteric-type variants. All cases were negative for PSA and PSAP, therefore immunoreactivity for P501S and PSMA should be interpreted with caution in such settings (Fig. 17-4, A-C). The lack of granular perinuclear staining for P501S and the absence of membranous PSMA staining both favor a bladder adenocarcinoma. Membranous PSMA staining indistinguishable from that seen in PCa can be seen in less than 10% of bladder adenocarcinoma.
A
B
SMALL CELL CARCINOMA OF URINARY BLADDER
Small cell carcinoma of the bladder occurs as rare aggressive tumors found either in pure form or more commonly admixed with urothelial CIS, invasive URCa, SCC, or an adenocarcinoma component. Clinically, small cell carcinoma is usually seen at an advanced stage with visceral and bone metastases, and it may be associated with paraneoplastic syndromes. In the largest series by Cheng and associates,74 a dismal 5-year survival rate of 16% was encountered despite adopting a multimodal therapeutic approach that included chemotherapy and radical cystectomy. In our experience, immunostains for neuroendocrine markers are only rarely needed (synaptophysin+, chromogranin+, and CD56+), especially when a non–small cell component is associated. The presence of typical small cell morphology similar to that
C Figure 17-4 Primary adenocarcinoma of urinary bladder (A) showing cytoplasmic positivity for prostate markers P501S (B) and prostatespecific membrane antigen (C).
encountered in the lung counterpart with characteristic brisk mitotic activity and extensive necrosis facilitate the diagnosis. In cases in which the differential diagnosis includes malignant lymphoma or other small blue cell tumors, pancytokeratins AE1/AE3 and CAM5.2, in
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms
addition to the above neuroendocrine markers, can help establish the diagnosis. Small cell carcinoma of bladder has a high number of genomic alterations. In their analysis of a single tumor having areas of both small cell and URCa, Cheng and associates75-77 revealed genetic evidence that strongly suggests that small cell carcinoma can develop from URCa through the acquisition of additional genetic alterations. Deletions are most frequent at 10q, 4q, 5q, and 13q. These regions may carry tumor suppressor genes with relevance for this particular tumor type. Gains at 8q, 5p, 6p, and 20q and amplifications at 1p22-32, 3q26.3, 8q24, and 12q14-21 suggest localization of oncogenes at these loci.77 BENIGN MIMICS OF BLADDER CARCINOMA
We will limit our discussion to two of the benign mimic of bladder tumors, nephrogenic adenoma as a mimic of both URCa and adenocarcinoma and IMT as a mimic of sarcomatoid carcinoma or sarcomas. Nephrogenic Adenoma
Typically, nephrogenic adenoma (NA) displays tubulopapillary structures lined by a single layer of bland
623
cuboidal epithelial cells with low mitotic activity. Tubular structures are frequently surrounded by a distinct ringlike basement membrane and may contain eosinophilic or mucinous secretions. The tubular lining cells frequently display hobnail nuclei. Other tubules can have a flattened lining, thus leading to a false impression of lymphatic structures. Rarely, intracytoplasmic lumina can form in single infiltrating cells that mimic signet-ring carcinoma. Finally, rare examples in which hyalinized myxoid stroma “suffocates” the compressed tubular structures, termed fibromyxoid variant of NA, can be confused with mucinous adenocarcinoma of bladder.78 When typical, NA is easily recognized in TURB samples. In difficult examples, the diagnosis of NA can be supported by their unique positivity for Pax-2 and Pax-8.79,80 NA is negative for HMWCK and p63 in two thirds of cases. A word of caution is merited in this setting regarding the fact that clear cell adenocarcinoma of bladder will share the above immunophenotype with NA and should be recognized by the higher degree of cytologic atypia and mitotic activity found in clear cell bladder adenocarcinoma, which can be further illustrated by a high Ki-67 index compared with NA (Fig. 17-5, A-D).79,80
A
B
C
D Figure 17-5 A to C, Nephrogenic adenoma of the urinary bladder. D, Nuclear positivity for Pax-8 is shown.
624
Immunohistology of the Bladder, Kidney, and Testis
KEY DIAGNOSTIC POINTS Nephrogenic Adenoma • Variable expression of high-molecular-weight cytokeratin/ p63 • Positive for Pax-2 and Pax-8 • Lacks malignant cytology of clear cell carcinoma • Low Ki-67
INFLAMMATORY MYOFIBROBLASTIC TUMOR
Inflammatory myofibroblastic tumor (IMT) of bladder may arise either spontaneously or as a result of a prior instrumentation of the bladder. IMTs are benign mesenchymal neoplasms composed of a proliferation of relatively monotonous myofibroblastic cells (typical tissue culture appearance) in a richly vascularized background with red blood cell extravasation and lymphoplasmacytic inflammatory infiltrate. Mitotic activity ranges from absent to brisk, and abnormal mitotic figures are not present. Although IMTs may occasionally recur (25%), only one case of malignant transformation of IMT has been reported in the genitourinary tract.35 As mentioned above in the section on anaplastic lymphoma kinase, two thirds of IMTs contain rearrangement of the ALK gene on chromosome 2p23 with different translocation partners. The latter can be demonstrated by splitapart interphase cytogenetic FISH techniques (Fig. 17-6, A-B). The translocation leads to ALK protein overexpression on IHC (Fig. 17-7, A-C). IMTs are frequently immunoreactive for pancytokeratin and CAM5.2, a fact worth remembering when attempting to differentiate IMT from sarcomatoid carcinoma. IMTs are also frequently positive for smooth muscle actin (SMA) and desmin, and presence of desmin may lead to their
misinterpretation as leiomyosarcoma. IMT is usually negative for CD34, S-100 protein, and CD117 (Table 17-4).
Genomic and Theranostic Applications Accumulating molecular genetic evidence supports two distinct broad pathogenetic pathways for bladder cancer (BC) development that seem to parallel the contrasting biologic and clinical phenotypes of non–muscle invasive (superficial) and muscle-invasive URCa. Whereas the majority of invasive URCas are thought to originate through progression from dysplasia to flat CIS and highgrade noninvasive lesions, superficial urothelial lesions are thought to originate from benign urothelium through a process of urothelial hyperplasia. Progression from non–muscle invasive to muscle-invasive disease accounts for only a small percentage (10% to 15%) of the entire pool of noninvasive lesions. Genetic instability is key in the accumulation of genetic alterations required for progression to muscle-invasive bladder cancer (MI-BC).81-84 Clinically, a significant proportion of non–muscle invasive bladder cancer (NMI-BC; pTa and pT1) are deemed to recur following TURB, and only a minority of cases endure progression to high-grade carcinoma that will ultimately progress to MI-BC. Three primary genetic alterations have consistently been associated with the pathogenesis pathway of NMI-BC. These include tyrosine kinase receptor FGFR3, H-RAS,85 and PIK3CA.83,86,87 Alterations in the RASMAPK and PIK3CA-Akt pathways are in large part responsible for promoting cell growth in urothelial neoplasia. Activating mutations in the RAS family of genes leads to activation of mitogen-activated kinase-like protein (MAPK) and PIK3CA pathways. Not surprisingly, activating mutations in upstream tyrosine kinase
1R1G1F
2F
A
B
Figure 17-6 A and B, Interphase fluorescence in situ hybridization analysis for ALK1 gene in inflammatory myofibroblastic tumor by using split-apart probes. The presence of a set of one green and one red signal in addition to a juxtaposed red-green (yellow overlap) set indicates a rearrangement in one of the two ALK1 alleles.
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms
A
625
B
Figure 17-7 A and B, Inflammatory myofibroblastic tumor of the urinary bladder. C, Positive ALK1 staining is shown.
receptor FGFR3 seems to be mutually exclusive with RAS mutations given that both signal through a common downstream pathway in urothelial oncogenesis. PIK3CA and FGFR3 mutations generally co-occur, suggesting a potential synergistic, additive, oncogenic effect for PIK3CA mutations. The pathogenic pathway for MI-BC primarily involves alterations in tumor suppressor genes involved in cell-cycle control, including TP53, CDKN2A, and RB1 (see Figs. 17-8 and 17-9).59,81,84 As illustrated in
C Figure 17-8, progression of the subset of NMI-BC into higher grade muscle-invasive disease is similarly based on alterations in TP53 and Rb tumor suppressor genes. Established clinicopathologic prognostic parameters for NMI-BC include pT stage, World Health Organization (WHO)/International Society of Urological Pathology (ISUP) grade, tumor size and multifocality, presence of CIS, and frequency and rate of prior recurrences.88 Prognostic parameters that can accurately predict progression in patients with NMI-BC tumors are actively
TABLE 17-4 Immunohistochemistry of Spindle Cell Neoplasms of the Urinary Bladder IMT
Sarcomatoid URCa
Leiomyosarcoma
Rhadomyosarcoma
Keratin
+
+
S
−
EMA
−
+
−
−
Vimentin
+
+
+
+
Desmin
+
−
+
+
MSA
+
S
+
+
Alk
S
−
−
−
Alk, Anaplastic lymphoma kinase; EMA, epithelial membrane antigen; IMT, inflammatory myofibroblastic tumor; MSA, muscle-specific actin; URCa, urothelial carcinoma. Reactivity: + almost always positive; S, sometimes positive; −, negative.
626
Immunohistology of the Bladder, Kidney, and Testis
Urothelial hyperplasia
9q / 9p
70% recurrence
LG URCa
HRAS/FGFR3 PIK3CA-Akt Normal urothelium
~15%
p53, Rb 8p , 11p , 13q , 14q ~50%
9q / 9p
Dysplasia/ CIS
HG URCa
Invasive URCa
p53, Rb, 8p 8p , 17p
E-cad MMP, VEGF COX2
MMP9, VEGF TSP, IL8, EGFR, IMP3, LAMC2
Metastasis
Figure 17-8 Divergent molecular pathways of oncogenesis in non–muscle-invasive and muscle-invasive urothelial carcinoma (URCa) of urinary bladder. Genetic alterations are depicted in key stages of disease progression. CIS, Carcinoma in situ; COX2, cyclooxygenase 2; E-cad, E-cadherin; EGFR, epithelial growth factor receptor; HG URCa, noninvasive high-grade URCa; IL8, interleukin 8; LG URCa, noninvasive low-grade URCa; MMP, matrix metalloproteinase; Rb, retinoblastoma; VEGF, vascular endothelial growth factor.
sought to further facilitate identification of those in need of vigilant surveillance and an aggressive treatment plan. The latter is especially pertinent in a disease in which the financial burden and loss of quality of life for patients under surveillance are significant. Per patient, bladder cancer is the most expensive single solid tumor in the United States, with a staggering $3 billion estimated annual cost to our health care system.89 Furthermore, given the current poor outcome of muscle-invasive disease (60% or less overall survival rate), markers that can improve prognostication in this group of patients are needed.90-92 As our understanding of molecular pathways involved in urothelial oncogenesis increasingly come into focus,
the translational field of molecular prognostication, theranostics, and targeted therapy in BC has sharply gained momentum.41,52,93-109 Evidently, a rigorous validation process ought to precede the incorporation of such molecular biomarkers in clinical management. Initial retrospective discovery studies need to be confirmed and validated in large independent cohorts. The subsequent crucial step is validating the robustness of the proposed biomarker in a well-controlled multiinstitutional randomized prospective study. Such a prospective study should support an additive role for the inclusion of the new biomarker over existing management algorithms.110,111 The lack of the latter crucial steps in biomarker development hindered the streamlining of
EGF EGFR
Ras
Raf
MEK
ERK
RASSF1A
C-Myc
Cyclin D1
Cyclin E CDK4 CDK2
p14
CDKN2
p16 Rb
Rb
E2F MDM2
p53
p21
E2F Proliferation DNA
Figure 17-9 Receptor tyrosine kinases (EGFR/Ras/Mek/ERK) and cell-cycle regulators (p14, p16, p53, p21, cyclins D1 and E, and Rb) pathways in urothelial carcinoma. Green and red arrows represent stimulation and inhibition, respectively.
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms
clinical utilization of several promising markers in BC patient management.112-115
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications CHROMOSOMAL NUMERICAL ALTERATION
Chromosome 9 alterations are the earliest genetic alterations in both of the divergent pathways of BC development described above. They are responsible for providing the necessary milieu of genetic instability that, in turn, allows for the accumulation of subsequent genetic defects.84 Several additional structural/numerical somatic chromosomal alterations are also a common occurrence in BC. Among these, gains of chromosomes 3q, 7p, and 17q and 9p21 deletions (CDKN2A locus) are of special interest given their potential diagnostic and prognostic value.116,117 A multitarget interphase FISH-based urine cytogenetic assay was developed118 based on the above numerical chromosomal alterations and is now commercially available and is commonly used in clinical management (Fig. 17-10). Initially approved by the Food and Drug Administration (FDA) for surveillance of recurrence in previously diagnosed BC patients, the test subsequently gained approval for screening in high-risk (smoking exposure) patients with hematuria. The multicolor FISH assay appears to enhance the sensitivity of routine urine cytology analysis and can be used in combination with routine cytology as a reflex testing in cases with atypical cytology. A sensitivity range of 69% to 87% and a specificity range of 89% to 96% have been reported with the multitarget interphase FISH assay.119 With the exception of one study,120 the multitarget FISH urine assay has been shown to be more sensitive than routine cytology. An additional advantage of urine-based FISH testing could be the anticipatory positive category of patients identified by such assay. This refers to patients in whom FISH assay detects molecular alteration of BC in urine cells several
Figure 17-10 Interphase fluorescence in situ hybridization urine cytology analysis by using Vysis UroVysion probe sets (Abbott Molecular, Abbott Park, IL) for chromosomes 3q (red), 7p (green), and 17q (aqua) and 9p21 deletions (p16 locus: gold). Note polysomy for 17q (aqua) and deletion of 9p21 loci (absence of gold signals) in two urothelial carcinoma cells. Modified from Moonen PM, Merkx GF, Peelen P, et al: UroVysion compared with cytology and quantitative cytology in the surveillance of non–muscle-invasive bladder cancer. Eur Urol 2007;51:1275-1280.
627
months before cancer detection by cystoscopy or routine cytology. In the study by Yoder and colleagues,121 two thirds of the 27% of patients categorized as “anticipatory positive” developed BC that was detected by cystoscopy up to 29 months later. Such encouraging results point to the great potential of molecular testing in early detection and allocation of vigorous, frequent follow-up cystoscopy in at-risk patients.122-125 Finally, several recent studies have pointed to the potential prognostic role for multitarget FISH analysis.116,117,126-128 Maffezini and colleagues127 were able to demonstrate that low-risk FISH-positive patients, defined as those with 9p21 loss and chromosome (Ch) 3 abnormalities, had a higher rate of recurrence compared with FISH-negative patients. The recurrence rate was even greater in patients with a high-risk positive FISH (Ch7/Ch17 abnormality). Kawauchi and colleagues116 used bladder washings, and Kruger and associates117 used formalin-fixed paraffin-embedded (FFPE) transurethral biopsy samples and both independently found loss of 9p21 to predict recurrence but not progression in NMI-BC. Furthermore, both Savic and associates126 and Whitson and colleagues128 found urine cytology and FISH in post-BCG bladder washings to be predictive of failure of BCG therapy in patients with non–muscle-invasive disease. Such a promising prognostic role for multitarget FISH awaits prospective randomized trials before clinical integration into a practice algorithm. Clear guidelines for interpretation and test performance parameters in terms of interobserver reproducibility are also needed.129 RECEPTOR TYROSINE KINASES
Recent studies have pointed to the potential prognostic value of evaluating the expression of receptor tyrosine kinases (RTKs) such as FGFR3, EGFR, and other ERB family members (ERBB2, formerly HER2/neu, and ERBB3)84,100,130-140 in non–muscle-invasive and muscleinvasive bladder cancer. FGFR3 mutations are a common occurrence in NMI-BC and can theoretically be used alone or combined with RAS and PIK3CA oncogenes as markers of early recurrence during surveillance. Both Zuiverloon and colleagues141 and Miyake and associates142 independently developed sensitive polymerase chain reaction (PCR) assays for detecting FGFR3 mutations in voided urine. A positive urine sample by the assay developed by Zuiverloon’s group was associated with concomitant or future recurrence in 81% of NMI-BC cases. An even higher positive predictive value of 90% was achieved in patients with consecutive FGFR3-positive urine samples. Similarly, Miyake and associates were able to detect FGFR3 mutations in 53% of their 45 patients and found their assay to be superior to cytology (78% vs. 0%) in detecting post-TURB recurrence in NMI-BC harboring FGFR3 mutations in primary tumors. Kompier and colleagues87 were recently able to develop a multiplex PCR assay for mutational analysis that detects the most frequent mutation hot spots of HRAS, KRAS, NRAS, FGFR3, and PIK3CA in FFPE TURB samples. They demonstrated evidence of at least
Immunohistology of the Bladder, Kidney, and Testis
one mutation in up to 88% of low-grade NMI-BC samples. Hernandez and colleagues143 revealed that FGFR3 mutations were more common among low malignant potential neoplasms (LMPNs; 77%) and TaG1 and TaG2 tumors (61% and 58%) than among TaG3 tumors (34%) and T1G3 tumors (17%). On multivariable analysis, mutations were associated with increased risk of recurrence in NMI-BC. Van Rhijn and associates144 previously proposed a molecular grade parameter based on a combination of FGFR3 gene mutation status and MIB-1 index as an alternative to pathologic grade in NMI-BC. Recently, the same group130 elegantly validated their previously proposed molecular grade parameter and compared it with the European Organization for Research and Treatment of Cancer (EORTC) NMI-BC risk calculator,145 a weighted score of six variables that includes 1973 WHO grade, stage, presence of CIS, multiplicity, size, and prior recurrence rate. The molecular grade was more reproducible than the pathologic grade (89% vs. 41% to 74%). FGFR3 mutations significantly correlated with favorable disease parameters, whereas increased MIB-1 was frequently seen with pT1, high grade, and high EORTC risk scores. EORTC risk score remained significant in multivariable analyses for recurrence and progression. Importantly, molecular grade also maintained independent significance for progression and disease-specific survival, and the addition of molecular grade to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7%. Several studies have suggested a negative prognostic role for ERBB2 amplification and/or overexpression in MI-BC.110,146-148 Most recently, Bolenz and colleagues138 found ERBB2-positive MI-BC patients to be at twice the risk for recurrence and cancer-specific mortality on multivariable analyses adjusted for pathologic stage, grade, lymphovascular invasion, lymph node metastasis, and adjuvant chemotherapy. TP53, CELL-CYCLE REGULATORS, AND PROLIFERATION INDEX MARKERS
Early studies by Sarkis and associates42,43,149,158 revealed TP53 alterations to be a strong independent predictor of disease progression in BC (NMI-BC, MI-BC, and CIS), and TP53 has also been shown to be predictive of increased sensitivity to chemotherapeutic agents that lead to DNA damage.49,50,150 Recent studies have further supported the prognostic role of TP53151 in pT1-pT2 patients following cystectomy, showing an independent role for TP53 alteration in predicting disease-free survival (DFS) and disease-specific survival (DSS). Among other G1-S phase cell-cycle regulators, cyclins D1 and D3, p16, p21, and p27 have also been evaluated as prognosticators in NMI-BC.15,53,109,150,152-154 Lopez-Beltran and associates152 confirmed their initial finding150 of the independent prognostic role of cyclin D1 and D3 overexpression in predicting progression in pTa and pT1 tumors. Their findings, however, are in contrast to subsequent findings by Shariat and colleagues,109 emphasizing the need for further validation in multiinstitutional large cohorts of patients.
A synergistic prognostic role for combining TP53 evaluation with other cell-cycle control elements such as pRb, cyclin E1, p21, and p27 is emerging both in NMI-BC and MI-BC.49,103,105,155,156 In a study by Shariat and colleagues,157 NMI-BC patients with TURB who demonstrated synchronous IHC alterations in all four tested markers—p53, p21, pRb, and p27—were at significantly lower likelihood of sustaining DFS compared with patients with only three markers. The negative predictive effect was decreased with decreasing number of altered markers (3 vs. 2 vs. 1; Fig. 17-11). Similarly, some of the same researchers later found that combining p53, p27, and Ki-67 assessment in pT1 radical cystectomy specimens improved the prediction of DFS and DSS.156 Chatterjee and associates103 demonstrated a similar synergistic prognostic role for the assessment of immunoexpression of multiple molecular markers (p53, pRb, and p21) in patients undergoing cystectomy for MI-BC. The superiority of a multimarker approach compared with the prior single-marker approach certainly merits further assessment. Such a multimarker approach of prognostication could soon be integrated in the standard of care in BC management once additional multiinstitutional prospective trials confirm these promising findings. Tumor proliferation index measured immunohistochemically by either Ki-67 or MIB-1 has been consistently shown to be a prognosticator in bladder cancer.53,130,144,150,159-162 As mentioned earlier, tumor proliferation index (MIB-1) in NMI-BC plays a prognostic role as one of the elements of the molecular grade parameter forwarded by Van Rhijn and colleagues.144 The independent prognostic role of proliferation index measured by Ki-67 has also been shown. In the study 100 Progression-free survival probability
628
None altered
90
Any one altered
80 70 60
Any two altered
50 40
Any three altered
30
P < .001 Log rank test pooled over strata
20 10
All four altered
0 0
12 24 36 48 60 72 84 96 108 120 132 144 Months after surgery 5 yr. + SE One altered 100 + 0% Any one altered 89 + 8% Any two altered 49 + 16% Any three altered 33 + 13% All four altered 0 + 0%
Figure 17-11 Synergistic prognostic role of immunohistochemical analysis of four markers—p53, p21, pRb, and p27—in superficial urothelial carcinoma. SE, Standard error.
Diagnostic Immunohistochemistry of Specific Bladder Neoplasms
by Quintero and colleagues,159 Ki-67 index in NMI-BC TURB was predictive of progression-free survival (PFS) and DSS. A similar role for proliferation index assessment as a prognosticator has been established in MI-BC. Building on initial findings of significance in an organ-confined subset of MI-BC by Margulis and colleagues,160 a recent report of the bladder consortium multiinstitutional trial (7 institution, 713 patients) again confirmed the role of the proliferation index, measured in cystectomy specimens.161 In the later study, Ki-67 improved prediction of both PFS and DSS when added to standard prediction models, supporting a role for proliferation index assessment in stratifying patients for perioperative systemic chemotherapy. This approach has certainly taken Ki-67 assessment a step closer to clinical applicability in MI-BC. GENE EXPRESSION AND GENOMIC ANALYSIS
Several recent gene-expression studies have highlighted sets of differentially expressed genes that may play a role in diagnosis and in predicting recurrence and progression in BC.81,94-96,106,108,163-171 In a landmark study by Sanchez-Carbayo and colleagues,94 oligonucleotide arrays were used to analyze transcript profiles of 105 cases of NMI-BC and MI-BC. Hierarchical clustering and supervised algorithms were used to stratify bladder tumors based on stage, nodal metastases, and overall survival. Predictive algorithms were 89% accurate for tumor staging by using genes differentially expressed in NMI versus MI tumors. Accuracies of 82% (entire cohort) and 90% (MI-BC) were also obtained for predicting overall survival. A genetic profile that consisted of 174 probes was able to identify patients with positive lymph nodes and poor survival. Furthermore, two independent global test runs confirmed the robust association of the suggested profile with lymph node metastases and overall survival simultaneously. As another layer of validation, one of the top-ranked genes to code for a soluble protein, synuclein, was selected from the gene-expression profile to evaluate associations with its prognostic significance. IHC analyses on tissue arrays confirmed the significant association of synuclein with tumor staging and clinical outcome independent of clinicopathologic parameters. Recently, Birkhahn and colleagues106 attempted to identify genes predictive for recurrence and progression using a quantitative pathway-specific approach in a set of 24 key genes and real-time PCR in tumor biopsies at initial presentation. They found CCND3 expression to be highly sensitive and specific for recurrence (97% and 63%, respectively). Whereas HRAS, E2F1, BIRC5/ survivin, and VEGFR2 were predictive for progression by univariate analysis, on multivariable analysis, the combination of HRAS, VEGFR2, and VEGF expression status predicted progression with an impressive 81% sensitivity and 94% specificity. In a recent study, Lindgren and colleagues169 suggested that a combined molecular and histopathologic classification of BC may prove more powerful in predicting outcome and stratifying treatment. The authors combined gene-expression analysis, whole genome array
629
comparative genomic hybridization (CGH) analysis, and mutational analysis of FGFR3, PIK3CA, KRAS, HRAS, NRAS, TP53, CDKN2A, and TSC1 to identify two intrinsic molecular signatures, MS1 and MS2. Genomic instability was the most distinguishing genomic feature of MS2 signature, independent of TP53/MDM2 alterations. Their genetic signatures were validated in two independent datasets that successfully classified URCas into low-grade and high-grade tumors and also distinguished NMI-BC and MI-BC with high precision and sensitivity. Furthermore, a gene-expression signature that independently predicts metastasis and DSF was also defined. This clearly supports the role of molecular grading as a complement to standard pathologic grading. Mengual and associates108 performed gene-expression analysis in 341 urine samples from NMI-BC and MI-BC patients and in 235 controls by TaqMan arrays. A 12+2 gene-expression signature demonstrated a staggering 98% sensitivity and 99% specificity in discriminating between BC and control and a 79% sensitivity and 92% specificity in predicting tumor aggressiveness (NMI-BC vs. MI-BC). The signature was then validated in voided urine samples and maintained accuracy. In an integrated genetic/epigenetic approach, Serizawa and colleagues168 prospectively performed mutational screening of a set of six genes—FGFR3, PIK3CA, TP53, HRAS, NRAS, and KRAS—and quantitatively assessed promoter methylation status of 11 additional genes (APC, ARF, DBC1, CDKN2A [formerly INK4A], RARB, RASSF1, SFRP1, SFRP2, SFRP4, SFRP5, and WIF1) in NMI-BC tumor biopsies and corresponding urine samples from 118 patients and 33 controls. A total of 95 oncogenic mutations and 189 hypermethylation events were detected. The total panel of markers provided a sensitivity of 93% and 70% in biopsies and urine samples, respectively. FGFR3 mutations in combination with three methylation markers—APC, RASSF1, and SFRP2—provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. Selecting patients for neoadjuvant chemotherapy on the basis of risk of node-positive disease has the potential to benefit high-risk patients while sparing other patients toxic effects and delay to cystectomy. Smith and colleagues172 recently reported a 20-gene expression model to predict the pathologic node status in primary tumor tissue from three independent cohorts that clinically lacked evidence of nodal metastasis. The model was developed in two separate training cohorts (90 and 66 patients); but most importantly, it was then assessed for its ability to predict node-positive disease in tissues from a separate phase III trial cohort (185 patients). The model was able to predict nodal status independent of standard clinicopathologic prognostic criteria. The validation step in an independent cohort is vital given recent reports that gene-expression tumor signatures are not portable across cohorts. With the impending cost and turnaround time advantages of next-generation sequencing technology, the power of a genomic approach in providing a noninvasive diagnostic and predictive tool should be actively pursued in a prospective large cohort.
630
Immunohistology of the Bladder, Kidney, and Testis
EPIGENETIC ALTERATIONS
Epigenetic analysis is also gaining momentum in BC as a noninvasive diagnostic tool for screening and surveillance. As a prognostic tool, epigenetic analysis has similarly shown promising potential in BC patients.168,173-185 In an early study by Catto and colleagues,180 hypermethylation analysis at 11 C-phosphate-G (CpG) promoter islands was performed by methylation-specific PCR (MSP-PCR) in 116 bladder and 164 upper urinary tract tumors. Promoter methylation was found in 86% of all tumors, and the incidence was relatively higher in upper-tract tumors compared with BC. Methylation was associated with advanced tumor stage and higher tumor progression and mortality rates. Most importantly, on multivariate analysis, methylation at the RASSF1 and DAPK1 gene promoters was associated with disease progression independent of tumor stage and grade. The same group185 using quantitative MSP-PCR at 17 candidate gene promoters found five loci to be associated with progression: RASSF1, E-cadherin, tumor necrosis factor (TNF) SR25, EDNRB, and APC. Multivariate analysis revealed that the overall degree of methylation was more significantly associated with subsequent progression and death than tumor stage. An epigenetic predictive model developed by using artificial intelligence techniques identified likelihood and timing of progression with 97% specificity and 75% sensitivity. Among the studies to evaluate the diagnostic role of promoter hypermethylation, the study by Lin and colleagues174 used MSP assay in four genes—CDH1, CDKN2A, p14 and RASSF1—in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. MSP detected hypermethylation in the urine of 80% of tested patients. Hypermethylation analysis of CDH1, p14, or RASSF1 in urine sediment DNA detected 85% of superficial and low-grade BC and 79% of high-grade and 75% of invasive BC. The study highlighted the great potential of such tests in detecting NMI-BC. A similar diagnostic role was also found by Cabello and associates176 using a novel technology, methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLAP), to analyze 25 tumor suppressor genes (TSGs) thought to play a role in BC oncogenesis. The TSGs included PTEN, CD44, WT1, GSTP1, BRCA2, RB1, TP53, BRCA1, TP73, RARB, VHL, ESR1, pax5, CDKN2A, and PAX6. The authors found BRCA1, WT1, and RARB to be the most frequently methylated TSGs, and receiver operating characteristic curve analyses revealed significant diagnostic accuracies in two additional validation sets. Finally, assessment of promoter hypermethylation offers additional insights into BC oncogenesis. Promoter hypermethylation of CpG islands and “shores” controlling microRNA (miRNA) expression is one such example.178 PLOIDY AND MORPHOMETRIC ANALYSIS
Several studies have pointed to the independent prognostic role of ploidy and S-phase analysis in
NMI-BC.162,186-192 Ploidy analysis can be performed by flow cytometry or automated image cytometry (ICM) and is applicable to urine cytology specimens as well as biopsy supernatant187 and disaggregated TURB FFPE specimens.188 In one of the largest studies to assess DNA ploidy in NMI-BC (377 test set; 156 validation set); Ali-El-Dein and colleagues186 found stage, DNA ploidy, tumor multiplicity, history of recurrence, tumor configuration, and type of adjuvant therapy to independently predict recurrence. Recurrence at 3 months, grade, and DNA ploidy were the only predictors of progression to muscle invasion. The constructed “predictive index” model successfully stratified patients in a second validation set into three risk groups. Likewise, Baak and colleagues188 were able to show ploidy status and S phase measured by ICM to be strong independent predictors of recurrence and progression in pTa and pT1 patients. Despite all this encouraging data, ploidy analysis still awaits prospective randomized trials to bring ICM and flow cytometry techniques into current standard management algorithms for NMI-BC. EMERGING BIOMARKERS
Other biomarkers with encouraging but less robust data on their potential prognostic role in BC include tumor microenvironment markers, such as cell-adhesion markers E-cadherin and N-cadherin,99,193 and angiogenesis modulators such as hypoxia-induced factors (HIFs) 1α and 2, vascular endothelial growth factor (VEGF), carbonic anhydrase IX (CAIX), and thrombospondin 1.97,98,194-198 In addition, we and others have demonstrated a potential prognostic role for MTOR pathway markers.99,193,199-201 Other markers such as Aurora-A kinase have also been investigated in this setting.202,203 Finally, miRNA profile alterations will certainly be a new area of heavy investigation as a noninvasive diagnostic and prognostic tool in BC patients.204-207 TARGETED THERAPY AND PREDICTIVE MARKERS IN BLADDER CANCER
RTK-HRAS-MAPK, MTOR, and angiogenesis pathways of the tumor microenvironment offer promising opportunities for new targeted treatments of BC.87,104,199,201,208-221 Among receptor tyrosine kinases, HER2 has been targeted in a multicenter phase II trial reported in 2007 by Hussain and associates.222 Fortyfour advanced BC patients with metastatic disease and evidence of tumor HER2 positivity by either IHC, FISH, or elevated serum extracellular HER2 domain levels were treated with a combination of carboplatin, paclitaxel, and gemcitabine with the humanized monoclonal anti-HER2 antibody trastuzumab. Approximately 70% of treated patients demonstrated partial (59%) or complete (11%) response with a median overall survival of 14.1 months. A higher response rate was associated in patients with 3+ HER2 expression by IHC or HER2 gene amplification by FISH compared with those who had 2+ HER2 expression and FISH-negative tumors. Interestingly, in contrast to the strongly correlated HER2
Immunohistology of Renal Neoplasms
gene amplification and 3+ IHC HER2 overexpression usually seen in breast cancer, the majority of HER2 overexpression in bladder URCas is not associated with HER2 gene amplification.223 A second, ongoing, randomized phase II trial is evaluating the role of anti-EGFR recombinant humanized murine monoclonal antibody cetuximab. Patients with metastatic, locally recurrent, or nonresectable disease are treated with standard gemcitabine and carboplatin (GC) chemotherapy with or without cetuximab. By blocking epithelial growth factor binding to the extracellular EGFR domain, cetuximab inhibits downstream signal transduction pathway, accounting for its antiproliferative activity in solid tumors. In BC, a potential added synergistic antiangiogenic effect could be also at play.224,225 A separate, phase II Cancer and Leukemia Group B trial investigated the role of a small molecule inhibitor of EGFR (gefitinib) in advanced BC patients. Gefitinib in combination with GC had no survival or time to progression advantage over GC alone.226,227 Based on the results of a phase II single-arm trial228 that suggested a therapeutic advantage for lapatinib, a tyrosine kinase inhibitor (TKI) that targets both EGFR and HER2 in EGFR- or HER2-positive BC tumors, a phase II/II randomized trial is underway to look at the role of maintenance with lapatinib versus placebo in patients with objective response to first-line chemotherapy who are positive for either marker by IHC or FISH studies. In an attempt to target BC dependence on angiogenesis, monoclonal antibodies and small molecule inhibitors of angiogenesis are under investigation in advanced disease. An initial phase II trial to evaluate the role of bevacizumab, a recombinant humanized monoclonal anti-VEGF antibody, in combination with GC as a firstline therapy in metastatic BC, revealed objective response in two thirds of patients, with 6 of 43 showing complete response, albeit with significant treatment-related toxicity.229 A Cancer and Leukemia Group B (CALBG) phase III randomized trial for GC with and without bevacizumab for metastatic URCa is now underway, in addition to other phase II trials for bevacizumab in combination with other chemotherapeutic agents such as methotrexate, vinblastine, adriamycin, and cisplatin (M-VAC).230 The role of multitarget TKIs in BC has also been investigated, with mixed results. Although sorafenib phase II trials have failed to show significant objective response (inhibition of Raf kinase, PDGFRB, VEGFR-2, and VEGFR-3), Sunitinib has shown a more promising effect (inhibition of VEGFR-2 and PDGFRB) in a recent phase II trial that involved 77 patients at Memorial Sloan–Kettering Cancer Center (MSKCC), where clinical benefit was observed in almost one third of patients. A subsequent randomized double-blind phase II trial was undertaken to investigate the efficacy of sunitinib in delaying progression as a maintenance agent in patients with initial response to standard chemotherapy.231 Finally, given the recent evidence suggesting the presence of MTOR pathway alterations in BC, a phase II trial to evaluate the potential role of everolimus, an inhibitor of the MTOR pathway, in advanced BC is underway.199-201
631
In summary, as our understanding of the complex molecular mechanisms involved in BC development has come into sharper focus, our approaches to diagnosis and management of bladder cancer continue to evolve. In the not so distant future, the current paradigm of the clinicopathologic-based prognostic approach to predicting progression in NMI-BC164,232-234 will be supplemented by a molecularly guided approach based on some of the markers listed in Table 17-5.88,90,103,112,113,156,180,185,196,198,199,201,235-237 Several new targeted therapy agents are under investigation in randomized trials in combination with standard chemotherapy agents, either as first-line treatment or on a maintenance basis, to prolong response in patients with advanced BC.
Immunohistology of Renal Neoplasms Renal carcinoma continues to be a major cause of morbidity and mortality worldwide. Last year, approximately 64,770 new renal tumor patients were diagnosed, and 15,570 deaths were ascribed to renal cancer in the United States. Renal cell carcinoma (RCC) is the seventh most common neoplasm in American males and the eighth most common neoplasm in females.1 A twofold to threefold male predominance of RCC incidence has been noted, but no obvious racial predilection is apparent. Recognized risk factors include tobacco smoking, obesity (BMI >29 may double the risk of RCC), and acquired or hereditary polycystic diseases. The classic clinical presentation symptom triad of flank pain, hematuria, and palpable mass is no longer the leading form of occurrence. Patient presentation as a result of RCC-associated paraneoplastic syndromes because of secreted parathyroid hormone, erythropoietin, prostaglandins, or adrenocorticotropic hormone (ACTH) is also unusual at the current time. This change in clinical presentation is mainly due to a marked increase in incidentally found smaller RCC lesions during imaging studies performed for a variety of other causes. The widespread adoption of partial nephrectomy procedures and the introduction of various forms of ablative treatment (cryoablation and radiofrequency ablation) have brought new challenges to the pathologist in terms of the need to render a diagnosis of RCC on small needle biopsy or during intraoperative consultation. Additionally, the introduction of specific forms of targeted systemic therapy to certain classes of RCC has further emphasized the need for proper classification of RCC on needle biopsy material. Therefore the recent rise in interest in the utilization of ancillary techniques for the diagnosis of RCC comes as no surprise. The following is a practical discussion of the current use of IHC markers in the diagnosis of RCC followed by exploration of the potential role of molecular markers in the prognostication and prediction of therapy response in this disease (Box 17-1).
632
Immunohistology of the Bladder, Kidney, and Testis
TABLE 17-5 Established Clinicopathologic and Potential Molecular Prognostic Parameters in Superficial and Muscle-Invasive Urothelial Carcinoma of Bladder Clinicopathologic Prognostic Parameters In Urothelial Carcinoma Superficial Urothelial Carcinoma
Muscle-Invasive Urothelial Carcinoma
WHO/ISUP Grade pT stage Presence of associated CIS/dysplasia Disease duration Time to and frequency of recurrence Multifocality Tumor size (>3 cm) Failure of prior BCG therapy Presence of LVI Depth of lamina propria invasion
pTNM LVI Resistance to neoadjuvant chemotherapy Divergent Histology Micropapillary Osteoclast rich Undifferentiated/giant cell Plasmacytoid
Emerging Molecular Prognostic Markers Superficial Non–Muscle-Invasive Urothelial Carcinoma
Muscle-Invasive Urothelial Carcinoma
Proliferation index (Ki-67, MIB-1, S phase) FGFR3 mutation/overexpression (protective) Molecular grade (FGFR#/MIB-1) p53 inactivation/accumulation DNA ploidy status Multitarget FISH HRAS ERBB3, ERBB4 overexpression (protective) Loss of E-cadherin Cell-Cycle Control Downregulation of Rb expression Downregulation of p21 expression Downregulation of p27 expression Cyclin D3 overexpression Cyclin D1 overexpression Multimarker Immunoexpression Analysis p53, p27, Ki-67,Rb, p21 Angiogenesis Markers VEGF overexpression HIF-1α overexpression TSP1 overexpression Genomic and Gene-Expression Array Panels: Epigenetic Alterations RASSF1 promoter hypermethylation DAPK promoter hypermethylation APC promoter hypermethylation CDH1 promoter hypermethylation EDNRB promoter hypermethylation
TP53 inactivation/accumulation Alterations of Rb expression Loss of p21 expression Alteration of p16 expression Loss of E-cadherin Receptor Tyrosine Kinases EGFR overexpression HER2 overexpression/amplification Angiogenesis Markers VEGF overexpression HIF-1α overexpression TSP1 overexpression MTOR-Akt Pathway MTOR Phos-S6 expression (protective) Genomic and Gene-Expression Array Panels Epigenetic Alterations RASSF1 promoter hypermethylation CDH1 promoter hypermethylation EDNRB promoter hypermethylation
From Netto GJ, Cheng L: Emerging critical role of molecular testing in diagnostic genitourinary pathology. Arch Pathol Lab Med 2012; 136:372-390. BCG, Bacillus Chalmette-Guérin; CIS, carcinoma in situ; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; hypoxia-induced factor 1α, HIF-1α; ISUP, International Society of Urological Pathology; LVI, lymphovascular invasion; MTOR, mechanistic target of rapamycin; pT, pathologic tumor; pTNM, pathologic tumor/node/metastasis; Rb, retinoblastoma; TSP1, thrombospondin 1; VEGF, vascular endothelial growth factor; WHO, World Health Organization.
Renal Tumors: Specific Antibodies RENAL CELL CARCINOMA ANTIBODY
RCC antibody binds to a 200-kD glycoprotein (gp200) shown to be expressed in epithelial cells lining normal renal proximal tubules and renal carcinoma cells.238 Several studies have established the utility of RCC in labeling clear cell and papillary variants of renal carcinoma.239,240 Avery and colleagues240 revealed membranous RCC reactivity in up to 85% of clear cell renal cell carcinoma (CCRCC). Almost all tested papillary renal
cell carcinoma (PRCC) was also strongly positive for RCC. In contrast, chromophobe renal cell carcinoma (ChRCC) and oncocytoma were completely negative. CD10/ACUTE LYMPHOCYTE LEUKEMIA ANTIGEN
CD10, also known as acute lymphocyte leukemia antigen (CALLA), is expressed on the brush border of renal tubular epithelial cells. In the previously cited study by Avery and colleagues,240 CD10 demonstrated a similar profile to that of RCC antibody, with 94% of CCRCC
Immunohistology of Renal Neoplasms
Box 17-1 WORLD HEALTH ORGANIZATION 2004 CLASSIFICATION OF RENAL EPITHELIAL TUMORS Benign Epithelial Tumors Papillary adenoma Oncocytoma Metanephric adenoma Renal Cell Carcinoma Clear cell renal cell carcinoma (60% to 80%) Multilocular cystic renal cell carcinoma Papillary renal cell carcinoma (10% to 18%) Chromophobe renal cell carcinoma (2% to 6%) Carcinoma of the collecting ducts of Bellini (<1%) Medullary carcinoma (<1%) Xp11 translocation carcinoma (<1%) Mucinous tubular and spindle cell carcinoma (<1%) Renal cell carcinoma, unclassified (<1%) From Eble JN, Sauter G, Epstein JI, and Sesterhenn IA, editors: Pathology and genetics of tumours of the urinary system and male genital organs. Lyon, France: IARC Press; 2004.
and the majority of PRCC studied showing positivity for CD10. A similar profile was encountered by Bazille and associates.239 Variable CD10 staining has been reported in ChRCC, and negative staining240 to an almost 45% positive CD10 staining rate has been described.239-241 Approximately one third of oncocytomas stain positively for CD10.241 PAX2/PAX8
PAX2 and PAX8 are members of the paired box (PAX) gene family, which includes nine transcription factors (PAX1 through PAX9) involved in the development of several organ systems242 by preventing terminal differentiation and maintaining a progenitor cell state while inducing cell-lineage commitment. Consequently, PAX gene expression is cell-lineage restricted; PAX8 is expressed in thyroid, and PAX8 and PAX2 are expressed in wolffian (nephric) ducts and müllerian ducts. PAX2 and PAX8 have also been detected in epithelial neoplasms arising in these areas, including renal cell and ovarian tumors.243,244 IHC expression of Pax-2 has been demonstrated in CCRCC, PRCC, and ChRCC subtypes in addition to collecting duct carcinoma and mucinous tubular and spindle cell carcinoma (MTSC) renal tumors.245 Gokden and colleagues246 showed that 85% of metastatic CCRCCs had nuclear immunoreactivity for Pax-2. The marker, however, is not entirely specific, because one third of CCRCC mimics—such as parathyroid carcinoma and ovarian clear cell carcinoma—also express PAX2. Furthermore, PAX2 expression has been demonstrated in other genital tumors such as serous ovarian tumors, endometrioid carcinoma, and epididymal tumors. Pax-2 protein is also a reliable marker for nephrogenic adenoma.247 PAX8 is structurally and functionally related to PAX2, and PAX8 is also expressed in normal and neoplastic tissues of renal tubular cell origin. IHC expression of Pax-8 has been demonstrated in CCRCC, PRCC,
633
and ChRCC subtypes and in collecting duct carcinoma, MTSC, and metastatic renal carcinomas (Fig. 17-12, A-D).248-255 As with PAX2, PAX8 expression has also been demonstrated in nephrogenic adenoma (NA) and clear cell adenocarcinoma of the lower urinary tract.256 We have found PAX8 to have higher sensitivity than PAX2 in identifying RCC. EPITHELIAL CELL-ADHESION MOLECULE
Epithelial cell-adhesion molecule (EpCAM)—also known as KSA, KS1/4, and 17-1 antigen—is a 34- to 40-kD glycosylated transmembrane cell-surface epithelial protein of 232 amino acids.257 Recently, EpCAM has gained interest as a potential therapeutic target because of its wide-spectrum expression in many epithelial malignancies.258 EpCAM is consistently expressed in the distal nephron on normal renal epithelium; CCRCC shows minimal and infrequent EpCAM expression. Almost half of PRCC is positive for EpCAM, whereas intense and frequent expression is the rule in ChRCC and collecting duct carcinoma.259 A recent study by Liu and associates241 confirmed the utility of EpCAM in differentiating eosinophilic variant of ChRCC from oncocytoma and CCRCC. EpCAM protein was expressed diffusely (>90% of cells) in all 22 cases of ChRCC analyzed, whereas less than one third of oncocytomas displayed positivity for EpCAM, and only in single cells or small cell clusters of distribution.260 Combining EpCAM with other markers such as vimentin, glutathione S-transferase alpha (GST-α), CD117, and CK7 can be of utility in resolving the differential diagnosis of ChRCC. However, EpCAM is not recommended for routine use because others have not reproduced these findings in clinical practice. KIDNEY-SPECIFIC CADHERIN
Cadherins are a large family of cell-to-cell adhesion molecules that act in a homotypic, homophilic manner and play an important role in the maintenance of tissue integrity. In the human kidney, several members of the cadherin family—epithelial (E) and neuronal (N) cadherin and cadherins 6, 8, and 11—are expressed in a controlled spatiotemporal pattern. Cadherin-16, also referred to as kidney-specific cadherin (Ksp-cadherin), is exclusively expressed in epithelial cells of the adult kidney. In RCCs, a complex pattern of cadherin expression is observed. In CCRCC, Thedieck and colleagues261 revealed loss of Ksp-cadherin, which was subsequently proposed to differentiate ChRCC from oncocytoma. However, additional studies failed to reveal any difference in Kspcadherin immunoreactivity between these two tumor types, and one study showed Ksp-cadherin at the mRNA and protein levels in approximately 80% of ChRCC and oncocytoma.262 CARBONIC ANHYDRASE IX
Carbonic anhydrase IX (CAIX) is an enzyme involved in maintaining intracellular and extracellular pH. In addition, CAIX plays a regulatory role in cell
634
Immunohistology of the Bladder, Kidney, and Testis
A
C
B
D
Figure 17-12 PAX8 immunoexpression in primary clear cell renal cell carcinoma (CCRCC; A), papillary RCC (B), chromophobe RCC (C), and metastatic CCRCC (D).
proliferation, oncogenesis, and tumor progression. CAIX expression is von Hippel–Lindau (vHL)/HIF pathway dependent. In normal renal epithelium, expression of CAIX is suppressed by wild-type vHL protein. Given loss of vHL gene function in the majority of CCRCC tumors, CAIX antigen overexpression ensues. Most IHC studies have used clone M75 as the primary CAIX antibody to show diffuse overexpression in CCRCC.263,264 CAIX expression has also been demonstrated in almost half of PRCC in a recent study by Gupta and colleagues,264 whereas other studies, including ours, revealed only rare PRCC staining (Fig. 17-13, A-C).265 Prognostically, low CAIX expression reportedly indicates poor survival and low response to interleukin therapy in CRCC. A new commercially available
antibody, clone NB100-417, was recently shown by Al-Ahmadie and associates263 to have a comparable expression profile. Although CAIX is of some utility in establishing a CCRCC origin of a metastatic carcinoma, it is not entirely specific, because CAIX staining has been demonstrated in normal gastric mucosa and in biliary ductules.266 GLUTATHIONE S-TRANSFERASE ALPHA
Glutathione S-transferase alpha (GST-α) protects cells by catalyzing the detoxification of xenobiotics and carcinogens. GST-α was recently found to be of diagnostic value in renal tumors. Based on cDNA microarray findings, IHC studies have so far shown GST-α to be highly
Immunohistology of Renal Neoplasms
A
C expressed in CCRCC (90%) but not in ChRCC or oncocytomas241,267 and only occasionally in PRCC.
Immunohistology of Specific Renal Tumors RENAL ONCOCYTOMA
Oncocytomas are benign renal neoplasms typically characterized grossly by a mahogany-brown color. Microscopically, the tumor is composed of islands of “oncocytic” cells with coarsely granular eosinophilic cytoplasm set in a typical edematous, myxoid, focally hyalinized stroma.268-271 Oncocytomas display round, vesicular, centrally located nuclei with conspicuous nucleoli and occasional marked nuclear polymorphism. A range of architectural features is encountered that includes nested, tubular, acinar, cystic, and solid patterns. Variable rates of mitotic activity can also be encountered. Areas of necrosis are not seen, with the exception of commonly observed central degenerative ischemic change. The diagnosis of oncocytoma should be called into question if an “oncocytic” tumor reveals papillary architecture or areas of bona fide optically clear cytoplasm. An oncoblastic pattern is well recognized in some oncocytomas, in which the tumor cells become smaller in size with increased nuclear/
635
B
Figure 17-13 A, Carbonic anhydrase IX immunoexpression in clear cell renal cell carcinoma (CCRCC). Negative staining is encountered in papillary (B) and chromophobe RCC (C).
cytoplasmic ratio; the latter does not carry any significant prognostic connotation.269,270 A cautionary note is warranted when making a diagnosis of oncocytoma on limited material, because other types of renal neoplasms—such as PRCC, ChRCC, and CCRCC—can display focal “oncocytic” areas that share the granular cytoplasmic appearance of oncocytoma. Immunohistochemically, oncocytomas are positive for pancytokeratin AE1/AE3 and low-molecular-weight cytokeratin (LMWCK) CAM5.2 and are negative for vimentin.241 In more than two thirds of oncocytomas, c-Kit (CD117) expression is encountered, a feature also shared by ChRCC.241,272,273 Similarly, Ksp-cadherin is expressed in the majority of both ChRCC and oncocytoma tumors. Unlike more diffuse CK7 staining seen in KEY DIAGNOSTIC POINTS Oncocytoma Immunohistology • Positive: AE1/AE3, cell-adhesion molecule 5.2 (CAM5.2), CD117, Ksp cadherin, and CD10 (sometimes positive) • Negative: Vimentin, carbonic anhydrase IX, CK7 (focal), EpCAM, and RCC • Hale’s colloidal iron with lumen staining (difficult stain with +/− utility)
636
Immunohistology of the Bladder, Kidney, and Testis
Box 17-2 KEY IMMUNOHISTOCHEMISTRY DIFFERENTIAL DIAGNOSES Eosinophilic Variant of Chromophobe Renal Cell Carcinoma Positive: Hale’s colloidal iron stain, in perinuclear location; CK 7, EpCAM, and Ksp-cadherin “Oncocytic” Papillary Renal Cell Carcinoma Positive: CK, vimentin Negative: Ksp-cadherin, CD117 Clear Cell Renal Cell Carcinoma Positive: Vimentin, CD10, RCC, CAIX Negative: CD117, Ksp-cadherin CAIX, Carbonic anhydrase IX; CK, cytokeratin; EpCAM, epithelial cell-adhesion molecule; Ksp-cadherin, kidney-specific cadherin; RCC, renal cell carcinoma.
chromophobe RCC, oncocytomas are typically negative or patchy for CK7, which can help in their differentiation. Oncocytoma is usually negative for RCC and only occasionally expresses antibody CD10 (Box 17-2). Commonly encountered cytogenetic findings in oncocytomas include loss of chromosome Y and chromosome 1.274-280
METANEPHRIC ADENOMA
Metanephric adenoma of the kidney is a unique form of renal adenoma characterized by a proliferation of tubular and micropapillary to glomeruloid structures lined by bland cuboidal epithelial cells. The relatively high nuclear/cytoplasmic ratio of metanephric adenoma cells and their slightly amphophilic cytoplasmic coloration impart a typical “blue” low-power appearance to the neoplastic nodule. The latter is in contrast to the lighter eosinophilic appearance of its main differential diagnosis, the solid variant of PRCC.281-284 Like PRCC, metanephric adenoma can feature foamy histiocytes in papillary cores and occasional psammomatous calcifications. Helpful IHC features of the tumor are its positivity for Wilms tumor 1 (WT1) and negative staining for epithelial membrane antigen (EMA) and CK7, a profile that contrasts with that of solid PRCC (WT1−, EMA+, CK7 +; Fig. 17-14, A-D).281-284 CLEAR CELL RENAL CELL CARCINOMA
Clear cell renal cell carcinoma (CCRCC) is the most common type of RCC2 and accounts for 70% to 80%. Microscopically, CCRCC is composed of cuboidal cells
A
B
C
D
Figure 17-14 Metanephric adenoma (A and B) showing positive nuclear staining for Wilms tumor 1 (C) with lack of cytokeratin 7 expression (D).
Immunohistology of Renal Neoplasms
with typical optically cleared cytoplasm arranged in nests, tubules, and acini. CCRCC are richly vascularized tumors with areas of hemorrhage that impart a “bleeding acini” characteristic morphology. “Granular” RCC with eosinophilic granular cytoplasm is no longer considered a unique variant and is now included in the CCRCC variant. As discussed below, at the genetic level, similar to their familial counterpart,285 almost two thirds of sporadic CCRCC demonstrate partial or complete chromosome 3 loss or mutation on the short arm of chromosome 3p, resulting in the loss of the VHL tumor suppressor gene, located at 3p25-26.2,285 Box 17-3 summarizes the IHC profile of CCRCC.269,283,286,287 CCRCC is usually positive for LMWCKs, such as CAM5.2, and cytokeratins AE1/AE3 but is negative for CK7 and CK20. It is positive for EMA and vimentin.283,287 CCRCC’s negative reactivity for HMWCKs such as cytokeratin CK5/6 and 34βE12 (keratin 903) and for p63 and GATA3 is a useful feature in the differential against upper urinary tract URCa, which is regularly HMWCK and p63 positive.283,287 It must be remembered that the range of CCRCC reactivity with many of the above markers varies among studies, making it necessary to exercise a judicial interpretation of any IHC panel when classifying a renal tumor. In the most recent study from the MSKCC
Box 17-3 IMMUNOHISTOLOGY OF CLEAR CELL RENAL CELL CARCINOMA Immunohistochemistry Positive: CAM5.2, AE1/AE3, EMA, vimentin, CAIX (diffuse, membranous), HIF-1α (nuclear), CD10 (diffuse, membranous), RCC, Pax-2, Pax-8 Negative: HMWCK, CK7, CK20, CEA, GATA3, p63 Key Differential Diagnoses Adrenocortical Carcinoma
Positive: Inhibin, calretinin Negative: EMA, AE1/AE3, CAM5.2, RCC Equivocal: CD10, synaptophysin (sometimes positive) Urothelial Carcinoma of the Renal Pelvis
Positive: HMWCK, CK7, CK20, uroplakin III, thrombomodulin, p63, GATA3 Negative: Vimentin Papillary Renal Cell Carcinoma
Positive: AMACR, diffuse; CK7, diffuse intense; CD10, patchy/luminal Negative: CAIX Chromophobe Carcinoma
Positive: Hale’s colloidal iron stain, perinuclear; CD117, CK7, EpCAM, Ksp-cadherin Negative: Vimentin AMACR, α-Methylacyl–Coenzyme-A racemase; CAIX, carbonic anhydrase IX; CAM5.2, cell-adhesion molecule 5.2; CEA, carcinoembryonic antigen; CK, cytokeratin; EMA, epithelial membrane antigen; EpCAM, epithelial cell-adhesion molecule; HIF-1α, hypoxia-induced factor α; HMWCK, high-molecular-weight cytokeratin; Ksp-cadherin, kidney-specific cadherin; RCC, renal cell carcinoma.
637
group to address the utility of IHC in needle biopsies of renal masses (taken ex vivo for the study),288 the extent and pattern of immunoexpression were highly useful in the diagnoses: diffuse, membranous CAIX expression was noted in CCRCC, diffuse positivity for α-methylacyl–Coenzyme-A racemase (AMACR) in PRCC, distinct peripheral cytoplasmic accentuation for CD117 in ChRCC, widespread and intense positivity for CK7 in ChRCC and PRCC, diffuse membranous reactivity in CCRCC, and patchy/luminal staining in PRCC for CD10. In conclusion, utilizing immunostains improves classification of renal tumors on needle biopsy, which may be of particular help for pathologists with limited experience. Both extent and pattern must be considered for a definitive diagnosis, and IHC results should always be integrated with the overall morphologic features of a given renal neoplasm. PAPILLARY RENAL CELL CARCINOMA
Papillary renal cell carcinoma (PRCC) is the second most common subtype of RCC. Microscopically, this subtype contains characteristic complex papillary formations often accompanied by foamy macrophages that infiltrate the fibrovascular cores. Two subtypes of PRCC are recognized: type 1, in which the papillae are lined by a single layer of cells with scant pale cytoplasm, and type 2, in which the papillae are lined by pseudostratified cuboidal to columnar epithelial cells with abundant eosinophilic cytoplasm and prominent eosinophilic nucleoli.289-293 Type 1 tumors are usually of lower Fuhrman nuclear grade and are associated with a more favorable prognosis than type 2 tumors. A solid variant of PRCC is well recognized, whereas distinct papillary structures are not easily discernable. Glomeruloid structures and overall cytologic features together with the presence of typical host-infiltrating histiocytes can point to the diagnosis. As mentioned previously, the solid variant of PRCC suggests the differential diagnosis of metanephric adenoma. Commonly encountered cytogenetic alterations in RCC include trisomy of chromosomes 7, 17, 3q, 8, 16, and 20 and loss of Y chromosome.294-296 A subset of sporadic PRCC (12%) exhibits c-met oncogene mutations similar to their familial counterparts in hereditary PRCC syndrome (HPRCC).285 Immunohistochemically,269,293 the majority of PRCCs are positive for AE1/AE3, vimentin, RCC, and AMACR.297 Differential EMA immunostaining was found to be useful in differentiating type 1 and type 2 tumors; polarized expression is seen in type 1, but only rare expression is seen in type 2 (Box 17-4).289,290 CHROMOPHOBE RENAL CELL CARCINOMA
Chromophobe renal cell carcinoma (ChRCC) is composed of characteristic large polygonal cells with clear to lightly eosinophilic reticulated cytoplasm and distinct “plantlike” cell membranes. Typical perinuclear halos are a unique feature in this type of RCC. Another helpful diagnostic feature is their diffuse cytoplasmic staining with Hale’s iron stain. Cytogenetically, ChRCCs are hypodiploid tumors because of commonly present
638
Immunohistology of the Bladder, Kidney, and Testis
Box 17-4 IMMUNOHISTOLOGY OF PAPILLARY RENAL CELL CARCINOMA Immunohistochemistry Positive: AE1/AE3; CAM5.2; CK7, diffuse intense; AMACR, diffuse; vimentin, EMA; CD10, patchy luminal Negative: HMWCK, Ulex europaeus lectin, CAIX Differential Diagnosis Collecting Duct Carcinoma
Positive: Mucin, HMWCK Metanephric Adenoma
Positive: WT1 Negative: EMA, CK7 AMACR, α-Methylacyl–Coenzyme-A racemase; CAIX, carbonic anhydrase IX; CAM5.2, cell-adhesion molecule 5.2; CK, cytokeratin; EMA, epithelial membrane antigen; HMWCK, high-molecularweight cytokeratin; WT1, Wilms tumor 1.
loss of chromosomes 1, 2, 6, 10, 13, 17, and 21 as shown by FISH and comparative genomic hybridization (CGH) analysis.277-280,298,299 Ultrastructural and IHC features of ChRCC point to differentiation toward the intercalated cells of renal collecting ducts (Box 17-5).2,239-241,243,272,273,283,300-306 COLLECTING DUCT CARCINOMA
Collecting duct carcinoma (CDC) of the kidney is a rare but aggressive type of RCC with presumed origin from Bellini collecting ducts. Previously reported cases of “low-grade collecting duct carcinoma” have been reclassified as tubulocystic carcinoma.
Box 17-5 CHROMOPHOBE RENAL CELL CARCINOMA: IMMUNOHISTOCHEMICAL PROFILE Immunohistochemistry Positive: EMA, LMWCK (CAM5.2), AE1/AE3; CK7, diffuse intense; CD117, distinct peripheral cytoplasmic accentuation; parvalbumin; Alcian blue; EpCAM; Ksp-cadherin; Hale’s colloidal iron stain Equivocal: HMWCK Negative: CD10, RCC, vimentin Differential Diagnosis Oncocytoma
Negative: CK7 (or only patchy positive), EpCAM, Hale’s colloidal iron stain Clear Cell Renal Cell Carcinoma
Positive: Vimentin, RCC, CD10, CAIX Negative: CD117, Hale’s colloidal iron stain CAIX, Carbonic anhydrase IX; CAM5.2, cell-adhesion molecule 5.2; CK, cytokeratin; EMA, epithelial membrane antigen; EpCAM, epithelial cell-adhesion molecule; HMWCK, high-molecular-weight cytokeratin; Ksp-cadherin, kidney-specific cadherin; LMWCK, low-molecular-weight cytokeratin; RCC, renal cell carcinoma.
CDC is typically centered in the medulla of the kidney and extends into the cortex. Histologic patterns include tubulopapillary, tubular, solid, and sarcomatoid types. Prominent stromal desmoplasia, angiolymphatic invasion, and host inflammatory response are commonly found. Associated “dysplastic” changes in entrapped nonneoplastic collecting ducts and the presence of intracytoplasmic or luminal mucin secretions in neoplastic glands are also helpful diagnostic features of this type of RCC. Cytogenetic features include loss of chromosomes 8p and 13. Monosomy of chromosomes 1, 6, 14, 15, and 22 are also observed.239,271,276,283,287,307,308 The CDC immunoprofile is that of positive reactivity with pancytokeratins AE1/AE3 and CAM5.2 and highmolecular-weight cytokeratins (HMWCKs) CK19 and 34βE12. CDCs are positive for EMA, vimentin, and the lectin Ulex europaeus agglutinin I (UEA-1). The diagnosis of CDC carries a poor prognosis, and a majority of patients die of metastatic disease within 2 years of presentation.239,271,276,283,287,307,308 We found the combination of GATA3 and PAX8 positivity and TP63 negativity in CDC to be helpful in distinguishing them from URCa of the renal pelvis (see Fig. 17-15, A-D; Box 17-6).250 MUCINOUS TUBULAR AND SPINDLE CELL CARCINOMA
Mucinous tubular and spindle cell carcinoma (MTSC) is one of the latest types of RCC to be recognized as a distinct variant. Before characterization of MTSC, such tumors were most likely classified as either a sarcomatoid variant of RCC or “low-grade CDC.” MTSC is composed of uniform cuboidal to spindle cells with eosinophilic, focally vacuolated cytoplasm and relatively bland ovoid nuclei. Tumor cells generally form interconnecting tubules with smaller areas of solid growth. The myxoid stroma is a distinguishing feature, and mucoid material deposits at times appear as secretions within tubular or intercellular spaces.
Box 17-6 IMMUNOHISTOLOGY OF COLLECTING DUCT CARCINOMA Immunohistochemistry Positive: Vimentin, EMA, UEA-1, CAM5.2, AE1/AE3, HMWCK, Mucin, Pax-8 Negative: GATA3, p63 Differential Diagnosis Papillary Renal Cell Carcinoma
Negative: UEA-1, HMWCK Urothelial Carcinoma of the Renal Pelvis
Positive: GATA3, p63 (in 60% to 70% of cases), HMWCK, uroplakin III, TM Negative: Pax-8, vimentin CAM5.2, Cell-adhesion molecule 5.2; EMA, epithelial membrane antigen; HMWCK, high-molecular-weight cytokeratin; TM, thrombomodulin; UEA-1, Ulex europaeus agglutinin 1.
Immunohistology of Renal Neoplasms
A
C
639
B
D
Figure 17-15 Renal collecting duct carcinoma (A and B) showing positive nuclear staining for Pax-8 (C) with lack of p63 expression (D).
Cytogenetically, MTSC shows multiple chromosomal losses (1, −4, −6, −8, −9, −13, −14, −15, −22). Their overall immunoprofile and ultrastructural features have suggested differentiation toward the loop of Henle, distal convoluted tubule, or collecting ducts. MTSCs are low-grade tumors in terms of their biologic behavior with occasional recurrence on record but no known distant metastases or death reported.309-315 Their relatively good prognosis highlights the importance of distinguishing these “spindle cell” variants of RCC from the aggressive, lethal, sarcomatoid phenotype. MTSC is typically positive for vimentin, CK7, AMACR, and EMA but is negative for HMWCK, CD10, and RCC. The presence of some morphologic and IHC similarities314 between MTSC and the solid variant of PRCC have led some to suggest a histogenetic relationship between the two subtypes, however, their distinct cytogenetic and gene-expression profile argues against such a relationship (Box 17-7).298,316 ANGIOMYOLIPOMA
Angiomyolipoma is a member of the group of tumors that contain perivascular epithelioid cells (PECs),
referred to as PEComas. Oncogenesis in PEComas is related to the genetic alterations of the tuberous sclerosis complex (TSC). Tuberous sclerosis is an autosomaldominant genetic disease that results from losses of the TSC1 (9q34) or TSC2 (16p13.3) genes involved in the regulation of the Rheb/MTOR/p70S6K pathway.
Box 17-7 IMMUNOHISTOLOGY OF MUCINOUS TUBULAR AND SPINDLE CELL CARCINOMA Immunohistochemistry Positive: CK7, EMA, AMACR, vimentin, Alcian blue Negative: HMWCK, RCC, CD10 Differential Diagnosis Papillary Renal Cell Carcinoma
Positive: CD10 (patchy luminal), CK7 (diffuse intense), RCC, EMA, AMACR, vimentin Negative: HMWCK AMACR, α-Methylacyl–coenzyme-A racemase; CK, cytokeratin; EMA, epithelial membrane antigen; HMWCK, high-molecularweight cytokeratin; RCC, renal cell carcinoma.
640
Immunohistology of the Bladder, Kidney, and Testis
PEComas of the kidney include “classic” angiomyolipoma (AML) and its recognized cystic, epithelioid, and oncocytoma-like variants.2,317 Classic AML represents the most common mesenchymal tumors of the kidney, which are composed of variable proportions of adipose cells and spindle and epithelioid smooth muscle cells admixed with, and at times appearing to emanate from, abnormal thickwalled blood vessels (Fig. 17-16, A-C). In patients with TSC, multiple bilateral renal AMLs are found during the third and fourth decades of life. Sporadic AMLs are larger solitary lesions that occur in older patients.2 The epithelioid variant of AML is composed of polygonal epithelioid cells arranged in sheets and usually lacks a fat tissue component. Epithelioid AML is reactive with human melanoma black 45 (HMB-45), melanA, tyrosinase, microphthalmia transcription factor (MiTF), and smooth muscle markers (α-SMA) but is negative for epithelial markers, including cytokeratins and EMA. Tumor cells are clear to eosinophilic with at times considerable nuclear atypia and associated necrosis. Epithelioid AML has been associated with recurrence and metastasis; however, it has not been possible to predict its malignant behavior based on morphologic criteria.318
A
C
Oncocytoma-like angiomyolipomas have distinct granular eosinophilic cytoplasm, which brings renal oncocytoma into the differential diagnosis. The differential diagnosis of AML also includes sarcomatoid RCC. This is especially the case with epithelioid AML with significant cytologic atypia. Expression of melanocytic markers and lack of epithelial markers in AML will help differentiate it from sarcomatoid RCC. Rarely, Xp11 translocation carcinoma can enter into the differential diagnosis of epithelioid AML. In this regard, expression of some melanocytic markers (HMB-45, melan-A) in rare Xp11 translocation carcinoma should be kept in mind. Positive transcription factor E3 (TFE3) reaction is unique to Xp11 translocation carcinomas (Box 17-8).319-321 CLEAR CELL PAPILLARY RENAL CELL CARCINOMA
This novel type of low-grade renal epithelial neoplasm exhibits diffuse cytoplasmic clarity and various combinations of tubular, papillary, and cystic architecture. A distinctive and diagnostically helpful conspicuous nuclear positioning, away from the basement membrane, is typical of clear cell PRCC. An occasional leiomyomatous stromal component may accompany the
B
Figure 17-16 A and B, Renal angiomyolipoma. C, Focal positive staining for human melanoma black 45 (HMB-45) is shown.
Immunohistology of Renal Neoplasms
Box 17-8 IMMUNOHISTOLOGY OF ANGIOMYOLIPOMA Immunohistochemistry Positive: HMB-45, melan-A, MiTF, α-SMA, desmin Negative: EMA, pancytokeratins, RCC, CD10, Pax-2/Pax-8, GATA3, p63 Differential Diagnosis Sarcomatoid Renal Cell Carcinoma
Positive: AE1/AE3, CAM5.2, CD10, RCC, EMA, Pax-8 Equivocal: α-SMA Negative: Desmin, HMB-45, melan-A, MiTF CAM5.2, Cell-adhesion molecule 5.2; EMA, epithelial membrane antigen; HMB-45, human melanoma black 45; MiTF, microphthalmic transcription factor; RCC, renal cell carcinoma; SMA, smooth muscle actin.
epithelial proliferation, raising the suspicion of overlap of this entity with previously described entities such as renal angiomyoadenomatous tumor or RCC with prominent leiomyomatous proliferation. The fibrovascular cores are typically thin, lined by a single layer of cells with the above characteristic nuclear arrangement, and they lack the foamy macrophages common in PRCC. Cystic change is also common in clear cell PRCC. In the largest series reported on clear cell PRCC, follow-up was available in 61% (20/33) of the patients for a mean of 27.4 (range 1 to 85) months. No patient had evidence of the disease after surgery.322 In a recent study by Rohan and associates323 from the MSKCC group, the typical immunostaining positivity for CK7, CAIX, HIF1α, and GLUT-1 and negative expression of CD10, AMACR, and TFE3 was further established and could assist in confirming the diagnosis in difficult cases. At the molecular level, none of the tumors in the latter study harbored VHL gene mutations, loss of chromosomal region 3p25, or trisomies of chromosomes 7 or 17. In fact, VHL mRNA was found to be overexpressed in clear cell PRCC compared with normal renal tissue and CCRCC. The latter and the coexpression of CAIX, HIF-1α, and GLUT-1 in the absence of VHL gene mutations or promoter hypermethylation suggest activation of the HIF pathway by non–VHL-dependent mechanisms in clear cell PRCC. Wolf and colleagues324 similarly found a lack of VHL gene mutations in their analyzed cases of clear cell PRCC, although clonal trisomy 7 was demonstrated (Box 17-9). SECONDARY TUMORS OF KIDNEY
Metastases to the kidney usually occur as part of a widespread dissemination. Renal involvement is frequently bilateral and in a multinodular fashion. Primary carcinoma sources include lung, melanoma, and contralateral kidney and GI tract.2 Rarely, metastasis to kidney is the presenting manifestation. Therefore, such a possibility should be considered in needle biopsy specimens, in which the tumor lacks the typical morphologic features of the usual subtypes of RCC or those of URCa. RCC markers, such as RCC antibody and CD10; Pax-2;
641
Pax-8; and urothelial markers such as uroplakin, thrombomodulin, and GATA3 can be of some utility only when combined with other tissue lineage–specific markers such as thyroid transcription factor 1 (TTF-1; lung and thyroid) and melanoma markers.
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications Current established prognostic parameters in RCC include pathologic tumor/node/metastasis (pTNM) stage, Fuhrman grade, histologic subtype, and clinical parameters such as Eastern Cooperative Oncology Group (ECOG) Performance Status, hemoglobin level, and lactate dehydrogenase levels among others.271,325,326 Continuous refinements of staging criteria and development of nomograms to integrate the above factors promise to yield better prognostic and management discriminators.327 A large number of biomarkers are under current intense investigation for their potential utility as prognosticators and or therapy predictors in RCC.327-334 Box 17-10 lists some of these markers. Kim and colleagues329 evaluated a set of IHC markers, including Ki-67, carbonic anhydrases (CAs) IX and XII, p53, PTEN, gelsolin, EpCAM, and vimentin in combination with established parameters. Their study suggested a new, combined molecular-clinicopathologic prognostic model that included CAIX, vimentin, p53, pTNM staging, and ECOG Performance Status to be superior to prior models of clinical and pathologic parameters alone, including the commonly used the UCLA Integrated Scoring System (UISS) clinical model. Parker and colleagues335 derived another biomarker-based scoring system from 634 patients with CCRCC. This weighted algorithm, or BioScore, integrated dichotomized expression of B7-H1, Ki-67, and survivin. Patients with high BioScores (>4) were 5 times more likely to die of RCC than those with low scores. In addition, the sequential use (as opposed to integration into a new model) of BioScore with existing clinicopathologic scoring systems (TNM; UISS; Mayo Clinic stage, size, grade, and necrosis score [SSIGN]) further enhance the predictive ability
Box 17-9 CLEAR CELL PAPILLARY RENAL CELL CARCINOMA Immunohistochemistry Positive: CK7, CAIX, HIF-1α, GLUT-1 Negative: AMACR, CD10, TFE3, RCC Differential Diagnosis Papillary Renal Cell Carcinoma
Positive: CD10 (patchy luminal), RCC, CK7 (diffuse intense), AMACR, vimentin Negative: HIF-1α, CAIX AMACR, α-Methylacyl–Coenzyme-A racemase; CAIX, carbonic anhydrase IX; CK, cytokeratin; GLUT-1, glucose uptake and transport 1; HIF-1α, hypoxia-induced factor 1α; RCC, renal cell carcinoma; TFE3, transcription factor E-3.
642
Immunohistology of the Bladder, Kidney, and Testis
Box 17-10 CURRENT AND FUTURE PROGNOSTIC PARAMETERS IN RENAL CELL CARCINOMA Current Prognostic Parameters • Patient factors: Age and gender • Anatomic factors: Pathologic tumor/node/metastasis staging • Histologic factors: Histologic type, Fuhrman grade necrosis, vascular invasion • Clinical factors: ECOG performance status, Hgb level, lactate dehydrogenase, etc. Future Molecular Parameters Prediction of Behavior and Prediction/Guidance of Therapy Hypoxia-Inducible Markers
• HIF-1 • Carbonic anhydrase IX • Carbonic anhydrase XII • CXCR4 • VEGF and VEGFR • IGF-1 Cell-Adhesion Markers
• EpCAM • E-cadherin • Alpha-catenin • Catenin-6 Proliferation Markers
• Ki-67 • MCM2 Cell-Cycle Regulators
• Cyclin • p27 Apoptosis Regulators
• p53 • Bcl-2 • DIABLO MTOR Pathway
• PTEN • Phos akt • Phos-S6k ECOG, Eastern Cooperative Oncology Group; EpCAM, epithelial cell-adhesion marker; Hgb, hemoglobin; HIF-1, hypoxia-induced factor 1; IGF-1, insulin-like growth factor 1; MCM2, minichromosome maintenance complex component 2; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
compared with each of these scoring systems alone, although prospective validation of these biomarker prognostic models is required before they become standard of care. A promising prognostic role for MTOR pathway members was initially pointed to by Pantuck and colleagues.336,337 Their studies revealed an independent negative prognostic role for PTEN loss and RPS6KB1 (formerly pS6k) overexpression. The same study showed increased pAKT cytoplasmic expression and loss of AKT1 nuclear expression to be negative predictors of survival. We recently analyzed immunoexpression status and prognostic significance of MTOR and HIF pathway members in 135 primary and 41 metastatic CCRCCs
using tissue microarrays. We found significantly lower PTEN levels in primary and metastatic CCRCCs compared with benign tissues. Phos-S6 and 4E binding protein 1 (4EBP1) levels were higher in primary CCRCC compared with benign tissues. HIF-1α levels were also significantly higher in primary and metastatic tumors. In primary CCRCC, levels of all MTOR and HIF pathway members were significantly associated with pT stage. Tumor size, HIF-1α, and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary CCRCC.338,339 Using a similar tissue microarray and IHC analysis approach, we recently were also able to demonstrate evidence of dysregulation of the MTOR and hypoxiainduced pathways in papillary RCC. However, the MTOR pathway and HIF-1α alterations lacked prognostic significance in our cohort for PRCC.338,339 A prognostic role for the angiogenesis pathway is illustrated in RCC. Bui and colleagues340 demonstrated that both low expression of CAIX and high Ki-67 proliferation index were independent negative predictors of survival in CCRCC. Interestingly, CAIX overexpression predicted response to interleukin 2 (IL-2) immune therapy in metastatic RCC, a finding also documented in the study by Atkins and colleagues.341 In another study by Jacobsen and associates,342 VEGF expression appeared to correlate with tumor size and pTNM stage in RCC. The authors found high VEGF expression to be a negative survival prognosticator on univariate but not multivariate analysis. Separately, Kluger and associates330 analyzed tissue microarrays that contained 330 CCRCCs and PRCCs using a novel method of automated quantitative analysis of VEGF and VEGF-R expression by fluorescent IHC. Unsupervised hierarchical clustering classified tumors by coordinated expression of VEGF and VEGF-R. The authors found high expression of VEGF and VEGF-R in tumor cells to be associated with poor survival. Finally, a study by Lidgren and associates343 revealed high expression of HIF-1α to be an independent negative prognosticator in CCRCC. Among cell-cycle control molecules, p27 (Kip1) and cyclin D1 appear to have a promising prognostic role in CCRCC. Migita and associates344 found loss of p27 expression to be an independent predictor of poor DSS. Hedberg and associates also documented similar p27 findings.345,346 Analysis of molecular expression profiles permits simultaneous measurement of thousands of genes to create a global picture of cellular function. Multiple groups have evaluated gene-expression profiling in RCC, with variable results. Kosari and colleagues347 found significant differences in gene expression between nonaggressive and aggressive CCRCC. Thirty-four of the 35 transcripts that displayed the most significant differential expression by microarray analysis also displayed significant differential expression in independent validation studies using quantitative reverse transcription polymerase chain reaction (RT-PCR). Hierarchical clustering of the quantitative RT-PCR data using candidate markers accurately grouped 88% of aggressive and 100% of nonaggressive CCRCC samples. The group showed the expression of one of their candidate markers (survivin) to inversely predict cancer-specific survival in
Immunohistology of Renal Neoplasms
a separate cohort of 183 CCRCC patients treated at Mayo Clinic.
Genomic and Theranostic Applications Renal tumors are unique among human neoplasms in terms of the tight correlation between their different morphologic phenotypes and underlying genetic alterations. Lessons learned from the relatively rare familial renal cancer syndromes have helped unlock the complex molecular oncogenic mechanisms involved in their sporadic counterparts. As a result, many potential new targets of therapy are now under investigation in the heretofore unsuccessful endeavor of treating advanced RCC. The following discussion of the molecular basis of Von Hippel–Lindau (VHL) disease best illustrates the great therapeutic and theranostic potentials of uncovering the molecular mechanisms of renal oncogenesis.348 VHL syndrome is a rare autosomal-dominant familial cancer syndrome that comprises retinal angiomas, hemangioblastomas, pheochromocytomas, and CCRCC. VHL syndrome patients are born with a germline VHL gene mutation that affects all their cellular elements. Inactivation or silencing of the remaining wild-type allele in the renal location facilitates the formation of CCRCC in VHL patients. The fact that similar defects in the VHL gene are found to be responsible for approximately 60% to 90% of sporadic CCRCC285,349 greatly widened the implication of our understanding the molecular mechanisms of VHL gene inactivation.350 VHL can be thought of as a cellular oxygen sensor. Under normoxic conditions, HIF-1α is inhibited by normal VHL protein that facilitates its elimination.351 Under hypoxic conditions, HIF1-α escapes destruction by VHL and is allowed to exert its crucial role in inducing an angiogenesis factor (VEGF), tumor growth factors (TGF-α and TGF-β), and factors involved in glucose uptake and acid–base balance (GLUT-1 and CAIX respectively). A defective VHL function in CCRCC leads to abnormal accumulation of HIF-1α even under normal conditions, in turn resulting in the overexpression of the above proteins that are normally inducible only during hypoxia. The overexpressed VEGF, PDGFB, and TGF-β act on neighboring vascular cells to promote tumor angiogenesis. The augmented tumor vasculature provides additional nutrients and oxygen to promote the growth of tumor cells. Furthermore, TGF-α acts in an autocrine manner on the tumor cells by signaling through the EGFR, which promotes tumor-cell proliferation and survival. More recently, mutations in a number of genes that affect chromatin remodeling have been identified in CCRCC. The most prevalent of these is the PBRM1 gene, which is mutated in approximately 40% of CCRCC tumors.350,352 Currently, several agents that target the VHL pathway, particularly components of VEGF signaling (sunitinib, sorafenib, pazopanib, bevacizumab) and the MTOR C1 complex (temsirolimus, everolimus), are approved by the U.S. FDA for the treatment of advanced kidney cancer.350,353-369
643
Currently, the majority of advanced CCRCC patients receive a VEGF pathway antagonist, either sunitinib or pazopanib, as first-line therapy for metastatic disease, based on the demonstration that these agents prolong progression-free survival (PFS) compared with interferon-α or placebo, respectively. Despite the availability of a number of agents with activity, the most effective second-line therapy, as well as the optimal sequencing of these agents, remains unclear. Both sorafenib and pazopanib are associated with an improved PFS compared with placebo in patients who have previously received cytokine therapy. In patients who have progressed on first-line therapy with a VEGF pathway antagonist, everolimus was the only agent shown to offer clinical benefit (modest prolongation of PFS compared with placebo in a randomized phase III study).355,356 Axitinib is a potent, selective, second-generation VEGFR (1, 2, and 3) inhibitor recently shown to offer a superior PFS compared with sorafenib, a first-generation VEGFR and RAF inhibitor, in the second-line setting in a phase III Axitinib as Second Line Therapy for Metastatic Renal Cell Cancer (AXIS) trial.354 Analysis of VHL mutation status and of plasma CAIX, VEGF, sVEGFR2, tissue inhibitor of metalloproteinase 1 (TIMP-1), and Ras p21 was performed in the Treatment Approaches in Renal Cancer Global Evaluation (TARGET) trial of sorafenib versus placebo in advanced RCC.370 On multivariate analysis that included ECOG performance status, MSKCC score, and the biomarkers assayed, only baseline TIMP-1 levels were prognostic for survival. No predictive markers were identified.369 Choueiri and colleagues371 evaluated tumor CAIX expression (IHC) in 94 patients treated with antiangiogenic therapies. Whereas CAIX expression was neither prognostic nor predictive of response to sunitinib, the sorafenib-treated patients, with high CAIX expression (>85%), were associated with decreased tumor size in response to treatment. Other targeted agents being pursued in advanced CCRCC include temsirolimus (CCI-779), a selective MTOR inhibitor. Partial response was noted in 7% of patients, and minor responses were seen in 26%. The median survival rate was 15 months, and the notable activity of the drug in patients with poor prognostic features prompted a phase III trial.341,372 Cho and Chung373 examined expression of CAIX, phos-S6, phosAkt, and PTEN in 20 patients with advanced CCRCC treated with temsirolimus in a phase II clinical trial. These investigators found a positive significant association between phos-S6 expression and objective response to temsirolimus. A similar trend was associated with positive expression of phos-Akt. Hereditary papillary renal cancer, the familial form of type 1 PRCC, is characterized by activating germline mutations of the MET oncogene located on chromosome 7q31. Somatic MET mutations have also been detected in 5% to 13% of sporadic PRCC.374 The MET oncogene encodes a transmembrane receptor tyrosine kinase for hepatocyte growth factor (HGF). MET undergoes autophosphorylation when stimulated by its ligand, HGF, leading to downstream activation of an intracellular cascade that includes PIK3CA and
644
Immunohistology of the Bladder, Kidney, and Testis
MTOR. MET has been implicated in motility, proliferation, angiogenesis, and cell survival. Several strategies that target the MET pathway are being explored in PRCC, including antibodies against HGF and MET as well as inhibitors of MET kinase activity. Foretinib is a dual MET and VEGFR-2 kinase inhibitor that is being currently evaluated in a phase II study of patients with PRCC (NCT00726323). Unlike previous trials of MET pathway antagonists, this trial is restricted to patients with papillary histology; both type 1 and 2 histologies are included. The presence of MET pathway activation is analyzed to determine whether MET status impacts on response to the agent. Foretinib appears to be well tolerated, and a majority of patients experience some degree of tumor regression on interim analysis.350
Immunohistology of Testicular Tumors Testicular germ cell tumors (GCTs) are the most common malignancy in men aged 15 to 44. In 2012 in the United States alone, 8590 cases were diagnosed. GCT incidence increases after puberty and peaks in the third decade of life. A significant geographic variation in GCT risk is observed in which the lifetime risk is estimated to be 0.4% to 0.7% in U.S. born males and 1% in Nordic European regions; risk is lowest in Asia and the Caribbean. A puzzling steep increase in incidence in the United States and Northern Europe was observed up to 2007. The great advances in our understanding of the biology of GCT and the unparalleled strides in refining the treatment of this group of testicular tumors is one of the great success stories in oncologic diseases, with only 360 deaths as a result of GCT recorded in the United States in 2012.1
Biology of Principal Antigens/Antibodies OCT4
Among several new markers of GCT, OCT4 is probably of the greatest utility given its great degree of sensitivity and specificity.375-377 OCT4, also referred to as OCT3/4, OTF3, and POU5F1, is a stem cell transcriptional regulator mapped to chromosome 6p21.3 locus. OCT4 maintains pluripotency in embryonic stem cells and germ cells.378 As originally demonstrated by Jones and colleagues, OCT4 is a very sensitive and specific marker of seminoma and embryonal carcinoma (ECa).379 In their analysis of 91 primary testicular neoplasms, the authors found OCT4 to be expressed in all 51 tested classic seminomas and in 53 of 54 ECas (98%). OCT4 staining was diffuse in distribution and nuclear in pattern. Equally important was their finding that none of the tested somatic carcinomas was positive for OCT4 expression. A separate study by the same group also established a great sensitivity of OCT4 for intratubular germ cell neoplasia, unclassified type (IGCNU).380
CD117 (c-Kit)
One transmembrane glycoprotein receptor tyrosine kinase with homologies to the platelet-derived growth factor (PDGF) and granulocyte macrophage colony-stimulating factor (GM-CSF) receptors is c-Kit. Intact signal transduction by c-Kit is crucial for the development and survival of germ cells, hematopoietic stem cells, melanocytes, mast cells, and interstitial cells of Cajal, making it a recent favorite target of molecular therapy. Nikolaou and colleagues384 revealed CD117 staining (membranous and/or cytoplasmic) in 77% of analyzed seminomas and in almost 50% of teratomas. Several studies have demonstrated activating c-Kit mutations (codons 11 and 17) in seminomas with contradictory conclusions in regard to its association with incidence risk of bilateral seminomas.381-385 SALL4
SALL4, a member of the SALL gene family, is a nuclear transcription factors that with POU5F1, NANOG, and SOX2 forms a regulatory network that maintains embryonic stem cell pluripotency and ability of selfrenewal.386-391 SALL4 is a zinc finger transcription factor located on chromosome 20q13.2.392 SALL4 has been recently shown to be a sensitive and relatively specific marker for GCT, including primary and metastatic gonadal and extragonadal GCTs.393-397 Several studies, including our own,398 have shown SALL4 expression in occasional somatic carcinomas, including primary (up to 95%)399,400 and metastatic (33%)395 gastric adenocarcinoma and less frequently metastatic esophageal and colonic adenocarcinomas (20% and 8%, respectively).395 This should be taken into consideration, especially when using this marker in the extragonadal setting and in the setting of investigating tumors of unknown primary site. Within GCTs, it appears that SALL4 may label a wider spectrum of GCT types compared with OCT4, including seminoma, ECa, yolk sac tumors (YSTs), and some choriocarcinomas. PODOPLANIN (D2-40/M2A)
Podoplanin is an oncofetal transmembrane mucoprotein expressed by fetal germ cells and testicular GCTs. Monoclonal antibody D2-40 labels podoplanin in a membranous staining pattern. Excellent sensitivity for IGCNU and seminoma, including a diffuse staining in metastatic/extratesticular sites, has been demonstrated. However, D2-40 has a lower sensitivity for nonseminomatous GCT and will also mark nontesticular neoplasms of lymphatic and vascular endothelial origin and epithelioid mesothelioma.401 ACTIVATOR PROTEIN 2 GAMMA
This nuclear transcription factor is involved in embryonic morphogenesis and is functionally related to c-Kit and placental alkaline phosphatase (PLAP) expression. Nuclear staining is demonstrated in IGCNU and
Immunohistology of Testicular Tumors
seminoma with a high degree of sensitivity. Positivity in nonseminomatous GCT is of lower sensitivity and, as with podoplanin, activator protein 2G is also expressed in nontesticular neoplasms, such as somatic malignant melanoma and mammary and ovarian carcinomas.376,402-404 PLACENTAL ALKALINE PHOSPHATASE
Human alkaline phosphatase activity results from three genetically distinct isoenzymes produced in tissues such as liver, bone, intestine, and placenta. The placental fraction of alkaline phosphatase is a membrane-bound enzyme of 120 kD, normally synthesized by placental syncytiotrophoblasts and released into maternal circulation after the twelfth week of pregnancy. However, it is also produced by many neoplasms and is a useful tumor marker. Physiologically, this enzyme is involved in cellular transport, proliferation and cellular differentiation, and regulation of metabolism and gene transcription. Despite its common use as a germ cell marker, PLAP lacks specificity for GCTs and is expressed in GI, gynecologic, lung, breast, and urologic tumors among others. Among GCTs, IGCNU, seminoma, and ECa are almost uniformly (more than 97%) positive for PLAP, whereas a lower rate of positivity is reported with YSTs (85%), choriocarcinoma, and teratomas (up to 50% positivity rates). The PLAP staining pattern is membranous and cytoplasmic. Normal seminiferous tubules lack PLAP expression, a fact that is exploited in illustration of IGCNU in testicular biopsies from cryptorchid testis, contralateral testis of a germ cell neoplasm, and infertility biopsies.405,406 α-FETOPROTEIN
α-fetoprotein (AFP) is a fetal serum protein normally produced by fetal yolk sac, liver, and GI epithelium, and it is elevated in up to 75% of patients with nonseminomatous GCTs (NSGCTs) and is expressed by both ECa and YST but not by pure seminomatous GCT. Staining is usually focal. Among non–GCTs, AFP is a sensitive serum marker for the diagnosis and surveillance of hepatocellular carcinoma (HCC). However, IHC expression of AFP in HCC tissue is limited to a minority of cases.2 HUMAN CHORIONIC GONADOTROPIN
Human chorionic gonadotropin (hCG) is a 37-kD glycoprotein; the hCG beta subunit is synthesized by benign and malignant syncytiotrophoblasts. As a serum marker, β-hCG serves an important role in the diagnosis, staging, therapeutic monitoring, and follow-up of patients with gestational trophoblastic disease and NSGCT testicular tumors. Serum β-hCG is elevated in a majority of the cases of choriocarcinoma (50% to 90%) and in up to 10% of patients with seminoma. In GCT other than choriocarcinoma, β-hCG is expressed immunohistochemically by associated syncytiotrophoblastic giant cells.2,407
645
HUMAN PLACENTAL LACTOGEN
Human placental lactogen (HPL) is a 22-kD protein with partial homology to growth hormone. HPL is secreted by syncytiotrophoblastic cells in choriocarcinoma and by intermediate trophoblasts in a testicular trophoblastic tumor that is a counterpart to uterine placental site trophoblastic tumor.407 GLYPICAN-3
Glypican-3 is a cell membrane proteoglycan expressed in normal trophoblast, fetal liver, and neoplastic liver but not in normal adult hepatocytes. It is also expressed in normal breast, ovary, lung, and kidney, where expression is usually lost with neoplastic transformation in these sites. Glypican-3 plays a role in embryonic cell growth and development, and it is expressed strongly and diffusely in YSTs, making it a very useful IHC marker in that setting. A majority of choriocarcinomas are also positive for glypican-3, whereas only weak staining is seen in rare ECas, and no staining is expected in IGCNU, classic seminoma, and teratoma.408-410 INHIBIN A
Inhibin is a 32-kD dimeric (alpha and beta subunits) glycoprotein produced by ovarian granulosa cells and testicular Sertoli cells and, to a lesser degree, by testicular Leydig cells. It inhibits the release of folliclestimulating hormone (FSH) from the pituitary to inhibit folliculogenesis. Inhibin A is a sensitive IHC marker of ovarian and testicular sex cord–stromal tumors that include Leydig and Sertoli cell tumors (66% to 90% strong cytoplasmic positivity).411,412 In nontesticular tumors, inhibin A labels benign and malignant adrenal neoplasms while being negative in other carcinomas, melanoma, and hematologic neoplasms. Although not expressed by seminoma cells, a cautionary note is warranted in regard to its expression by accompanying syncytiotrophoblasts. In fact, inhibin A is a useful IHC marker for intermediate trophoblasts and syncytiotrophoblasts in hydatidiform mole, placental site trophoblastic tumor, and choriocarcinoma.407,412
Diagnostic Immunohistochemistry of Specific Testicular Neoplasms GERM CELL TUMORS Intratubular Germ Cell Neoplasia
Intratubular germ cell neoplasia, unclassified type (IGCNU), is the precursor of the majority of invasive testicular GCTs with the exception of spermatocytic seminoma and infantile GCTs. Isolated IGCNU can be found in testicular biopsies of cryptorchid testes taken during orchiopexy procedures and in biopsies of oligospermic infertility, dysgenetic testes, and contralateral testis of patients with GCT.
646
Immunohistology of the Bladder, Kidney, and Testis
Given the reported higher risk of developing invasive GCT (up to 50% risk within 5 years) following a diagnosis of IGCNU,413-416 ancillary techniques should be sought in equivocal cases. In general, seminiferous tubules involved by IGCNU rarely show spermatogenesis and often have decreased tubular diameter with thickening of the peritubular basement membrane. IGCNU cells are basally located and have enlarged hyperchromatic nuclei with one or more nucleoli, clear cytoplasm that contains periodic acid–Schiff (PAS)–positive material (glycogen), and distinct cytoplasmic borders. As mentioned previously, PLAP is a very sensitive and specific marker for IGCNU in such settings, with up to 98% membranous staining in IGCNU cells, in contrast to negative staining in normal spermatogonia and Sertoli cells (Fig. 17-17, A-C).417 Newer markers of utility include c-Kit protooncogene protein product (CD117) and OCT4, and both have a high degree of sensitivity and specificity for IGCNU.380,413,415,418,419 Special attention is warranted in prepubertal testes, in which occasional spermatogonia can be multinucleated and may show nuclear enlargement (giant gonadocytes), thus mimicking IGCNU and can be positive for CD117, PLAP, and OCT-4.419
A
C
Immunohistochemistry of Germ Cell Tumors
Proper classification of testicular GCTs is crucial to their management and prognostication. Detailed listing of the histologic types and their proportion in a given testicular tumor is a requirement for adequate surgical pathology reporting. Pure seminomas are treated differently than pure nonseminomatous or mixed seminomatous/nonseminomatous GCT. Furthermore, the percentages of different GCT components are to be included in the report. A 50% cutoff of ECa is considered a negative prognosticator, therefore in cases in which distinction of seminoma from ECa or YST is not certain on H&E sections, immunostains can be of utility and should be used. Classic Seminoma Versus Nonseminomatous Germ Cell Tumors
Generally, in a testicular location, the diagnosis of seminoma is usually achievable on morphologic grounds alone given their characteristic histologic features. The tumor cells have abundant clear cytoplasm, distinct cytoplasmic membranes, round vesicular nuclei, and prominent nucleoli. The fibrous septa that separate
B
Figure 17-17 A and B, Placental alkaline phosphatase immunoexpression in intratubular germ cell neoplasia. C, Infiltrating classic seminoma cells are also positive.
Immunohistology of Testicular Tumors
tumor cell clusters contain lymphoplasmacytic and/or granulomatous infiltrate. Areas of seminoma with increased nuclear atypia, increased apoptotic rate, and increased mitotic activity (especially when combined with sectioning artifactual changes) can mimic ECa. In general, ECa cells display more primitive nuclear features than seminoma and are frequently arranged in tubular and papillary structures. On the other side of the spectrum, given that the tubular and papillary architectural features are not restricted to YST, we find the pronounced degree of nuclear anaplasia in ECa to be of help in distinguishing ECa from YST, in which less primitive nuclear features are usually displayed. Finally, variants of classic seminoma such as tubular/cystic seminoma can mimic YST. Although PLAP, c-Kit, SALL4, and OCT4 are sensitive markers for seminoma (Fig. 17-18, A-B),376,379,419-422 all three are also positive in ECa, making them of little utility in distinguishing seminoma from ECa. Immunoreactivity for PLAP has been reported in 86% to 97% of ECas and tends to be more intense and focal than in seminomas. OCT4 stains more than 90% of ECa. More than 80% of ECas are positive for CD30 (Ki-1, BerH2). CD30 immunoreactivity is rarely seen in other GCTs, making it useful in a differential diagnosis. Immunoreactivity for AFP and β-hCG may be seen in scattered tumor cells in a minority (21% to 33%) of ECa. EMA, carcinoembryonic antigen (CEA), and vimentin are generally negative in ECa.376,377,379,405,418-427 Reports on pancytokeratin (AE1/AE3) expression in seminomas range from 0% to 73% positivity rates, but staining is usually weak and is seen only in isolated cells or in small clusters. Low-molecular-weight cytokeratin CAM5.2 (CK8 and CK18) has been demonstrated in some seminomas, and dotlike staining is reported in up to 80% of mediastinal seminomas.428 In contrast, both pancytokeratins AE1/AE3 and CAM5.2 demonstrate diffuse and strong staining in NSGCT including ECa, YST, choriocarcinoma, and teratoma. For all practical purposes, diffuse and strong reactivity for AE1/AE3 or CAM5.2 should argue against the diagnosis of seminoma, whereas negative staining of
A
647
AE1/AE3 should support such a diagnosis. Adding CD30 to AE1/AE3 can further help in differentiating seminoma (CD30 negative) from ECa (CD30 positive).375,377,384,418-421,428-431 Negative AE1/AE3, AFP, and β-hCG reactivity in seminoma—except for syncytiotrophoblasts, when present—is also helpful in distinguishing seminoma from nonseminomatous YST and choricarcinoma.432 PLAP is positive in only half of choriocarcinomas, which also lack OCT4 expression.376,377,407,419,422-424,433 Finally, in the rare situations in choriocarcinoma foci, including the monophasic cytotrophoblastic variant, are difficult to differentiate from seminoma, YST, or ECa with syncytiotrophoblast, we find that demonstrating cytokeratin, β-hCG, inhibin, CEA, and/or HPL positivity in the biphasic syncytiotrophoblastic and cytotrophoblastic proliferation to be helpful (Boxes 17-11 and 17-12 and Table 17-6).434,435 Extratesticular Primary and Metastatic Germ Cell Tumors
In extratesticular primary sites (e.g., mediastinal) and in metastatic tumors of unknown origin, distinguishing GCTs from somatic carcinoma is crucial to prognosis and therapy. Retroperitoneal GCT metastases and metastases to other sites can be encountered in the presence of a completely regressed primary testicular tumor, leading to a low clinical index of suspicion for a GCT.432 Dense host reaction to a seminoma metastasis combined with negative AE1/AE3 reactivity can also lead to misinterpretation as a lymphoid malignancy. In such settings, adding a germ cell marker such as PLAP, c-Kit, and OCT4 to the initial IHC panel in the workup of a midline or retroperitoneal mass in a young male is highly recommended. Nevertheless, it is important to remember that PLAP can be positive in non-GCTs (somatic carcinomas).375-377,418,419,422,423,428,433 Immunoreactivity for PLAP and c-Kit should be coupled with positive reactivity with germ cell–specific markers such as OCT4 to further lend support to a diagnosis of ECa over a somatic malignancy. Negative reactivity for EMA and CD30 positivity would also favor a diagnosis of ECa
B Figure 17-18 Expression in classic seminoma of c-Kit (A) and OCT4 (B).
648
Immunohistology of the Bladder, Kidney, and Testis
Box 17-11 IMMUNOHISTOLOGY OF GERM CELL TUMOR
Box 17-12 IMMUNOHISTOLOGY OF TESTICULAR SEX CORD TUMORS
Classic Seminoma Positive: PLAP, OCT4, SALL4, c-kit Equivocal: CAM5.2 Negative: AE1/AE3, AFP, β-hCG, inhibin, EMA, CD30
Leydig Cell Tumor Positive: Inhibin, vimentin, S-100 Equivocal: CD99, CAM5.2 Negative: EMA, PLAP
Embryonal Carcinoma Positive: PLAP, AE1/AE3, CAM5.2, OCT4, SALL4, CD30 Negative: EMA
Sertoli Cell Tumor Positive: Inhibin A, vimentin, S-100, synaptophysin, chromogranin, NSE Equivocal: CAM5.2, CD99 Negative: PLAP
Yolk Sac Tumor Positive: SALL4, glypican-A, AFP, AE1/AE3, CAM5.2 Equivocal: α1-Antitrypsin, CD34, PLAP Negative: CD30, EMA
CAM5.2, Cell-adhesion molecule 5.2; EMA, epithelial membrane antigen; NSE, neuron-specific enolase; PLAP, placental alkaline phosphatase.
Choriocarcinoma Positive: CEA, PLAP, pancytokeratins (AE1/AE3 and CAM5.2), HPL (in intermediate cytotrophoblasts); glypican-3 Positive in syncytiotrophoblasts: Inhibin, β-hCG, EMA Equivocal: SALL4 AFP, α-Fetoprotein; β-hCG, beta human chorionic gonadotropin; CAM5.2, cell-adhesion molecule 5.2; CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen; HPL, human placental lactogen; PLAP, placental alkaline phosphatase ; SALL4, sal-like protein 4.
over a somatic carcinoma in the presence of AE1/AE3 and CAM5.2 pancytokeratin positivity. Although not entirely specific, a profile of AE1/AE3, CAM5.2, PLAP, AFP, and SALL4 positivity with negative CD30 and EMA staining is compatible with YST diagnosis in extratesticular sites.375-377,418,419,422,423,428,433 Spermatocytic Seminoma
Spermatocytic seminoma (SS) is unique among GCTs in terms of older age of the patient at presentation, their
lack of association with other GCTs, and the usual risk factors of GCT: cryptorchid testis and dysgenesis. Primary SS does not occur outside the testis and is considered benign with the exception of very rare cases that undergo sarcomatous transformation. The differential diagnosis of spermatocytic seminoma includes malignant lymphoma, classic seminoma, and solid ECa. In difficult cases, IHC can play a role. SS is negative for cytokeratins AE1/AE3, AFP, β-hCG, CD30, EMA and CD45. PLAP is focally expressed but only in rare cases. CAM5.2 and c-Kit has been found in 40% of cases of SS. SALL4 expression is also encountered, in addition to Chk2, a regulatory protein in the transition of gonocytes to spermatogonia and from a mitotic phenotype to a meiotic one, and MAGE-A4, a protein involved in DNA repair, which have been found to be positive in more than 90% and 100% of SS, respectively.419,423,433,436-441
TABLE 17-6 Immunohistochemistry in Intratubular Germ Cell Neoplasia, Unspecified Type, and Germ Cell Tumor IGCNU
Classic Seminoma
Spermatocytic Seminoma
C-kit (CD117)
+
+
S
OCT3/4
+
+
−
PLAP
+
+
AE1/AE3
−
CD30
Marker
Embryonal Carcinoma
YST
Choriocarcinoma
−
−
−
+
−
−
S
+
+
+
−
−
+
+
+
−
−
−
+
−
−
α-Fetoprotein
−
−
−
−
+
−
SALL4
+
+
S
+
+
+
Glypican-3
−
−
−
−
+
+
hCG
−
−
−
−
−
+
Data from Ulbright TM: Germ cell tumors of the gonads: a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod Pathol 2005; 18 Suppl 2:S61-S79; and also Emerson RE, Ulbright TM: Intratubular germ cell neoplasia of the testis and its associated cancers: The use of novel biomarkers. Pathology 2010;42:344-355. Reactivity: +, positive; S, sometimes positive; −, negative. hCG, Human chorionic gonadotropin; IGCNU, intratubular germ cell neoplasia, unspecified type; PLAP, placental alkaline phosphatase; YST, yolk sac tumor.
Immunohistology of Testicular Tumors
TESTICULAR SEX CORD TUMORS Leydig Cell Tumor
IHC can occasionally be of value in differentiating Leydig cell tumors from entities such as malakoplakia, malignant lymphoma, plasmacytoma, seminoma, and metastatic carcinoma. Leydig cell tumors are usually diffusely and strongly positive for inhibin, and they lack PLAP immunoreactivity. Similar to their ovarian counterparts, positive immunoreactivity for a number of antibodies has been found in testicular Leydig cell tumors, including CAM5.2 (variable), vimentin, S-100 protein (negative HMB-45), and desmin. IHC coexpression of inhibin and vimentin, lack of reactivity for EMA, and variable cytokeratin immunoreactivity supports the diagnosis of Leydig cell tumor over metastatic carcinoma. CD99, a transmembrane glycoprotein encoded by the MIC-2 gene can be useful in identifying Leydig cell tumors, although it is inferior to inhibin.423,424,433,440,442-448 Sertoli Cell Tumor
Inhibin A is also a sensitive marker of Sertoli cell differentiation (over 90% positive). Sertoli cell tumor, including its large-cell calcifying variant, stains positively with antibodies to vimentin, cytokeratin, S-100, synaptophysin, chromogranin, and neuron-specific enolase (NSE). When present, cytokeratin immunoreactivity in Sertoli cell tumors is usually stronger than that seen in Leydig cell tumors but is often negative in both. Immunoreactivity for PLAP is not seen in Sertoli cell tumors. Tubular seminomas, which often mimic Sertoli cell tumors architecturally, can be separated from Sertoli cell tumors by their PLAP immunoreactivity and lack of staining for inhibin and cytokeratins. Like Leydig cell tumors, CD99 can be a useful adjunct in identifying Sertoli cell tumors.423,424,433,440,442-448 SECONDARY TUMORS OF TESTIS
Compared with primary tumors, secondary testicular tumors are typically found in an older age group (over 50 years of age); however, one third of cases occur before age 40. PCa (50%) dominates the list of primary sources, in large part because of the prior practice of bilateral orchiectomy as a part of hormone-deprivation therapy for PCa. Other primary sources, in descending order, include kidney, melanoma, and lung primaries.2 Metastatic carcinomas infiltrate the testicular interstitium while usually sparing seminiferous tubules. Rare intratubular growth patterns have been reported. Extensive vascular involvement and bilaterality are other features that are more likely to be encountered in metastatic tumors. As mentioned previously, germ cell markers such as c-Kit and OCT4 can be used in difficult cases. EMA negativity and CD30 positivity can also be of help in cases in which the differential diagnosis includes ECa versus metastatic somatic carcinoma.419,449,450 Primary and secondary involvement of testes by malignant lymphomas and leukemias can be supported by a panel of lymphoid and hematologic markers that include CD45,
649
CD20, CD79a, CD3, CD5, CD33, c-Kit, and myeloperoxidase among others.451,452
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications Tremendous advances have been achieved in our understanding of the biology and genetic molecular events involved in the pathogenesis of GCT. Current GCT treatment success rates are the envy of other solid tumors disciplines. Nevertheless, given the young age of the affected GCT patient population and the gravity of potential side effects (e.g., secondary malignancy) associated with current treatment protocols, new biomarkers that will help better identify patients in need of such aggressive treatment are invaluable. Likewise, identification of new molecular targets of GCT therapy would be advantageous.453-457 As shown in Figure 17-19,458 during fetal life, following an initiating event in diploid primordial germ cells, loss of imprinting and aneuploidization lead to the formation of the IGCNU precursor lesions. The extensive chromosomal instability in IGCNU leads to further transformation into invasive GCT, primarily through loss of DNA and gain of 12p, which results in the development of seminoma. Additional events—such as loss of N-myc and c-Kit activity and activation of pRb, HER2, and p53—result in histogenetic differentiation toward NSGCT.458,459 Overrepresentation of the chromosome 12p region is a consistently present chromosomal structural aberration in GCT. Isochromosome 12p is present in up to 80% of GCTs, including extratesticular tumors, therefore the identification of isochromosome 12p by interphase FISH-based cytogenetics is occasionally resorted to in cases in which the aforementioned IHC approach fails to resolve the differential diagnosis of GCT versus somatic carcinoma.460-464 Recently, we performed the first comprehensive genome-wide analysis of copy-number variation (CNV) and loss of heterozygosity (LOH) in 25 primary seminomas using high-resolution single-nucleotide polymorphism (SNP) array.465 Our study confirmed several previously reported genomic alterations and discovered eight novel alterations that included amplifications and homozygous deletions (Table 17-7). Moreover, a comparison of genomic alterations of early and late-stage seminoma identified CNVs that appear to correlate with progression, which included deletions in chromosomes 4q, 5p, 9q, 13q, and 20p and amplifications in chromosomes 9q and 13q associated with advanced stage (stages II and III vs. stage I). A deletion of chromosome 13q13.3, which encodes for DCLK1 and SOHLH2, was altered in 20% of the samples. SOHLH2 is a germ cell–specific transcription factor that may be a critical regulator of early germ cell development.465 At the epigenetic level, alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5mC) are a hallmark of cancer cells, including testicular GCTs. Smiraglia and associates466 have demonstrated significant epigenetic differences between seminomas and nonseminomas using
650
Immunohistology of the Bladder, Kidney, and Testis
Seminoma <3N+>
Distortion Aneuploidization (loss or relaxation) (cell fusion, of imprinting endoreduplication) Primitive germ cell (PGC) (2N)
Initiated PGC (2N) Initiation
Promoted PGC (2N)
Promotion
Invasive growth phase
Carcinoma in situ (CIS) (3–4N)
Net loss of DNA
+X,+7,+8,+12 –11,–13,–18,–Y l12p multiplication
Progression Net loss of DNA
Genetically unstable
Net loss of DNA (–15,–22) Switch-off: CKIT, SCF, NMYC, PTHLH
Histogenetic differentiation
Switch-on: HST1, RB1, CERB-B1/2 Increase: TP53
Nonseminoma <3N–> Figure 17-19 Proposed model of pathogenetic steps in germ cell tumor. Modified from Sandberg AA, Meloni AM, Suijkerbuijk RF: Reviews of chromosome studies in urological tumors. III. Cytogenetics and genes in testicular tumors. J Urol 1996;155:1531-1556.
restriction landmark genomic scanning. Seminomas show almost no CpG island methylation, in contrast to nonseminomas, which show it at a level similar to that of other solid tumors. The group proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone global 5mC erasure (loss of imprinting), and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation
after the original erasure. More recently, we used IHC staining against 5mC to evaluate global methylation in GCT467 and found staining for 5mC to be undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet staining in nonseminomatous GCTs was robust. Our findings supported that testicular GCTs are derived in most cases from IGCNU cells that have undergone developmentally programmed 5mC erasure and that the degree of subsequent de novo methylation
TABLE 17-7 Novel Alterations of Chromosomal Regions in Seminoma Region
CNV Alteration
Frequency
Genes
Candidate Gene Function
2q24.1
Amp
13%
UPP2
Folliculogenesis, hormone secretion regulation
2q14.3
Amp
26%
Amp
22%
CYP27C1, ERCC3, MAP3K2 MKI67IP, TSN
CYP27C1: only expressed in testis; ERCC3: DNA repair enzyme NER TSN: chromosomal translocations and regulation of RNA expression
2q22.1
Amp
17%
THSD7B
Unknown
2q23.3
Amp
17% 26%
NEB, ARL5A XIRP2
NEB: Calcium/CaM regulation DNA repair
2q32.1
Amp
22% 26%
ELF2P4, FSIP2 SLC23A3, ABCB6
FSIP2: spermatocyte development STK16: involved in VEGF expression regulation ABCB6: drug transporter, ATP dependent, expressed in testis and linked with breast cancer
2q33.3
Amp
26%
CRYGEP, CRYGC, CRYGB, CRYGA
Stress response
2q34
Amp
26%
IKZF2
Aberrant expression in Hodgkin and non-Hodgkin lymphoma
20p12.1
Del
13%
ESF1, C20ORF7, SEL1L2, MACROD2
ESF1: pre-RNA processing
Modified from LeBron C, Pal P, Brait M, et al: Genome-wide analysis of genetic alterations in testicular primary seminoma using highresolution single nucleotide polymorphism arrays. Genomics 2011;97:341-349. ATP, Adenosine triphosphate; CNV, copy-number variation; VEGF, vascular endothelial growth factor.
IN UTERO
Birth
Immunohistology of Testicular Tumors
651
PREPUBERTY/ADULT
Spermatogonial stem cell
Gametes
NS-GCT PGC with de novo methylation marks
IGCNU
Seminoma 5 mC 5 mC
PGC with methylation erasure
5 mC
IGCNU
Seminoma
PGC Figure 17-20 Proposed model for global methylation in germ cell tumors (GCTs). Primordial germ cells (PGCs) undergo physiologic erasure of 5mC with subsequent de novo methylation to become PGCs and then spermatogonial stem cells committed to sperm cell differentiation. In the development of intratubular germ cell neoplasia, unspecified type (IGCNU), abnormal retention of PGCs with methylation erasure persists after birth. Such cells are prone to undergo transformation into IGCNU cells in response to environmental insults (lower right). In most cases, it is these cells that give rise to seminomas, which remain unmethylated. In mixed GCTs, seminoma cells undergo de novo methylation as they differentiate into other neoplastic cell types (e.g., yolk sac tumor, teratoma, embryonal carcinoma). In rare cases IGCNU cells may be derived from PGCs that have already undergone de novo methylation, in which these cells are prone to undergo development into nonseminomatous GCTs. Solid boxes show neoplastic cells, and dashed boxes indicate the transient and/or infrequent nature of the methylated state. Black arrows indicate infrequent events. Modified from Netto GJ, Nakai Y, Nakayama M, et al: Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors. Mod Pathol 2008;21:1337-1344.
is most closely related to the differentiation state of the neoplastic cells. In such a proposed linear pathogenesis model, it appears that IGCNU and subsequently developed seminoma cells remain unmethylated, whereas nonseminomatous histologic types appear to arise mainly after a de novo methylation of seminoma cells destined to give rise to nonseminomas (Fig. 17-20). Most recently, we found a differential methylation pattern between seminomas and nonseminomatous GCTs using MS-PCR. Although MGMT, VGF, ESR2 (formerly ER-β), and FKBP4 appear to be predominantly methylated in nonseminomas; APC and MLH1 genes are methylated in both groups.467-469 Among therapy-predictive markers, DNA damage detection and apoptosis initiation programs are thought to play a strong role in the exquisite chemosensitivity enjoyed by GCT. Wild-type p53 overexpression has been associated with chemosensitivity in GCT, whereas overexpression of MDM2 (p53 inhibitor) was associated with resistance to therapy.454,470-472 On the other hand, defective mismatch repair pathways that lead to microsatellite instability (MSI) have been related to resistance in a subset of cisplatin-refractory seminoma. Mayer and colleagues were able to illustrate a correlation between
MSI and IHC loss of MLH1, MSH2, and MSH6 in 50% of unstable tumors, demonstrating for the first time a positive correlation between MSI and treatment resistance in GCT.473,474 Mutations of BRAF are associated with MSI and have also been shown to be frequent in GCTs with cisplatin chemotherapy resistance.409,475 In another study, Dimov and associates476 were able to show that a high level of type II topoisomerase alpha (topo-2α) is expressed by most seminomas, ECas, YSTs, and choriocarcinomas, which suggests a possible mechanism for the sensitivity of these components to topo-2α inhibitors. Interestingly, teratomas with mature and immature elements expressed low levels of topo-2α, which might have contributed to their relative chemoresistance, which further implies that the variable chemoresponsiveness of testicular GCTs could have an underlying molecular basis.476 Mazumdar and colleagues477 recently suggested that a subgroup of ECa (with or without other histologies) with a cluster profile of high Ki-67, low apoptosis, and low p53 had a better survival beyond what would be predicted by their International Germ Cell Consensus Cancer Group (IGCCCG) prognostic category, reflecting their increased tendency to respond to treatment.477
652
Immunohistology of the Bladder, Kidney, and Testis
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that has been extensively targeted therapeutically in solid tumors, including lung and colonic adenocarcinoma. IHC EGFR expression is observed in almost half of chemotherapy-refractory metastatic ECas, and it appears to correlate with EGFR gene amplification or polysomy observed by FISH. The therapeutic role of EGFR-targeted therapy remains to be determined.478 As mentioned above, so far, conflicting results have been found regarding the role of the presence of exon 11 and 17 c-Kit–activating mutations in predicting incidence risk of bilateral seminoma in patients with prior orchiectomy.381-385
Summary Neoplasms of the bladder, kidney, and testis represent extraordinary and exciting venues of genomic and theranostic applications in medicine. The role of IHC is equally exciting, translating the genomic and molecular data into actionable patient therapies.
REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin. 62:10–29, 2012. 2. Eble JN, Sauter G, Epstein JI, et al, editors: Pathology and genetics of tumours of the urinary system and male genital organs, 2004, IARC Press. 3. Jiang J, Ulbright TM, Younger C, et al: Cytokeratin 7 and cytokeratin 20 in primary urinary bladder carcinoma and matched lymph node metastasis. Arch Pathol Lab Med. 125:921–923, 2001. 4. Bassily NH, Vallorosi CJ, Akdas G, et al: Coordinate expression of cytokeratins 7 and 20 in prostate adenocarcinoma and bladder urothelial carcinoma. Am J Clin Pathol. 113:383–388, 2000. 5. Desai S, Lim SD, Jimenez RE, et al: Relationship of cytokeratin 20 and CD44 protein expression with WHO/ISUP grade in pTa and pT1 papillary urothelial neoplasia. Mod Pathol. 13:1315– 1323, 2000. 6. Cabibi D, Licata A, Barresi E, et al: Expression of cytokeratin 7 and 20 in pathological conditions of the bile tract. Pathol Res Pract. 199:65–70, 2003. 7. Matros E, Bailey G, Clancy T, et al: Cytokeratin 20 expression identifies a subtype of pancreatic adenocarcinoma with decreased overall survival. Cancer. 106:693–702, 2006. 8. Vang R, Gown AM, Barry TS, et al: Cytokeratins 7 and 20 in primary and secondary mucinous tumors of the ovary: Analysis of coordinate immunohistochemical expression profiles and staining distribution in 179 cases. Am J Surg Pathol. 30:1130– 1139, 2006. 9. Saad RS, Cho P, Silverman JF, et al: Usefulness of Cdx2 in separating mucinous bronchioloalveolar adenocarcinoma of the lung from metastatic mucinous colorectal adenocarcinoma. Am J Clin Pathol. 122:421–427, 2004. 10. Wang HL, Lu DW, Yerian LM, et al: Immunohistochemical distinction between primary adenocarcinoma of the bladder and secondary colorectal adenocarcinoma. Am J Surg Pathol. 25:1380–1387, 2001. 11. Chuang AY, DeMarzo AM, Veltri RW, et al: Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma. Am J Surg Pathol. 31:1246–1255, 2007. 12. Harnden P, Eardley I, Joyce AD, et al: Cytokeratin 20 as an objective marker of urothelial dysplasia. Br J Urol. 78:870–875, 1996. 13. McKenney JK, Desai S, Cohen C, et al: Discriminatory immunohistochemical staining of urothelial carcinoma in situ and non-neoplastic urothelium: An analysis of cytokeratin 20, p53, and CD44 antigens. Am J Surg Pathol. 25:1074–1078, 2001. 14. Yin H, He Q, Li T, et al: Cytokeratin 20 and ki-67 to distinguish carcinoma in situ from flat non-neoplastic urothelium. Appl Immunohistochem Mol Morphol. 14:260–265, 2006. 15. Yin M, Bastacky S, Parwani AV, et al: P16ink4 immunoreactivity is a reliable marker for urothelial carcinoma in situ. Hum Pathol. 39:527–535, 2008. 16. Burger M, Denzinger S, Hartmann A, et al: Mcm2 predicts recurrence hazard in stage Ta/T1 bladder cancer more accurately than CK20, Ki67 and histological grade. Br J Cancer. 96:1711– 1715, 2007. 17. Ramos D, Navarro S, Villamon R, et al: Cytokeratin expression patterns in low-grade papillary urothelial neoplasms of the urinary bladder. Cancer. 97:1876–1883, 2003. 18. Barbisan F, Santinelli A, Mazzucchelli R, et al: Strong immunohistochemical expression of fibroblast growth factor receptor 3, superficial staining pattern of cytokeratin 20, and low proliferative activity define those papillary urothelial neoplasms of low malignant potential that do not recur. Cancer. 112:636–644, 2008. 19. Kaufmann O, Volmerig J, Dietel M: Uroplakin III is a highly specific and moderately sensitive immunohistochemical marker for primary and metastatic urothelial carcinomas. Am J Clin Pathol. 113:683–687, 2000. 20. Ohtsuka Y, Kawakami S, Fujii Y, et al: Loss of uroplakin III expression is associated with a poor prognosis in patients with urothelial carcinoma of the upper urinary tract. BJU Int. 97:1322–1326, 2006.
652.e1
21. Parker DC, Folpe AL, Bell J, et al: Potential utility of uroplakin III, thrombomodulin, high molecular weight cytokeratin, and cytokeratin 20 in noninvasive, invasive, and metastatic urothelial (transitional cell) carcinomas. Am J Surg Pathol. 27:1–10, 2003. 22. Huang HY, Shariat SF, Sun TT, et al: Persistent uroplakin expression in advanced urothelial carcinomas: Implications in urothelial tumor progression and clinical outcome. Hum Pathol. 38:1703–1713, 2007. 23. Moll R, Wu XR, Lin JH, et al: Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas. Am J Pathol. 147:1383– 1397, 1995. 24. Matsumoto K, Satoh T, Irie A, et al: Loss expression of uroplakin III is associated with clinicopathologic features of aggressive bladder cancer. Urology. 72:444–449, 2008. 25. Logani S, Oliva E, Amin MB, et al: Immunoprofile of ovarian tumors with putative transitional cell (urothelial) differentiation using novel urothelial markers: Histogenetic and diagnostic implications. Am J Surg Pathol. 27:1434–1441, 2003. 26. Olsburgh J, Harnden P, Weeks R, et al: Uroplakin gene expression in normal human tissues and locally advanced bladder cancer. J Pathol. 199:41–49, 2003. 27. Ogawa K, Johansson SL, Cohen SM: Immunohistochemical analysis of uroplakins, urothelial specific proteins, in ovarian brenner tumors, normal tissues, and benign and neoplastic lesions of the female genital tract. Am J Pathol. 155:1047–1050, 1999. 28. Ordonez NG: Thrombomodulin expression in transitional cell carcinoma. Am J Clin Pathol. 110:385–390, 1998. 29. Comperat E, Camparo P, Haus R, et al: Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma. A tissue microarray study of 158 cases. Virchows Arch. 448:319– 324, 2006. 30. Parwani AV, Kronz JD, Genega EM, et al: Prostate carcinoma with squamous differentiation: An analysis of 33 cases. Am J Surg Pathol. 28:651–657, 2004. 31. Helpap B, Kollermann J: Assessment of basal cell status and proliferative patterns in flat and papillary urothelial lesions: A contribution to the new WHO classification of the urothelial tumors of the urinary bladder. Hum Pathol. 31:745–750, 2000. 32. Higgins JP, Kaygusuz G, Wang L, et al: Placental S100 (S100P) and GATA3: Markers for transitional epithelium and urothelial carcinoma discovered by complementary DNA microarray. Am J Surg Pathol. 31:673–680, 2007. 33. Esheba GE, Longacre TA, Atkins KA, et al: Expression of the urothelial differentiation markers GATA3 and placental S100 (S100P) in female genital tract transitional cell proliferations. Am J Surg Pathol. 33:347–353, 2009. 34. Liu H, Shi J, Wilkerson ML, et al: Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: A useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol. 138:57–64, 2012. 35. Montgomery EA, Shuster DD, Burkart AL, et al: Inflammatory myofibroblastic tumors of the urinary tract: A clinicopathologic study of 46 cases, including a malignant example inflammatory fibrosarcoma and a subset associated with high-grade urothelial carcinoma. Am J Surg Pathol. 30:1502–1512, 2006. 36. Cook JR, Dehner LP, Collins MH, et al: Anaplastic lymphoma kinase (ALK) expression in the inflammatory myofibroblastic tumor: A comparative immunohistochemical study. Am J Surg Pathol. 25:1364–1371, 2001. 37. Tsuzuki T, Magi-Galluzzi C, Epstein JI: ALK-1 expression in inflammatory myofibroblastic tumor of the urinary bladder. Am J Surg Pathol. 28:1609–1614, 2004. 38. Sukov WR, Cheville JC, Carlson AW, et al: Utility of ALK-1 protein expression and ALK rearrangements in distinguishing inflammatory myofibroblastic tumor from malignant spindle cell lesions of the urinary bladder. Mod Pathol. 20:592–603, 2007. 39. Sengupta S, Harris CC: p53: Traffic cop at the crossroads of DNA repair and recombination. Nat Rev Mol Cell Biol. 6:44–55, 2005. 40. Fujimoto K, Yamada Y, Okajima E, et al: Frequent association of p53 gene mutation in invasive bladder cancer. Cancer Res. 52:1393–1398, 1992.
652.e2
Immunohistology of the Bladder, Kidney, and Testis
41. Miyamoto H, Kubota Y, Shuin T, et al: Analyses of p53 gene mutations in primary human bladder cancer. Oncol Res. 5:245– 249, 1993. 42. Sarkis AS, Bajorin DF, Reuter VE, et al: Prognostic value of p53 nuclear overexpression in patients with invasive bladder cancer treated with neoadjuvant MVAC. J Clin Oncol. 13:1384–1390, 1995. 43. Sarkis AS, Dalbagni G, Cordon-Cardo C, et al: Association of P53 nuclear overexpression and tumor progression in carcinoma in situ of the bladder. J Urol. 152:388–392, 1994. 44. Lianes P, Orlow I, Zhang ZF, et al: Altered patterns of MDM2 and TP53 expression in human bladder cancer. J Natl Cancer Inst. 86:1325–1330, 1994. 45. Brunner A, Verdorfer I, Prelog M, et al: Large-scale analysis of cell cycle regulators in urothelial bladder cancer identifies p16 and p27 as potentially useful prognostic markers. Pathobiology. 75:25–33, 2008. 46. McShane LM, Aamodt R, Cordon-Cardo C, et al: Reproducibility of p53 immunohistochemistry in bladder tumors. national cancer institute, bladder tumor marker network. Clin Cancer Res. 6:1854–1864, 2000. 47. Watanabe J, Nishiyama H, Okubo K, et al: Clinical evaluation of p53 mutations in urothelial carcinoma by IHC and FASAY. Urology. 63:989–993, 2004. 48. Salinas-Sanchez AS, Atienzar-Tobarra M, Lorenzo-Romero JG, et al: Sensitivity and specificity of p53 protein detection by immunohistochemistry in patients with urothelial bladder carcinoma. Urol Int. 79:321–327, 2007. 49. Garcia del Muro X, Condom E, Vigues F, et al: p53 and p21 expression levels predict organ preservation and survival in invasive bladder carcinoma treated with a combined-modality approach. Cancer. 100:1859–1867, 2004. 50. Tzai TS, Tsai YS, Chow NH: The prevalence and clinicopathologic correlate of p16INK4a, retinoblastoma and p53 immunoreactivity in locally advanced urinary bladder cancer. Urol Oncol. 22:112–118, 2004. 51. Orlow I, Lacombe L, Hannon GJ, et al: Deletion of the p16 and p15 genes in human bladder tumors. J Natl Cancer Inst. 87:1524–1529, 1995. 52. Miyamoto H, Kubota Y, Fujinami K, et al: Infrequent somatic mutations of the p16 and p15 genes in human bladder cancer: P16 mutations occur only in low-grade and superficial bladder cancers. Oncol Res. 7:327–330, 1995. 53. Kruger S, Mahnken A, Kausch I, et al: P16 immunoreactivity is an independent predictor of tumor progression in minimally invasive urothelial bladder carcinoma. Eur Urol. 47:463–467, 2005. 54. Miyamoto H, Shuin T, Torigoe S, et al: Retinoblastoma gene mutations in primary human bladder cancer. Br J Cancer. 71:831–835, 1995. 55. Cordon-Cardo C, Wartinger D, Petrylak D, et al: Altered expression of the retinoblastoma gene product: Prognostic indicator in bladder cancer. J Natl Cancer Inst. 84:1251–1256, 1992. 56. Cordon-Cardo C: p53 and RB: Simple interesting correlates or tumor markers of critical predictive nature? J Clin Oncol. 22:975–977, 2004. 57. Cairns P, Proctor AJ, Knowles MA: Loss of heterozygosity at the RB locus is frequent and correlates with muscle invasion in bladder carcinoma. Oncogene. 6:2305–2309, 1991. 58. Cote RJ, Dunn MD, Chatterjee SJ, et al: Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53. Cancer Res. 58:1090– 1094, 1998. 59. Kubota Y, Miyamoto H, Noguchi S, et al: The loss of retinoblastoma gene in association with c-myc and transforming growth factor-beta 1 gene expression in human bladder cancer. J Urol. 154:371–374, 1995. 60. Bates AW, Baithun SI: Secondary neoplasms of the bladder are histological mimics of nontransitional cell primary tumours: Clinicopathological and histological features of 282 cases. Histopathology. 36:32–40, 2000. 61. Raspollini MR, Nesi G, Baroni G, et al: Immunohistochemistry in the differential diagnosis between primary and secondary intestinal adenocarcinoma of the urinary bladder. Appl Immunohistochem Mol Morphol. 13:358–362, 2005.
62. Jacobs LB, Brooks JD, Epstein JI: Differentiation of colonic metaplasia from adenocarcinoma of urinary bladder. Hum Pathol. 28:1152–1157, 1997. 63. Silver SA, Epstein JI: Adenocarcinoma of the colon simulating primary urinary bladder neoplasia. A report of nine cases. Am J Surg Pathol. 17:171–178, 1993. 64. Chuang AY, DeMarzo AM, Veltri RW, et al: Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma. Am J Surg Pathol. 31:1246–1255, 2007. 65. Varma M, Morgan M, Amin MB, et al: High molecular weight cytokeratin antibody (clone 34betaE12): A sensitive marker for differentiation of high-grade invasive urothelial carcinoma from prostate cancer. Histopathology. 42:167–172, 2003. 66. Genega EM, Hutchinson B, Reuter VE, et al: Immunophenotype of high-grade prostatic adenocarcinoma and urothelial carcinoma. Mod Pathol. 13:1186–1191, 2000. 67. Mhawech P, Uchida T, Pelte MF: Immunohistochemical profile of high-grade urothelial bladder carcinoma and prostate adenocarcinoma. Hum Pathol. 33:1136–1140, 2002. 68. Fritsche HM, Burger M, Denzinger S, et al: Plasmacytoid urothelial carcinoma of the bladder: Histological and clinical features of 5 cases. J Urol. 180:1923–1927, 2008. 69. McKenney JK, Amin MB: The role of immunohistochemistry in the diagnosis of urinary bladder neoplasms. Semin Diagn Pathol. 22:69–87, 2005. 70. Patriarca C, Di Pasquale M, Giunta P, et al: CD138-positive plasmacytoid urothelial carcinoma of the bladder. Int J Surg Pathol. 16:215–217, 2008. 71. Ro JY, Shen SS, Lee HI, et al: Plasmacytoid transitional cell carcinoma of urinary bladder: A clinicopathologic study of 9 cases. Am J Surg Pathol. 32:752–757, 2008. 72. Sato K, Ueda Y, Kawamura K, et al: Plasmacytoid urothelial carcinoma of the urinary bladder: A case report and immunohistochemical study. Pathol Res Pract. 2008. 73. Lane Z, Hansel DE, Epstein JI: Immunohistochemical expression of prostatic antigens in adenocarcinoma and villous adenoma of the urinary bladder. Am J Surg Pathol. 32:1322–1326, 2008. 74. Cheng L, Pan CX, Yang XJ, et al: Small cell carcinoma of the urinary bladder: A clinicopathologic analysis of 64 patients. Cancer. 101:957–962, 2004. 75. Cheng L, Huang WB, Chen J: Recent advances in pathology and molecular genetics of small cell carcinoma of the urinary bladder. Zhonghua Bing Li Xue Za Zhi. 36:700–703, 2007. 76. Cheng L, Jones TD, McCarthy RP, et al: Molecular genetic evidence for a common clonal origin of urinary bladder small cell carcinoma and coexisting urothelial carcinoma. Am J Pathol. 166:1533–1539, 2005. 77. Terracciano L, Richter J, Tornillo L, et al: Chromosomal imbalances in small cell carcinomas of the urinary bladder. J Pathol. 189:230–235, 1999. 78. Hansel DE, Nadasdy T, Epstein JI: Fibromyxoid nephrogenic adenoma: A newly recognized variant mimicking mucinous adenocarcinoma. Am J Surg Pathol. 31:1231–1237, 2007. 79. Tong GX, Melamed J, Mansukhani M, et al: PAX2: A reliable marker for nephrogenic adenoma. Mod Pathol. 19:356–363, 2006. 80. Tong GX, Weeden EM, Hamele-Bena D, et al: Expression of PAX8 in nephrogenic adenoma and clear cell adenocarcinoma of the lower urinary tract: Evidence of related histogenesis? Am J Surg Pathol. 32:1380–1387, 2008. 81. Mitra AP, Datar RH, Cote RJ: Molecular pathways in invasive bladder cancer: New insights into mechanisms, progression, and target identification. J Clin Oncol. 24:5552–5564, 2006. 82. Mitra AP, Cote RJ: Molecular screening for bladder cancer: Progress and potential. Nat Rev Urol. 7:11–20, 2010. 83. Mitra AP, Cote RJ: Molecular pathogenesis and diagnostics of bladder cancer. Annu Rev Pathol. 4:251–285, 2009. 84. Wu XR: Urothelial tumorigenesis: A tale of divergent pathways. Nat Rev Cancer. 5:713–725, 2005. 85. Oxford G, Theodorescu D: The role of ras superfamily proteins in bladder cancer progression. J Urol. 170:1987–1993, 2003. 86. Lopez-Knowles E, Hernandez S, Malats N, et al: PIK3CA mutations are an early genetic alteration associated with FGFR3 mutations in superficial papillary bladder tumors. Cancer Res. 66:7401–7404, 2006.
References 87. Kompier LC, Lurkin I, van der Aa MN, et al: FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy. PLoS One. 5:e13821, 2010. 88. O’Donnell MA: Advances in the management of superficial bladder cancer. Semin Oncol. 34:85–97, 2007. 89. Eble JN, Sauter G, Epstein JI, et al: The world health organization classification of tumours of the urinary system and male genital system, Lyon, France, 2004, IARC Press, pp 209–211. 90. Stein JP, Lieskovsky G, Cote R, et al: Radical cystectomy in the treatment of invasive bladder cancer: Long-term results in 1,054 patients. J Clin Oncol. 19:666–675, 2001. 91. Shariat SF, Karakiewicz PI, Palapattu GS, et al: Outcomes of radical cystectomy for transitional cell carcinoma of the bladder: A contemporary series from the bladder cancer research consortium. J Urol. 176:2414,22; discussion 2422, 2006. 92. Shariat SF, Chade DC, Karakiewicz PI, et al: Combination of multiple molecular markers can improve prognostication in patients with locally advanced and lymph node positive bladder cancer. J Urol. 183:68–75, 2010. 93. Rabbani F, Koppie TM, Charytonowicz E, et al: Prognostic significance of p27(Kip1) expression in bladder cancer. BJU Int. 100:259–263, 2007. 94. Sanchez-Carbayo M, Socci ND, Lozano J, et al: Defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays. J Clin Oncol. 24:778– 789, 2006. 95. Sanchez-Carbayo M, Socci ND, Charytonowicz E, et al: Molecular profiling of bladder cancer using cDNA microarrays: Defining histogenesis and biological phenotypes. Cancer Res. 62:6973– 6980, 2002. 96. Sanchez-Carbayo M, Cordon-Cardo C: Applications of array technology: Identification of molecular targets in bladder cancer. Br J Cancer. 89:2172–2177, 2003. 97. Ioachim E, Michael M, Salmas M, et al: Hypoxia-inducible factors HIF-1alpha and HIF-2alpha expression in bladder cancer and their associations with other angiogenesis-related proteins. Urol Int. 77:255–263, 2006. 98. Ioachim E, Michael MC, Salmas M, et al: Thrombospondin-1 expression in urothelial carcinoma: Prognostic significance and association with p53 alterations, tumour angiogenesis and extracellular matrix components. BMC Cancer. 6:140, 2006. 99. Lascombe I, Clairotte A, Fauconnet S, et al: N-cadherin as a novel prognostic marker of progression in superficial urothelial tumors. Clin Cancer Res. 12:2780–2787, 2006. 100. Rotterud R, Nesland JM, Berner A, et al: Expression of the epidermal growth factor receptor family in normal and malignant urothelium. BJU Int. 95:1344–1350, 2005. 101. Highshaw RA, McConkey DJ, Dinney CP: Integrating basic science and clinical research in bladder cancer: Update from the first bladder specialized program of research excellence (SPORE). Curr Opin Urol. 14:295–300, 2004. 102. Clairotte A, Lascombe I, Fauconnet S, et al: Expression of E-cadherin and alpha-, beta-, gamma-catenins in patients with bladder cancer: Identification of gamma-catenin as a new prognostic marker of neoplastic progression in T1 superficial urothelial tumors. Am J Clin Pathol. 125:119–126, 2006. 103. Chatterjee SJ, Datar R, Youssefzadeh D, et al: Combined effects of p53, p21, and pRb expression in the progression of bladder transitional cell carcinoma. J Clin Oncol. 22:1007–1013, 2004. 104. Beekman KW, Bradley D, Hussain M: New molecular targets and novel agents in the treatment of advanced urothelial cancer. Semin Oncol. 34:154–164, 2007. 105. Shariat SF, Ashfaq R, Sagalowsky AI, et al: Predictive value of cell cycle biomarkers in nonmuscle invasive bladder transitional cell carcinoma. J Urol. 177:481,7; discussion 487, 2007. 106. Birkhahn M, Mitra AP, Williams AJ, et al: Predicting recurrence and progression of noninvasive papillary bladder cancer at initial presentation based on quantitative gene expression profiles. Eur Urol. 57:12–20, 2010. 107. Cheng L, Davidson DD, Maclennan GT, et al: The origins of urothelial carcinoma. Expert Rev Anticancer Ther. 10:865–880, 2010.
652.e3
108. Mengual L, Burset M, Ribal MJ, et al: Gene expression signature in urine for diagnosing and assessing aggressiveness of bladder urothelial carcinoma. Clin Cancer Res. 16:2624–2633, 2010. 109. Shariat SF, Ashfaq R, Sagalowsky AI, et al: Association of cyclin D1 and E1 expression with disease progression and biomarkers in patients with nonmuscle-invasive urothelial cell carcinoma of the bladder. Urol Oncol. 25:468–475, 2007. 110. Bolenz C, Lotan Y: Translational research in bladder cancer: From molecular pathogenesis to useful tissue biomarkers. Cancer Biol Ther. 10:407–415, 2010. 111. Bensalah K, Montorsi F, Shariat SF: Challenges of cancer biomarker profiling. Eur Urol. 52:1601–1609, 2007. 112. Netto GJ: Molecular diagnostics in urologic malignancies: A work in progress. Arch Pathol Lab Med. 135:610–621, 2011. 113. Netto GJ, Epstein JI: Theranostic and prognostic biomarkers: Genomic applications in urological malignancies. Pathology. 42:384–394, 2010. 114. Netto GJ: Molecular biomarkers in urothelial carcinoma of the bladder: Are we there yet? Nat Rev Urol. 9:41–51, 2011. 115. Netto GJ, Cheng L: Emerging critical role of molecular testing in diagnostic genitourinary pathology. Arch Pathol Lab Med. 136:372–390, 2012. 116. Kawauchi S, Sakai H, Ikemoto K, et al: 9p21 index as estimated by dual-color fluorescence in situ hybridization is useful to predict urothelial carcinoma recurrence in bladder washing cytology. Hum Pathol. 40:1783–1789, 2009. 117. Kruger S, Mess F, Bohle A, et al: Numerical aberrations of chromosome 17 and the 9p21 locus are independent predictors of tumor recurrence in non-invasive transitional cell carcinoma of the urinary bladder. Int J Oncol. 23:41–48, 2003. 118. Skacel M, Fahmy M, Brainard JA, et al: Multitarget fluorescence in situ hybridization assay detects transitional cell carcinoma in the majority of patients with bladder cancer and atypical or negative urine cytology. J Urol. 169:2101–2105, 2003. 119. Sarosdy MF, Kahn PR, Ziffer MD, et al: Use of a multitarget fluorescence in situ hybridization assay to diagnose bladder cancer in patients with hematuria. J Urol. 176:44–47, 2006. 120. Moonen PM, Merkx GF, Peelen P, et al: UroVysion compared with cytology and quantitative cytology in the surveillance of non-muscle-invasive bladder cancer. Eur Urol. 51:1275,80; discussion 1280, 2007. 121. Yoder BJ, Skacel M, Hedgepeth R, et al: Reflex UroVysion testing of bladder cancer surveillance patients with equivocal or negative urine cytology: A prospective study with focus on the natural history of anticipatory positive findings. Am J Clin Pathol. 127:295–301, 2007. 122. Fritsche HM, Burger M, Dietmaier W, et al: Multicolor FISH (UroVysion) facilitates follow-up of patients with high-grade urothelial carcinoma of the bladder. Am J Clin Pathol. 134:597– 603, 2010. 123. Karnwal A, Venegas R, Shuch B, et al: The role of fluorescence in situ hybridization assay for surveillance of non-muscle invasive bladder cancer. Can J Urol. 17:5077–5081, 2010. 124. Schlomer BJ, Ho R, Sagalowsky A, et al: Prospective validation of the clinical usefulness of reflex fluorescence in situ hybridization assay in patients with atypical cytology for the detection of urothelial carcinoma of the bladder. J Urol. 183:62– 67, 2010. 125. Ferra S, Denley R, Herr H, et al: Reflex UroVysion testing in suspicious urine cytology cases. Cancer. 117:7–14, 2009. 126. Savic S, Zlobec I, Thalmann GN, et al: The prognostic value of cytology and fluorescence in situ hybridization in the follow-up of nonmuscle-invasive bladder cancer after intravesical bacillus calmette-guerin therapy. Int J Cancer. 124:2899–2904, 2009. 127. Maffezzini M, Campodonico F, Capponi G, et al: Prognostic significance of fluorescent in situ hybridisation in the follow-up of non-muscle-invasive bladder cancer. Anticancer Res. 30:4761– 4765, 2010. 128. Whitson J, Berry A, Carroll P, et al: A multicolour fluorescence in situ hybridization test predicts recurrence in patients with high-risk superficial bladder tumours undergoing intravesical therapy. BJU Int. 104:336–339, 2009. 129. Renshaw AA: UroVysion, urine cytology, and the college of american pathologists: Where should we go from here? Arch Pathol Lab Med. 134:1106–1107, 2010.
652.e4
Immunohistology of the Bladder, Kidney, and Testis
130. van Rhijn BW, Zuiverloon TC, Vis AN, et al: Molecular grade (FGFR3/MIB-1) and EORTC risk scores are predictive in primary non-muscle-invasive bladder cancer. Eur Urol. 58:433– 441, 2010. 131. Mason RA, Morlock EV, Karagas MR, et al: EGFR pathway polymorphisms and bladder cancer susceptibility and prognosis. Carcinogenesis. 30:1155–1160, 2009. 132. Simonetti S, Russo R, Ciancia G, et al: Role of polysomy 17 in transitional cell carcinoma of the bladder: Immunohistochemical study of HER2/neu expression and fish analysis of c-erbB-2 gene and chromosome 17. Int J Surg Pathol. 17:198–205, 2009. 133. Latif Z, Watters AD, Dunn I, et al: HER2/neu gene amplification and protein overexpression in G3 pT2 transitional cell carcinoma of the bladder: A role for anti-HER2 therapy? Eur J Cancer. 40:56–63, 2004. 134. Gandour-Edwards R, Lara PN, Jr, Folkins AK, et al: Does HER2/ neu expression provide prognostic information in patients with advanced urothelial carcinoma? Cancer. 95:1009–1015, 2002. 135. Eissa S, Ali HS, Al Tonsi AH, et al: HER2/neu expression in bladder cancer: Relationship to cell cycle kinetics. Clin Biochem. 38:142–148, 2005. 136. Billerey C, Chopin D, Aubriot-Lorton MH, et al: Frequent FGFR3 mutations in papillary non-invasive bladder (pTa) tumors. Am J Pathol. 158:1955–1959, 2001. 137. Leibl S, Zigeuner R, Hutterer G, et al: EGFR expression in urothelial carcinoma of the upper urinary tract is associated with disease progression and metaplastic morphology. APMIS. 116:27–32, 2008. 138. Bolenz C, Shariat SF, Karakiewicz PI, et al: Human epidermal growth factor receptor 2 expression status provides independent prognostic information in patients with urothelial carcinoma of the urinary bladder. BJU Int. 106:1216–1222, 2010. 139. Al-Ahmadie HA, Iyer G, Janakiraman M, et al: Somatic mutation of fibroblast growth factor receptor-3 (FGFR3) defines a distinct morphological subtype of high-grade urothelial carcinoma. J Pathol. 224:270–279, 2011. 140. Ling S, Chang X, Schultz L, et al: An EGFR-ERK-SOX9 signaling cascade links urothelial development and regeneration to cancer. Cancer Res. 71:3812–3821, 2011. 141. Zuiverloon TC, van der Aa MN, van der Kwast TH, et al: Fibroblast growth factor receptor 3 mutation analysis on voided urine for surveillance of patients with low-grade non-muscle-invasive bladder cancer. Clin Cancer Res. 16:3011–3018, 2010. 142. Miyake M, Sugano K, Sugino H, et al: Fibroblast growth factor receptor 3 mutation in voided urine is a useful diagnostic marker and significant indicator of tumor recurrence in non-muscle invasive bladder cancer. Cancer Sci, 101:250–258, 2010. 143. Hernandez S, Lopez-Knowles E, Lloreta J, et al. Prospective study of FGFR3 mutations as a prognostic factor in nonmuscle invasive urothelial bladder carcinomas. J Clin Oncol. 24:3664– 3671, 2006. 144. van Rhijn BW, Vis AN, van der Kwast TH, et al: Molecular grading of urothelial cell carcinoma with fibroblast growth factor receptor 3 and MIB-1 is superior to pathologic grade for the prediction of clinical outcome. J Clin Oncol. 21:1912–1921, 2003. 145. Sylvester RJ, van der Meijden AP, Oosterlinck W, et al: Predicting recurrence and progression in individual patients with stage ta T1 bladder cancer using EORTC risk tables: A combined analysis of 2596 patients from seven EORTC trials0. Eur Urol. 49:466,5; discussion 475–477, 2006. 146. Chakravarti A, Winter K, Wu CL, et al: Expression of the epidermal growth factor receptor and her-2 are predictors of favorable outcome and reduced complete response rates, respectively, in patients with muscle-invading bladder cancers treated by concurrent radiation and cisplatin-based chemotherapy: A report from the radiation therapy oncology group. Int J Radiat Oncol Biol Phys. 62:309–317, 2005. 147. Jimenez RE, Hussain M, Bianco FJ, Jr, et al: Her-2/neu overexpression in muscle-invasive urothelial carcinoma of the bladder: Prognostic significance and comparative analysis in primary and metastatic tumors. Clin Cancer Res. 7:2440–2447, 2001. 148. Ravery V, Grignon D, Angulo J, et al: Evaluation of epidermal growth factor receptor, transforming growth factor alpha, epidermal growth factor and c-erbB2 in the progression of invasive bladder cancer. Urol Res. 25:9–17, 1997.
149. Sarkis AS, Dalbagni G, Cordon-Cardo C, et al: Nuclear overexpression of p53 protein in transitional cell bladder carcinoma: A marker for disease progression. J Natl Cancer Inst. 85:53–59, 1993. 150. Lopez-Beltran A, Luque RJ, Alvarez-Kindelan J, et al: Prognostic factors in stage T1 grade 3 bladder cancer survival: The role of G1-S modulators (p53, p21Waf1, p27kip1, cyclin D1, and cyclin D3) and proliferation index (ki67-MIB1). Eur Urol. 45:606–612, 2004. 151. Shariat SF, Bolenz C, Karakiewicz PI, et al: P53 expression in patients with advanced urothelial cancer of the urinary bladder. BJU Int. 105:489–495, 2010. 152. Lopez-Beltran A, Requena MJ, Luque RJ, et al: Cyclin D3 expression in primary Ta/T1 bladder cancer. J Pathol. 209:106– 113, 2006. 153. Fu TY, Tu MS, Tseng HH, et al: Overexpression of p27kip1 in urinary bladder urothelial carcinoma. Int J Urol. 14:1084–1087, 2007. 154. Sgambato A, Migaldi M, Faraglia B, et al: Loss of P27Kip1 expression correlates with tumor grade and with reduced disease-free survival in primary superficial bladder cancers. Cancer Res. 59:3245–3250, 1999. 155. Shariat SF, Karakiewicz PI, Ashfaq R, et al: Multiple biomarkers improve prediction of bladder cancer recurrence and mortality in patients undergoing cystectomy. Cancer. 112:315–325, 2008. 156. Shariat SF, Bolenz C, Godoy G, et al: Predictive value of combined immunohistochemical markers in patients with pT1 urothelial carcinoma at radical cystectomy. J Urol. 182:78,84; discussion 84, 2009. 157. Shariat SF, Ashfaq R, Sagalowsky AI, et al: Predictive value of cell cycle biomarkers in nonmuscle invasive bladder transitional cell carcinoma. J Urol. 177:481,7; discussion 487, 2007. 158. Dalbagni G, Presti JC, Jr, Reuter VE, et al: Molecular genetic alterations of chromosome 17 and p53 nuclear overexpression in human bladder cancer. Diagn Mol Pathol. 2:4–13, 1993. 159. Quintero A, Alvarez-Kindelan J, Luque RJ, et al: Ki-67 MIB1 labelling index and the prognosis of primary TaT1 urothelial cell carcinoma of the bladder. J Clin Pathol. 59:83–88, 2006. 160. Margulis V, Shariat SF, Ashfaq R, et al: Ki-67 is an independent predictor of bladder cancer outcome in patients treated with radical cystectomy for organ-confined disease. Clin Cancer Res. 12:7369–7373, 2006. 161. Margulis V, Lotan Y, Karakiewicz PI, et al: Multi-institutional validation of the predictive value of ki-67 labeling index in patients with urinary bladder cancer. J Natl Cancer Inst. 101:114–119, 2009. 162. Ramos D, Ruiz A, Morell L, et al: Prognostic value of morphometry in low grade papillary urothelial bladder neoplasms. Anal Quant Cytol Histol. 26:285–294, 2004. 163. Cheng L, Zhang S, Maclennan GT, et al: Bladder cancer: Translating molecular genetic insights into clinical practice. Hum Pathol. 2010. 164. Miyamoto H, Brimo F, Schultz L, et al: Low-grade papillary urothelial carcinoma of the urinary bladder: A clinicopathologic analysis of a post-world health Organization/International society of urological pathology classification cohort from a single academic center. Arch Pathol Lab Med. 134:1160–1163, 2010. 165. Mitra AP, Pagliarulo V, Yang D, et al: Generation of a concise gene panel for outcome prediction in urinary bladder cancer. J Clin Oncol. 27:3929–3937, 2009. 166. Rothman N, Garcia-Closas M, Chatterjee N, et al: A multi-stage genome-wide association study of bladder cancer identifies multiple susceptibility loci. Nat Genet. 42:978–984, 2010. 167. Sanchez-Carbayo M, Cordon-Cardo C: Molecular alterations associated with bladder cancer progression. Semin Oncol. 34:75– 84, 2007. 168. Serizawa RR, Ralfkiaer U, Steven K, et al: Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events. Int J Cancer. 2010. 169. Lindgren D, Frigyesi A, Gudjonsson S, et al: Combined gene expression and genomic profiling define two intrinsic molecular subtypes of urothelial carcinoma and gene signatures for molecular grading and outcome. Cancer Res. 70:3463–3472, 2010. 170. Heidenblad M, Lindgren D, Jonson T, et al: Tiling resolution array CGH and high density expression profiling of urothelial
References carcinomas delineate genomic amplicons and candidate target genes specific for advanced tumors. BMC Med Genomics. 1:3, 2008. 171. Lindgren D, Liedberg F, Andersson A, et al: Molecular characterization of early-stage bladder carcinomas by expression profiles, FGFR3 mutation status, and loss of 9q. Oncogene. 25:2685–2696, 2006. 172. Smith SC, Baras AS, Dancik G, et al: A 20-gene model for molecular nodal staging of bladder cancer: Development and prospective assessment. Lancet Oncol. 12:137–143, 2011. 173. Nishiyama N, Arai E, Chihara Y, et al: Genome-wide DNA methylation profiles in urothelial carcinomas and urothelia at the precancerous stage. Cancer Sci. 101:231–240, 2010. 174. Lin HH, Ke HL, Huang SP, et al: Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine. Urol Oncol. 28:597–602, 2010. 175. Vinci S, Giannarini G, Selli C, et al: Quantitative methylation analysis of BCL2, hTERT, and DAPK promoters in urine sediment for the detection of non-muscle-invasive urothelial carcinoma of the bladder: A prospective, two-center validation study. Urol Oncol. 2009. 176. Cabello MJ, Grau L, Franco N, et al: Multiplexed methylation profiles of tumor suppressor genes in bladder cancer. J Mol Diagn. 13:29–40, 2011. 177. Vallot C, Stransky N, Bernard-Pierrot I, et al: A novel epigenetic phenotype associated with the most aggressive pathway of bladder tumor progression. J Natl Cancer Inst. 103:47–60, 2011. 178. Dudziec E, Miah S, Choudhry H, et al: Hypermethylation of CpG islands and shores around specific MicroRNAs and mirtrons is associated with the phenotype and presence of bladder cancer. Clin Cancer Res. 2011. 179. Wiklund ED, Bramsen JB, Hulf T, et al: Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. Int J Cancer. 128:1327–1334, 2011. 180. Catto JW, Azzouzi AR, Rehman I, et al: Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma. J Clin Oncol. 23:2903– 2910, 2005. 181. Friedrich MG, Weisenberger DJ, Cheng JC, et al: Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients. Clin Cancer Res. 10:7457–7465, 2004. 182. Chan MW, Chan LW, Tang NL, et al: Hypermethylation of multiple genes in tumor tissues and voided urine in urinary bladder cancer patients. Clin Cancer Res. 8:464–470, 2002. 183. Yates DR, Rehman I, Meuth M, et al: Methylational urinalysis: A prospective study of bladder cancer patients and age stratified benign controls. Oncogene. 25:1984–1988, 2006. 184. Hoque MO, Begum S, Topaloglu O, et al: Quantitation of promoter methylation of multiple genes in urine DNA and bladder cancer detection. J Natl Cancer Inst. 98:996–1004, 2006. 185. Yates DR, Rehman I, Abbod MF, et al: Promoter hypermethylation identifies progression risk in bladder cancer. Clin Cancer Res. 13:2046–2053, 2007. 186. Ali-El-Dein B, Sarhan O, Hinev A, et al: Superficial bladder tumours: Analysis of prognostic factors and construction of a predictive index. BJU Int. 92:393–399, 2003. 187. Loughman NT, Lin BP, Dent OF, et al: DNA ploidy of bladder cancer using bladder biopsy supernate specimens. Anal Quant Cytol Histol. 25:146–158, 2003. 188. Baak JP, Bol MG, van Diermen B, et al: DNA cytometric features in biopsies of TaT1 urothelial cell cancer predict recurrence and stage progression more accurately than stage, grade, or treatment modality. Urology. 61:1266–1272, 2003. 189. Bol MG, Baak JP, Diermen B, et al: Correlation of grade of urothelial cell carcinomas and DNA histogram features assessed by flow cytometry and automated image cytometry. Anal Cell Pathol. 25:147–153, 2003. 190. Bellaoui H, Chefchaouni MC, Lazrak N, et al: Flow cytometric DNA analysis and cytology in diagnosis and prognosis of bladder tumors: Preliminary results of a comparative study of bladder lavage]. Ann Urol (Paris). 36:45–52, 2002. 191. Caraway NP, Khanna A, Payne L, et al: Combination of cytologic evaluation and quantitative digital cytometry is reliable in detecting recurrent disease in patients with urinary diversions. Cancer. 111:323–329, 2007.
652.e5
192. Falkman K, Tribukait B, Nyman CR, et al: S-phase fraction in superficial urothelial carcinoma of the bladder–a prospective, long-term, follow-up study. Scand J Urol Nephrol. 38:278–284, 2004. 193. Lin J, Dinney CP, Grossman HB, et al: E-cadherin promoter polymorphism (C-160A) and risk of recurrence in patients with superficial bladder cancer. Clin Genet. 70:240–245, 2006. 194. Palit V, Phillips RM, Puri R, et al: Expression of HIF-1alpha and glut-1 in human bladder cancer. Oncol Rep. 14:909–913, 2005. 195. Chai CY, Chen WT, Hung WC, et al: Hypoxia-inducible factor1alpha expression correlates with focal macrophage infiltration, angiogenesis and unfavourable prognosis in urothelial carcinoma. J Clin Pathol. 61:658–664, 2008. 196. Crew JP, O’Brien T, Bicknell R, et al: Urinary vascular endothelial growth factor and its correlation with bladder cancer recurrence rates. J Urol. 161:799–804, 1999. 197. Crew JP, O’Brien T, Bradburn M, et al: Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer. Cancer Res. 57:5281–5285, 1997. 198. Turner KJ, Crew JP, Wykoff CC, et al: The hypoxia-inducible genes VEGF and CA9 are differentially regulated in superficial vs invasive bladder cancer. Br J Cancer. 86:1276–1282, 2002. 199. Tickoo SK, Milowsky MI, Dhar N, et al: Hypoxia-inducible factor and mammalian target of rapamycin pathway markers in urothelial carcinoma of the bladder: Possible therapeutic implications. BJU Int. 107:844–849, 2011. 200. Platt FM, Hurst CD, Taylor CF, et al: Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer. Clin Cancer Res. 15:6008–6017, 2009. 201. Schultz L, Albadine R, Hicks J, et al: Expression status and prognostic significance of mammalian target of rapamycin pathway members in urothelial carcinoma of urinary bladder after cystectomy. Cancer. 116:5517–5526, 2010. 202. Comperat E, Camparo P, Haus R, et al: Aurora-A/STK-15 is a predictive factor for recurrent behaviour in non-invasive bladder carcinoma: A study of 128 cases of non-invasive neoplasms. Virchows Arch. 450:419–424, 2007. 203. Mhawech-Fauceglia P, Fischer G, Beck A, et al: Raf1, aurora-A/ STK15 and E-cadherin biomarkers expression in patients with pTa/pT1 urothelial bladder carcinoma; a retrospective TMA study of 246 patients with long-term follow-up. Eur J Surg Oncol. 32:439–444, 2006. 204. Veerla S, Panagopoulos I, Jin Y, et al: Promoter analysis of epigenetically controlled genes in bladder cancer. Genes Chromosomes Cancer. 47:368–378, 2008. 205. Dudziec E, Miah S, Choudhry H, et al: Hypermethylation of CpG islands and shores around specific MicroRNAs and mirtrons is associated with the phenotype and presence of bladder cancer. Clin Cancer Res. 2011. 206. Wszolek MF, Rieger-Christ KM, Kenney PA, et al: A MicroRNA expression profile defining the invasive bladder tumor phenotype. Urol Oncol. 2009. 207. Catto JW, Miah S, Owen HC, et al: Distinct microRNA alterations characterize high- and low-grade bladder cancer. Cancer Res. 69:8472–8481, 2009. 208. Iyer G, Milowsky MI, Bajorin DF: Novel strategies for treating relapsed/refractory urothelial carcinoma. Expert Rev Anticancer Ther. 10:1917–1932, 2010. 209. Wallerand H, Reiter RR, Ravaud A: Molecular targeting in the treatment of either advanced or metastatic bladder cancer or both according to the signalling pathways. Curr Opin Urol. 18:524–532, 2008. 210. Amit D, Tamir S, Birman T, et al: Development of targeted therapy for bladder cancer mediated by a double promoter plasmid expressing diphtheria toxin under the control of IGF2-P3 and IGF2-P4 regulatory sequences. Int J Clin Exp Med 4:91–102, 2011. 211. Gerullis H, Ecke TH, Janusch B, et al: Long-term response in advanced bladder cancer involving the use of temsirolimus and vinflunine after platin resistance. Anticancer Drugs. 2011. 212. Yafi FA, North S, Kassouf W: First- and second-line therapy for metastatic urothelial carcinoma of the bladder. Curr Oncol. 18:e25–34, 2011. 213. Zhang X, Godbey WT: Preclinical evaluation of a gene therapy treatment for transitional cell carcinoma. Cancer Gene Ther. 18:34–41, 2011.
652.e6
Immunohistology of the Bladder, Kidney, and Testis
214. Smaldone MC, Davies BJ: BC-819, a plasmid comprising the H19 gene regulatory sequences and diphtheria toxin A, for the potential targeted therapy of cancers. Curr Opin Mol Ther. 12:607–616, 2010. 215. Kramer MW, Krege S, Peters I, et al: Targeted therapy of urological tumours. experimental field or established therapeutic approach? Urologe A. 49:1260–1265, 2010. 216. Ching CB, Hansel DE: Expanding therapeutic targets in bladder cancer: The PI3K/Akt/mTOR pathway. Lab Invest. 90:1406– 1414, 2010. 217. Black PC, Agarwal PK, Dinney CP: Targeted therapies in bladder cancer–an update. Urol Oncol. 25:433–438, 2007. 218. Black PC, Dinney CP: Bladder cancer angiogenesis and metastasis–translation from murine model to clinical trial. Cancer Metastasis Rev. 26:623–634, 2007. 219. Downward J: Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 3:11–22, 2003. 220. Bellmunt J, Hussain M, Dinney CP: Novel approaches with targeted therapies in bladder cancer. therapy of bladder cancer by blockade of the epidermal growth factor receptor family. Crit Rev Oncol Hematol. 46(Suppl):S85–104, 2003. 221. Wallerand H, Robert G, Bernhard JC, et al: Targeted therapy for locally advanced and/or metastatic bladder cancer. Prog Urol. 18:407–417, 2008. 222. Hussain MH, MacVicar GR, Petrylak DP, et al: Trastuzumab, paclitaxel, carboplatin, and gemcitabine in advanced human epidermal growth factor receptor-2/neu-positive urothelial carcinoma: Results of a multicenter phase II national cancer institute trial. J Clin Oncol. 25:2218–2224, 2007. 223. Hansel DE, Swain E, Dreicer R, et al: HER2 overexpression and amplification in urothelial carcinoma of the bladder is associated with MYC coamplification in a subset of cases. Am J Clin Pathol. 130:274–281, 2008. 224. Inoue K, Slaton JW, Perrotte P, et al: Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma. Clin Cancer Res 6:4874–4884, 2000. 225. Perrotte P, Matsumoto T, Inoue K, et al: Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin Cancer Res. 5:257–265, 1999. 226. Philips GK, Halabi S, Sanford BL, et al: A phase II trial of cisplatin, fixed dose-rate gemcitabine and gefitinib for advanced urothelial tract carcinoma: Results of the cancer and leukaemia group B 90102. BJU Int. 101:20–25, 2008. 227. Philips GK, Halabi S, Sanford BL, et al: A phase II trial of cisplatin (C), gemcitabine (G) and gefitinib for advanced urothelial tract carcinoma: Results of cancer and leukemia group B (CALGB) 90102. Ann Oncol. 20:1074–1079, 2009. 228. Wulfing C, Machiels JP, Richel DJ, et al: A single-arm, multicenter, open-label phase 2 study of lapatinib as the second-line treatment of patients with locally advanced or metastatic transitional cell carcinoma. Cancer. 115:2881–2890, 2009. 229. Hahn NM, Stadler WM, Zon RT, et al: Phase II trial of cisplatin, gemcitabine, and bevacizumab as first-line therapy for metastatic urothelial carcinoma: Hoosier oncology group GU 04-75. J Clin Oncol. 29:1525–1530, 2011. 230. Elfiky AA, Rosenberg JE: Targeting angiogenesis in bladder cancer. Curr Oncol Rep. 11:244–249, 2009. 231. Bradley DA, Dunn R, Nanus D, et al: Randomized, double-blind, placebo-controlled phase II trial of maintenance sunitinib versus placebo after chemotherapy for patients with advanced urothelial carcinoma: Scientific rationale and study design. Clin Genitourin Cancer. 5:460–463, 2007. 232. Miyamoto H, Miller JS, Fajardo DA, et al: Non-invasive papillary urothelial neoplasms: The 2004 WHO/ISUP classification system. Pathol Int. 60:1–8, 2010. 233. Millan-Rodriguez F, Chechile-Toniolo G, Salvador-Bayarri J, et al: Multivariate analysis of the prognostic factors of primary superficial bladder cancer. J Urol. 163:73–78, 2000. 234. Soloway MS, Sofer M, Vaidya A: Contemporary management of stage T1 transitional cell carcinoma of the bladder. J Urol. 167:1573–1583, 2002. 235. Malekzadeh K, Sobti RC, Nikbakht M, et al: Methylation patterns of Rb1 and casp-8 promoters and their impact on their expression in bladder cancer. Cancer Invest. 27:70–80, 2009.
236. van der Kwast TH, Bapat B: Predicting favourable prognosis of urothelial carcinoma: Gene expression and genome profiling. Curr Opin Urol 19:516–521, 2009. 237. Wilhelm-Benartzi CS, Koestler DC, Houseman EA, et al: DNA methylation profiles delineate etiologic heterogeneity and clinically important subgroups of bladder cancer. Carcinogenesis. 31:1972–1976, 2010. 238. Yoshida SO, Imam A: Monoclonal antibody to a proximal nephrogenic renal antigen: Immunohistochemical analysis of formalin-fixed, paraffin-embedded human renal cell carcinomas. Cancer Res. 49:1802–1809, 1989. 239. Bazille C, Allory Y, Molinie V, et al: Immunohistochemical characterisation of the main histologic subtypes of epithelial renal tumours on tissue-microarrays. study of 310 cases. Ann Pathol. 24:395–406, 2004. 240. Avery AK, Beckstead J, Renshaw AA, et al: Use of antibodies to RCC and CD10 in the differential diagnosis of renal neoplasms. Am J Surg Pathol. 24:203–210, 2000. 241. Liu L, Qian J, Singh H, et al: Immunohistochemical analysis of chromophobe renal cell carcinoma, renal oncocytoma, and clear cell carcinoma: An optimal and practical panel for differential diagnosis. Arch Pathol Lab Med. 131:1290–1297, 2007. 242. Mansouri A, Hallonet M, Gruss P: Pax genes and their roles in cell differentiation and development. Curr Opin Cell Biol. 8:851–857, 1996. 243. Mazal PR, Exner M, Haitel A, et al: Expression of kidney-specific cadherin distinguishes chromophobe renal cell carcinoma from renal oncocytoma. Hum Pathol. 36:22–28, 2005. 244. Mazal PR, Stichenwirth M, Koller A, et al: Expression of aquaporins and PAX-2 compared to CD10 and cytokeratin 7 in renal neoplasms: A tissue microarray study. Mod Pathol. 18:535–540, 2005. 245. Daniel L, Lechevallier E, Giorgi R, et al: Pax-2 expression in adult renal tumors. Hum Pathol. 32:282–287, 2001. 246. Gokden N, Gokden M, Phan DC, et al: The utility of PAX-2 in distinguishing metastatic clear cell renal cell carcinoma from its morphologic mimics: An immunohistochemical study with comparison to renal cell carcinoma marker. Am J Surg Pathol. 32:1462–1467, 2008. 247. Tong GX, Melamed J, Mansukhani M, et al: PAX2: A reliable marker for nephrogenic adenoma. Mod Pathol. 19:356–363, 2006. 248. Carney EM, Banerjee P, Ellis CL, et al: PAX2(−)/PAX8(−)/ inhibin A(+) immunoprofile in hemangioblastoma: A helpful combination in the differential diagnosis with metastatic clear cell renal cell carcinoma to the central nervous system. Am J Surg Pathol. 35:262–267, 2011. 249. Cimino-Mathews A, Sharma R, Netto GJ: Diagnostic use of PAX8, CAIX, TTF-1, and TGB in metastatic renal cell carcinoma of the thyroid. Am J Surg Pathol. 35:757–761, 2011. 250. Albadine R, Schultz L, Illei P, et al: PAX8 (+)/p63 (−) immunostaining pattern in renal collecting duct carcinoma (CDC): A useful immunoprofile in the differential diagnosis of CDC versus urothelial carcinoma of upper urinary tract. Am J Surg Pathol. 34:965–969, 2010. 251. Jadallah S, Albadine R, Sharma RB, et al: PAX-8 expression in clear cell, papillary and chromophobe RCC and urothelial carcinoma of renal pelvis, Vol 22, 2009, pp 174A. 252. Knoepp SM, Kunju LP, Roh MH: Utility of PAX8 and PAX2 immunohistochemistry in the identification of renal cell carcinoma in diagnostic cytology. Diagn Cytopathol. 40:667–672, 2012. 253. Ordonez NG: Value of PAX2 immunostaining in tumor diagnosis: A review and update. Adv Anat Pathol. 19:401–409, 2012. 254. Ozcan A, de la Roza G, Ro JY, et al: PAX2 and PAX8 expression in primary and metastatic renal tumors: A comprehensive comparison. Arch Pathol Lab Med. 136:1541–1551, 2012. 255. Tacha D, Qi W, Zhou D, et al: PAX8 mouse monoclonal antibody [BC12] recognizes a restricted epitope and is highly sensitive in renal cell and ovarian cancers but does not cross-react with B cells and tumors of pancreatic origin. Appl Immunohistochem Mol Morphol. 2012. 256. Tong GX, Weeden EM, Hamele-Bena D, et al: Expression of PAX8 in nephrogenic adenoma and clear cell adenocarcinoma of the lower urinary tract: Evidence of related histogenesis? Am J Surg Pathol. 32:1380–1387, 2008.
References 257. Calabrese G, Crescenzi C, Morizio E, et al: Assignment of TACSTD1 (alias TROP1, M4S1) to human chromosome 2p21 and refinement of mapping of TACSTD2 (alias TROP2, M1S1) to human chromosome 1p32 by in situ hybridization. Cytogenet Cell Genet. 92:164–165, 2001. 258. Shetye J, Christensson B, Rubio C, et al: The tumor-associated antigens BR55-2, GA73-3. and GICA 19-9 in normal and corresponding neoplastic human tissues, especially gastrointestinal tissues. Anticancer Res. 9:395–404, 1989. 259. Seligson DB, Pantuck AJ, Liu X, et al: Epithelial cell adhesion molecule (KSA) expression: Pathobiology and its role as an independent predictor of survival in renal cell carcinoma. Clin Cancer Res. 10:2659–2669, 2004. 260. Went P, Dirnhofer S, Salvisberg T, et al: Expression of epithelial cell adhesion molecule (EpCam) in renal epithelial tumors. Am J Surg Pathol. 29:83–88, 2005. 261. Thedieck C, Kuczyk M, Klingel K, et al: Expression of kspcadherin during kidney development and in renal cell carcinoma. Br J Cancer. 92:2010–2017, 2005. 262. Adley BP, Gupta A, Lin F, et al: Expression of kidney-specific cadherin in chromophobe renal cell carcinoma and renal oncocytoma. Am J Clin Pathol. 126:79–85, 2006. 263. Al-Ahmadie HA, Alden D, Qin LX, et al: Carbonic anhydrase IX expression in clear cell renal cell carcinoma: An immunohistochemical study comparing 2 antibodies. Am J Surg Pathol. 32:377–382, 2008. 264. Gupta R, Balzer B, Picken M, et al: Diagnostic implications of transcription factor pax 2 protein and transmembrane enzyme complex carbonic anhydrase IX immunoreactivity in adult renal epithelial neoplasms. Am J Surg Pathol. 33:241–247, 2009. 265. Illei PB, Albadine R, Sharma R, et al: Immunohistochemical analysis of hypoxia inducible protein 2 (HIG2), KSP-cadherin and carbonic anhydrase IX (CAIX) expression in papillary, clear cell and chromophobe renal cell carcinoma, Vol 22, 2009, pp 174A. 266. Uemura H, Nakagawa Y, Yoshida K, et al: MN/CA IX/G250 as a potential target for immunotherapy of renal cell carcinomas. Br J Cancer. 81:741–746, 1999. 267. Grignon DJ, Abdel-Malak M, Mertens WC, et al: Glutathione S-transferase expression in renal cell carcinoma: A new marker of differentiation. Mod Pathol. 7:186–189, 1994. 268. Perez-Ordonez B, Hamed G, Campbell S, et al: Renal oncocytoma: A clinicopathologic study of 70 cases. Am J Surg Pathol. 21:871–883, 1997. 269. Reuter VE: The pathology of renal epithelial neoplasms. Semin Oncol. 33:534–543, 2006. 270. Reuter VE: Renal tumors exhibiting granular cytoplasm. Semin Diagn Pathol. 16:135–145, 1999. 271. Amin MB, Amin MB, Tamboli P, et al: Prognostic impact of histologic subtyping of adult renal epithelial neoplasms: An experience of 405 cases. Am J Surg Pathol. 26:281–291, 2002. 272. Huo L, Sugimura J, Tretiakova MS, et al: C-kit expression in renal oncocytomas and chromophobe renal cell carcinomas. Hum Pathol. 36:262–268, 2005. 273. Pan CC, Chen PC, Chiang H: Overexpression of KIT (CD117) in chromophobe renal cell carcinoma and renal oncocytoma. Am J Clin Pathol. 121:878–883, 2004. 274. Al-Saleem T, Cairns P, Dulaimi EA, et al: The genetics of renal oncocytosis: A possible model for neoplastic progression. Cancer Genet Cytogenet. 152:23–28, 2004. 275. Paner GP, Lindgren V, Jacobson K, et al: High incidence of chromosome 1 abnormalities in a series of 27 renal oncocytomas: Cytogenetic and fluorescence in situ hybridization studies. Arch Pathol Lab Med. 131:81–85, 2007. 276. Sibony M, Vieillefond A: Non clear cell renal cell carcinoma. 2008 update in renal tumor pathology. Ann Pathol. 28:381–401, 2008. 277. Storkel S: Epithelial tumors of the kidney. pathological subtyping and cytogenetic correlation. Urologe A. 38:425–432, 1999. 278. Verdorfer I, Hobisch A, Hittmair A, et al: Cytogenetic characterization of 22 human renal cell tumors in relation to a histopathological classification. Cancer Genet Cytogenet. 111:61–70, 1999. 279. Barocas DA, Mathew S, DelPizzo JJ, et al: Renal cell carcinoma sub-typing by histopathology and fluorescence in situ hybridization on a needle-biopsy specimen. BJU Int. 99:290–295, 2007.
652.e7
280. Furge KA, Lucas KA, Takahashi M, et al: Robust classification of renal cell carcinoma based on gene expression data and predicted cytogenetic profiles. Cancer Res. 64:4117–4121. 2004. 281. Argani P: Metanephric neoplasms: The hyperdifferentiated, benign end of the wilms tumor spectrum? Clin Lab Med. 25:379–392, 2005. 282. Olgac S, Hutchinson B, Tickoo SK, et al: Alpha-methylacyl-CoA racemase as a marker in the differential diagnosis of metanephric adenoma. Mod Pathol. 19:218–224, 2006. 283. Skinnider BF, Folpe AL, Hennigar RA, et al: Distribution of cytokeratins and vimentin in adult renal neoplasms and normal renal tissue: Potential utility of a cytokeratin antibody panel in the differential diagnosis of renal tumors. Am J Surg Pathol. 29:747–754, 2005. 284. Galmiche L, Vasiliu V, Poiree S, et al: Diagnosis of renal metanephric adenoma: Relevance of immunohistochemistry and biopsy. Ann Pathol. 27:365–368, 2007. 285. Cohen HT, McGovern FJ: Renal-cell carcinoma. N Engl J Med. 353:2477–2490, 2005. 286. Reuter VE, Presti JC, Jr: Contemporary approach to the classification of renal epithelial tumors. Semin Oncol. 27:124–137, 2000. 287. Skinnider BF, Amin MB: An immunohistochemical approach to the differential diagnosis of renal tumors. Semin Diagn Pathol. 22:51–68, 2005. 288. Al-Ahmadie HA, Alden D, Fine SW, et al: Role of immunohistochemistry in the evaluation of needle core biopsies in adult renal cortical tumors: An ex vivo study. Am J Surg Pathol. 35:949–961, 2011. 289. Leroy X, Zini L, Leteurtre E, et al: Morphologic subtyping of papillary renal cell carcinoma: Correlation with prognosis and differential expression of MUC1 between the two subtypes. Mod Pathol. 15:1126–1130, 2002. 290. Langner C, Ratschek M, Rehak P, et al: Expression of MUC1 (EMA) and E-cadherin in renal cell carcinoma: A systematic immunohistochemical analysis of 188 cases. Mod Pathol. 17:180–188, 2004. 291. Delahunt B, Eble JN: Papillary renal cell carcinoma: A clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol. 10:537–544, 1997. 292. Renshaw AA, Corless CL: Papillary renal cell carcinoma. histology and immunohistochemistry. Am J Surg Pathol. 19:842–849, 1995. 293. Renshaw AA, Zhang H, Corless CL, et al: Solid variants of papillary (chromophil) renal cell carcinoma: Clinicopathologic and genetic features. Am J Surg Pathol. 21:1203–1209, 1997. 294. Corless CL, Aburatani H, Fletcher JA, et al: Papillary renal cell carcinoma: Quantitation of chromosomes 7 and 17 by FISH, analysis of chromosome 3p for LOH, and DNA ploidy. Diagn Mol Pathol. 5:53–64, 1996. 295. Kattar MM, Grignon DJ, Wallis T, et al: Clinicopathologic and interphase cytogenetic analysis of papillary (chromophilic) renal cell carcinoma. Mod Pathol. 10:1143–1150, 1997. 296. Henke RP, Erbersdobler A: Numerical chromosomal aberrations in papillary renal cortical tumors: Relationship with histopathologic features. Virchows Arch. 440:604–609, 2002. 297. Tretiakova MS, Sahoo S, Takahashi M, et al: Expression of alphamethylacyl-CoA racemase in papillary renal cell carcinoma. Am J Surg Pathol. 28:69–76, 2004. 298. Couturier J: Genomic classification of renal cell tumors in adults. Ann Pathol. 28:402–408, 2008. 299. Meyer PN, Cao Y, Jacobson K, et al: Chromosome 1 analysis in chromophobe renal cell carcinomas with tissue microarray (TMA)-facilitated fluorescence in situ hybridization (FISH) demonstrates loss of 1p/1 which is also present in renal oncocytomas. Diagn Mol Pathol. 17:141–144, 2008. 300. Adley BP, Papavero V, Sugimura J, et al: Diagnostic value of cytokeratin 7 and parvalbumin in differentiating chromophobe renal cell carcinoma from renal oncocytoma. Anal Quant Cytol Histol. 28:228–236, 2006. 301. Abrahams NA, MacLennan GT, Khoury JD, et al: Chromophobe renal cell carcinoma: A comparative study of histological, immunohistochemical and ultrastructural features using high throughput tissue microarray. Histopathology. 45:593–602, 2004. 302. Amin MB, Paner GP, Alvarado-Cabrero I, et al: Chromophobe renal cell carcinoma: Histomorphologic characteristics and
652.e8
Immunohistology of the Bladder, Kidney, and Testis
evaluation of conventional pathologic prognostic parameters in 145 cases. Am J Surg Pathol. 32:1822–1834, 2008. 303. Pan CC, Chen PC, Ho DM: The diagnostic utility of MOC31, BerEP4, RCC marker and CD10 in the classification of renal cell carcinoma and renal oncocytoma: An immunohistochemical analysis of 328 cases. Histopathology. 45:452–459, 2004. 304. Rao Q, Zhou XJ, Shi QL, et al: Expression of ksp-cadherin in renal epithelial neoplasm and its clinicopathologic significance. Zhonghua Bing Li Xue Za Zhi. 36:15–18, 2007. 305. Shen SS, Krishna B, Chirala R, et al: Kidney-specific cadherin, a specific marker for the distal portion of the nephron and related renal neoplasms. Mod Pathol. 18:933–940, 2005. 306. Tickoo SK, Amin MB, Zarbo RJ: Colloidal iron staining in renal epithelial neoplasms, including chromophobe renal cell carcinoma: Emphasis on technique and patterns of staining. Am J Surg Pathol. 22:419–424, 1998. 307. Nese N, Paner GP, Mallin K, et al: Renal cell carcinoma: Assessment of key pathologic prognostic parameters and patient characteristics in 47,909 cases using the national cancer data base. Ann Diagn Pathol. 13:1–8, 2009. 308. Srigley JR, Eble JN: Collecting duct carcinoma of kidney. Semin Diagn Pathol. 15:54–67, 1998. 309. Brandal P, Lie AK, Bassarova A, et al: Genomic aberrations in mucinous tubular and spindle cell renal cell carcinomas. Mod Pathol. 19:186–194, 2006. 310. Cossu-Rocca P, Eble JN, Delahunt B, et al: Renal mucinous tubular and spindle carcinoma lacks the gains of chromosomes 7 and 17 and losses of chromosome Y that are prevalent in papillary renal cell carcinoma. Mod Pathol. 19:488–493, 2006. 311. Furge KA, Dykema K, Petillo D, et al: Combining differential expression, chromosomal and pathway analyses for the molecular characterization of renal cell carcinoma. Can Urol Assoc J. 1:S21–S27, 2007. 312. Kuroda N, Hes O, Michal M, et al: Mucinous tubular and spindle cell carcinoma with fuhrman nuclear grade 3: A histological, immunohistochemical, ultrastructural and FISH study. Histol Histopathol. 23:1517–1523, 2008. 313. Owens CL, Argani P, Ali SZ: Mucinous tubular and spindle cell carcinoma of the kidney: Cytopathologic findings. Diagn Cytopathol. 35:593–596, 2007. 314. Paner GP, Srigley JR, Radhakrishnan A, et al: Immunohistochemical analysis of mucinous tubular and spindle cell carcinoma and papillary renal cell carcinoma of the kidney: Significant immunophenotypic overlap warrants diagnostic caution. Am J Surg Pathol. 30:13–19, 2006. 315. Trabelsi A, Stita W, Yacoubi MT, et al: Renal mucinous tubular and spindle cell carcinoma. Can Urol Assoc J. 2:635–636, 2008. 316. Argani P, Netto GJ, Parwani AV: Papillary renal cell carcinoma with low-grade spindle cell foci: A mimic of mucinous tubular and spindle cell carcinoma. Am J Surg Pathol. 32:1353–1359, 2008. 317. Martignoni G, Pea M, Reghellin D, et al: PEComas: The past, the present and the future. Virchows Arch. 452:119–132, 2008. 318. Eble JN, Amin MB, Young RH: Epithelioid angiomyolipoma of the kidney: A report of five cases with a prominent and diagnostically confusing epithelioid smooth muscle component. Am J Surg Pathol. 21:1123–1130, 1997. 319. Kato I, Inayama Y, Yamanaka S, et al: Epithelioid angiomyolipoma of the kidney. Pathol Int. 59:38–43, 2009. 320. Argani P, Aulmann S, Karanjawala Z, et al: Melanotic Xp11 translocation renal cancers: A distinctive neoplasm with overlapping features of PEComa, carcinoma, and melanoma. Am J Surg Pathol. 2008. 321. Argani P, Hawkins A, Griffin CA, et al: A distinctive pediatric renal neoplasm characterized by epithelioid morphology, basement membrane production, focal HMB45 immunoreactivity, and t(6;11)(p21.1;q12) chromosome translocation. Am J Pathol. 158:2089–2096, 2001. 322. Aydin H, Chen L, Cheng L, et al: Clear cell tubulopapillary renal cell carcinoma: A study of 36 distinctive low-grade epithelial tumors of the kidney. Am J Surg Pathol. 34:1608–1621, 2010. 323. Rohan SM, Xiao Y, Liang Y, et al: Clear-cell papillary renal cell carcinoma: Molecular and immunohistochemical analysis with emphasis on the von hippel-lindau gene and hypoxia-inducible factor pathway-related proteins. Mod Pathol. 24:1207–1220, 2011.
324. Wolfe A, Dobin SM, Grossmann P, et al: Clonal trisomies 7,10 and 12, normal 3p and absence of VHL gene mutation in a clear cell tubulopapillary carcinoma of the kidney. Virchows Arch. 459:457–463, 2011. 325. Patard JJ, Leray E, Rioux-Leclercq N, et al: Prognostic value of histologic subtypes in renal cell carcinoma: A multicenter experience. J Clin Oncol. 23:2763–2771, 2005. 326. Cheville JC, Lohse CM, Zincke H, et al: Comparisons of outcome and prognostic features among histologic subtypes of renal cell carcinoma. Am J Surg Pathol. 27:612–624, 2003. 327. Lane BR, Kattan MW: Prognostic models and algorithms in renal cell carcinoma. Urol Clin North Am. 35:613,25; vii, 2008. 328. Kim HL, Seligson D, Liu X, et al: Using tumor markers to predict the survival of patients with metastatic renal cell carcinoma. J Urol. 173:1496–1501, 2005. 329. Kim HL, Seligson D, Liu X, et al: Using protein expressions to predict survival in clear cell renal carcinoma. Clin Cancer Res. 10:5464–5471, 2004. 330. Kluger HM, Siddiqui SF, Angeletti C, et al: Classification of renal cell carcinoma based on expression of VEGF and VEGF receptors in both tumor cells and endothelial cells. Lab Invest. 88:962– 972, 2008. 331. Hager M, Haufe H, Kemmerling R, et al: Increased activated akt expression in renal cell carcinomas and prognosis. J Cell Mol Med. 2008. 332. Eichelberg C, Junker K, Ljungberg B, et al: Diagnostic and prognostic molecular markers for renal cell carcinoma: A critical appraisal of the current state of research and clinical applicability. Eur Urol. 2009. 333. Djordjevic G, Mozetic V, Mozetic DV, et al: Prognostic significance of vascular endothelial growth factor expression in clear cell renal cell carcinoma. Pathol Res Pract. 203:99–106, 2007. 334. Bensalah K, Pantuck AJ, Crepel M, et al: Prognostic variables to predict cancer-related death in incidental renal tumours. BJU Int. 102:1376–1380, 2008. 335. Parker AS, Leibovich BC, Lohse CM, et al: Development and evaluation of BioScore: A biomarker panel to enhance prognostic algorithms for clear cell renal cell carcinoma. Cancer. 115:2092–2103, 2009. 336. Pantuck AJ, Seligson DB, Klatte T, et al: Prognostic relevance of the mTOR pathway in renal cell carcinoma: Implications for molecular patient selection for targeted therapy. Cancer. 109:2257–2267, 2007. 337. Pantuck AJ, Thomas G, Belldegrun AS, et al: Mammalian target of rapamycin inhibitors in renal cell carcinoma: Current status and future applications. Semin Oncol. 33:607– 613, 2006. 338. Chaux A, Schultz L, Albadine R, et al: Immunoexpression status and prognostic value of mammalian target of rapamycin and hypoxia-induced pathway members in papillary cell renal cell carcinomas. Hum Pathol. 43:2129–2137, 2012. 339. Schultz L, Chaux A, Albadine R, et al: Immunoexpression status and prognostic value of mTOR and hypoxia-induced pathway members in primary and metastatic clear cell renal cell carcinomas. Am J Surg Pathol. 35:1549–1556, 2011. 340. Bui MH, Seligson D, Han KR, et al: Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: Implications for prognosis and therapy. Clin Cancer Res. 9:802–811, 2003. 341. Atkins M, Regan M, McDermott D, et al: Carbonic anhydrase IX expression predicts outcome of interleukin 2 therapy for renal cancer. Clin Cancer Res. 11:3714–3721, 2005. 342. Jacobsen J, Grankvist K, Rasmuson T, et al: Expression of vascular endothelial growth factor protein in human renal cell carcinoma. BJU Int. 93:297–302, 2004. 343. Lidgren A, Hedberg Y, Grankvist K, et al: The expression of hypoxia-inducible factor 1alpha is a favorable independent prognostic factor in renal cell carcinoma. Clin Cancer Res. 11:1129–1135, 2005. 344. Migita T, Oda Y, Naito S, et al: Low expression of p27(Kip1) is associated with tumor size and poor prognosis in patients with renal cell carcinoma. Cancer. 94:973–979, 2002. 345. Hedberg Y, Davoodi E, Ljungberg B, et al: Cyclin E and p27 protein content in human renal cell carcinoma: Clinical outcome and associations with cyclin D. Int J Cancer. 102:601– 607, 2002.
References 346. Hedberg Y, Ljungberg B, Roos G, et al: Expression of cyclin D1, D3, E, and p27 in human renal cell carcinoma analysed by tissue microarray. Br J Cancer. 88:1417–1423, 2003. 347. Kosari F, Parker AS, Kube DM, et al: Clear cell renal cell carcinoma: Gene expression analyses identify a potential signature for tumor aggressiveness. Clin Cancer Res. 11:5128–5139, 2005. 348. Linehan WM, Pinto PA, Srinivasan R, et al: Identification of the genes for kidney cancer: Opportunity for disease-specific targeted therapeutics. Clin Cancer Res. 13:671s–679s, 2007. 349. Kim WY, Kaelin WG: Role of VHL gene mutation in human cancer. J Clin Oncol. 22:4991–5004, 2004. 350. Singer EA, Gupta GN, Srinivasan R: Targeted therapeutic strategies for the management of renal cell carcinoma. Curr Opin Oncol. 24:284–290, 2012. 351. Iliopoulos O, Levy AP, Jiang C, et al: Negative regulation of hypoxia-inducible genes by the von hippel-lindau protein. Proc Natl Acad Sci U S A. 93:10595–10599, 1996. 352. Varela I, Tarpey P, Raine K, et al: Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature. 469:539–542, 2011. 353. Motzer RJ, Hutson TE, Tomczak P, et al: Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med. 356:115–124, 2007. 354. Rini BI, Escudier B, Tomczak P, et al: Comparative effectiveness of axitinib versus sorafenib in advanced renal cell carcinoma (AXIS): A randomised phase 3 trial. Lancet. 378:1931–1939, 2011. 355. Motzer RJ, Escudier B, Oudard S, et al: Phase 3 trial of everolimus for metastatic renal cell carcinoma : Final results and analysis of prognostic factors. Cancer. 116:4256–4265, 2010. 356. Motzer RJ, Escudier B, Oudard S, et al: Efficacy of everolimus in advanced renal cell carcinoma: A double-blind, randomised, placebo-controlled phase III trial. Lancet. 372:449–456, 2008. 357. Motzer RJ, Bukowski RM, Figlin RA, et al: Prognostic nomogram for sunitinib in patients with metastatic renal cell carcinoma. Cancer. 113:1552–1558, 2008. 358. Dormoy V, Jacqmin D, Lang H, et al: From development to cancer: Lessons from the kidney to uncover new therapeutic targets. Anticancer Res. 32:3609–3617, 2012. 359. Maher ER: Genomics and epigenomics of renal cell carcinoma. Semin Cancer Biol. 2012. 360. Kruck S, Bedke J, Kuczyk MA, et al: Second-line systemic therapy for the treatment of metastatic renal cell cancer. Expert Rev Anticancer Ther. 12:777–785, 2012. 361. Fisher R, Gore M, Larkin J: Current and future systemic treatments for renal cell carcinoma. Semin Cancer Biol. 2012. 362. Thillai K, Allan S, Powles T, et al: Neoadjuvant and adjuvant treatment of renal cell carcinoma. Expert Rev Anticancer Ther. 12:765–776, 2012. 363. Barrisford GW, Singer EA, Rosner IL, et al: Familial renal cancer: Molecular genetics and surgical management. Int J Surg Oncol. 2011:658767, 2011. 364. Eisengart LJ, MacVicar GR, Yang XJ: Predictors of response to targeted therapy in renal cell carcinoma. Arch Pathol Lab Med. 136:490–495, 2012. 365. Coppin C, Kollmannsberger C, Le L, et al: Targeted therapy for advanced renal cell cancer (RCC): A cochrane systematic review of published randomised trials. BJU Int. 108:1556–1563, 2011. 366. Bitting RL, Madden J, Armstrong AJ: Therapy for non-clear cell histologies in renal cancer. Curr Clin Pharmacol. 6:169–180, 2011. 367. Allory Y, Culine S, de la Taille A: Kidney cancer pathology in the new context of targeted therapy. Pathobiology. 78:90–98, 2011. 368. Bex A, Larkin J, Blank C: Non-clear cell renal cell carcinoma: How new biological insight may lead to new therapeutic modalities. Curr Oncol Rep. 13:240–248, 2011. 369. Tang PA, Vickers MM, Heng DY: Clinical and molecular prognostic factors in renal cell carcinoma: What we know so far. Hematol Oncol Clin North Am. 25:871–891, 2011. 370. Pena C, Lathia C, Shan M, et al: Biomarkers predicting outcome in patients with advanced renal cell carcinoma: Results from sorafenib phase III treatment approaches in renal cancer global evaluation trial. Clin Cancer Res. 16:4853–4863, 2010. 371. Choueiri TK, Regan MM, Rosenberg JE, et al: Carbonic anhydrase IX and pathological features as predictors of outcome in
652.e9
patients with metastatic clear-cell renal cell carcinoma receiving vascular endothelial growth factor-targeted therapy. BJU Int. 106:772–778, 2010. 372. Bhatia S, Thompson JA: Temsirolimus in patients with advanced renal cell carcinoma: An overview. Adv Ther. 2009. 373. Cho IC, Chung J: Current status of targeted therapy for advanced renal cell carcinoma. Korean J Urol. 53:217–228, 2012. 374. Schmidt L, Duh FM, Chen F, et al: Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet. 16:68–73, 1997. 375. Cheng L: Establishing a germ cell origin for metastatic tumors using OCT4 immunohistochemistry. Cancer. 101:2006–2010, 2004. 376. Iczkowski KA, Butler SL, Shanks JH, et al: Trials of new germ cell immunohistochemical stains in 93 extragonadal and metastatic germ cell tumors. Hum Pathol. 39:275–281, 2008. 377. Sung MT, Jones TD, Beck SD, et al: OCT4 is superior to CD30 in the diagnosis of metastatic embryonal carcinomas after chemotherapy. Hum Pathol. 37:662–667, 2006. 378. Looijenga LH, Stoop H, de Leeuw HP, et al: POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Res 63:2244–2250, 2003. 379. Jones TD, Ulbright TM, Eble JN, et al: OCT4 staining in testicular tumors: A sensitive and specific marker for seminoma and embryonal carcinoma. Am J Surg Pathol. 28:935–940, 2004. 380. Jones TD, Ulbright TM, Eble JN, et al: OCT4: A sensitive and specific biomarker for intratubular germ cell neoplasia of the testis. Clin Cancer Res. 10:8544–8547, 2004. 381. Biermann K, Goke F, Nettersheim D, et al: c-KIT is frequently mutated in bilateral germ cell tumours and down-regulated during progression from intratubular germ cell neoplasia to seminoma. J Pathol. 213:311–318, 2007. 382. Coffey J, Linger R, Pugh J, et al: Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: Report of 220 tumors and review of literature. Genes Chromosomes Cancer. 47:34–42, 2008. 383. Kemmer K, Corless CL, Fletcher JA, et al: KIT mutations are common in testicular seminomas. Am J Pathol. 164:305–313, 2004. 384. Nikolaou M, Valavanis C, Aravantinos G, et al: Kit expression in male germ cell tumors. Anticancer Res. 27:1685–1688, 2007. 385. Willmore-Payne C, Holden JA, Chadwick BE, et al: Detection of c-kit exons 11- and 17-activating mutations in testicular seminomas by high-resolution melting amplicon analysis. Mod Pathol. 19:1164–1169, 2006. 386. Loh YH, Wu Q, Chew JL, et al: The OCT4 and nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet. 38:431–440, 2006. 387. Zhang J, Tam WL, Tong GQ, et al: SALL4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol. 8:1114–1123, 2006. 388. Li SS, Liu YH, Tseng CN, et al: Characterization and gene expression profiling of five new human embryonic stem cell lines derived in taiwan. Stem Cells Dev. 15:532–555, 2006. 389. Wu Q, Chen X, Zhang J, et al: SALL4 interacts with nanog and co-occupies nanog genomic sites in embryonic stem cells. J Biol Chem. 281:24090–24094, 2006. 390. Zhou Q, Chipperfield H, Melton DA, et al: A gene regulatory network in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 104:16438–16443, 2007. 391. Wong CC, Gaspar-Maia A, Ramalho-Santos M, et al: Highefficiency stem cell fusion-mediated assay reveals SALL4 as an enhancer of reprogramming. PLoS One. 3:e1955, 2008. 392. Kohlhase J, Heinrich M, Schubert L, et al: Okihiro syndrome is caused by SALL4 mutations. Hum Mol Genet. 11:2979–2987, 2002. 393. Cao D, Li J, Guo CC, et al: SALL4 is a novel diagnostic marker for testicular germ cell tumors. Am J Surg Pathol. 33:1065–1077, 2009. 394. Cao D, Guo S, Allan RW, et al: SALL4 is a novel sensitive and specific marker of ovarian primitive germ cell tumors and is particularly useful in distinguishing yolk sac tumor from clear cell carcinoma. Am J Surg Pathol. 33:894–904, 2009. 395. Cao D, Humphrey PA, Allan RW: SALL4 is a novel sensitive and specific marker for metastatic germ cell tumors, with particular
652.e10
Immunohistology of the Bladder, Kidney, and Testis
utility in detection of metastatic yolk sac tumors. Cancer. 115:2640–2651, 2009. 396. Mei K, Liu A, Allan RW, et al: Diagnostic utility of SALL4 in primary germ cell tumors of the central nervous system: A study of 77 cases. Mod Pathol. 22:1628–1636, 2009. 397. Wang F, Liu A, Peng Y, et al: Diagnostic utility of SALL4 in extragonadal yolk sac tumors: An immunohistochemical study of 59 cases with comparison to placental-like alkaline phosphatase, alpha-fetoprotein, and glypican-3. Am J Surg Pathol. 33:1529–1539, 2009. 398. Gonzalez-Roibon N, Katz B, Chaux A, et al: Immunohistochemical expression of SALL4 in hepatocellular carcinoma, a potential pitfall in the differential diagnosis of yolk sac tumors. Hum Pathol. in press. 399. Ushiku T, Shinozaki A, Shibahara J, et al: SALL4 represents fetal gut differentiation of gastric cancer, and is diagnostically useful in distinguishing hepatoid gastric carcinoma from hepatocellular carcinoma. Am J Surg Pathol. 34:533–540, 2010. 400. Ikeda H, Sato Y, Yoneda N, et al: α-Fetoprotein-producing gastric carcinoma and combined hepatocellular and cholangiocarcinoma show similar morphology but different histogenesis with respect to SALL4 expression. Hum Pathol. 2012. 401. Lau SK, Weiss LM, Chu PG: D2-40 immunohistochemistry in the differential diagnosis of seminoma and embryonal carcinoma: A comparative immunohistochemical study with KIT (CD117) and CD30. Mod Pathol. 20:320–325, 2007. 402. Hoei-Hansen CE, Nielsen JE, Almstrup K, et al: Transcription factor AP-2gamma is a developmentally regulated marker of testicular carcinoma in situ and germ cell tumors. Clin Cancer Res. 10:8521–8530, 2004. 403. Hong SM, Frierson HF, Jr, Moskaluk CA: AP-2gamma protein expression in intratubular germ cell neoplasia of testis. Am J Clin Pathol. 124:873–877, 2005. 404. Pauls K, Jager R, Weber S, et al: Transcription factor AP-2gamma, a novel marker of gonocytes and seminomatous germ cell tumors. Int J Cancer. 115:470–477, 2005. 405. Manivel JC, Jessurun J, Wick MR, et al: Placental alkaline phosphatase immunoreactivity in testicular germ-cell neoplasms. Am J Surg Pathol. 11:21–29, 1987. 406. Javadpour N: The role of biologic tumor markers in testicular cancer. Cancer. 45:1755–1761, 1980. 407. Ulbright TM, Young RH, Scully RE: Trophoblastic tumors of the testis other than classic choriocarcinoma: “monophasic” choriocarcinoma and placental site trophoblastic tumor: A report of two cases. Am J Surg Pathol. 21:282–288, 1997. 408. Zynger DL, Dimov ND, Luan C, et al: Glypican 3: A novel marker in testicular germ cell tumors. Am J Surg Pathol. 30:1570–1575, 2006. 409. Emerson RE, Ulbright TM: Intratubular germ cell neoplasia of the testis and its associated cancers: The use of novel biomarkers. Pathology. 42:344–355, 2010. 410. Ota S, Hishinuma M, Yamauchi N, et al: Oncofetal protein glypican-3 in testicular germ-cell tumor. Virchows Arch. 449: 308–314, 2006. 411. McCluggage WG, Shanks JH, Whiteside C, et al: Immunohistochemical study of testicular sex cord-stromal tumors, including staining with anti-inhibin antibody. Am J Surg Pathol. 22:615– 619, 1998. 412. McCluggage WG, Ashe P, McBride H, et al: Localization of the cellular expression of inhibin in trophoblastic tissue. Histopathology. 32:252–256, 1998. 413. Skakkebaek NE: Carcinoma in situ of the testis: Frequency and relationship to invasive germ cell tumours in infertile men. N. E. skakkebaek. histopathology 1978; 2; 157-170. Histopathology. 41:2,3; discussion 3–4, 2002. 414. Ulbright TM: Testis risk and prognostic factors. the pathologist’s perspective. Urol Clin North Am. 26:611–626, 1999. 415. van Casteren NJ, de Jong J, Stoop H, et al: Evaluation of testicular biopsies for carcinoma in situ: Immunohistochemistry is mandatory. Int J Androl. 2008. 416. Akre O, Pettersson A, Richiardi L: Risk of contralateral testicular cancer among men with unilaterally undescended testis: A meta analysis. Int J Cancer. 124:687–689, 2009. 417. Burke AP, Mostofi FK: Intratubular malignant germ cells in testicular biopsies: Clinical course and identification by staining for placental alkaline phosphatase. Mod Pathol. 1:475–479, 1988.
418. Cheng L, Sung MT, Cossu-Rocca P, et al: OCT4: Biological functions and clinical applications as a marker of germ cell neoplasia. J Pathol. 211:1–9, 2007. 419. Ulbright TM: Germ cell tumors of the gonads: A selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod Pathol. 18(Suppl 2):S61–S79, 2005. 420. Leroy X, Augusto D, Leteurtre E, et al: CD30 and CD117 (c-kit) used in combination are useful for distinguishing embryonal carcinoma from seminoma. J Histochem Cytochem. 50:283– 285, 2002. 421. Liu DL, Lu YP, Shi HY, et al: Expression of CD117 in human testicular germ cell tumors and its diagnostic value for seminoma and nonseminoma. Zhonghua Nan Ke Xue. 14:38–41, 2008. 422. Iczkowski KA, Butler SL: New immunohistochemical markers in testicular tumors. Anal Quant Cytol Histol. 28:181–187, 2006. 423. Emerson RE, Ulbright TM: The use of immunohistochemistry in the differential diagnosis of tumors of the testis and paratestis. Semin Diagn Pathol. 22:33–50, 2005. 424. Emerson RE, Ulbright TM: Morphological approach to tumours of the testis and paratestis. J Clin Pathol. 60:866–880, 2007. 425. de Jong J, Looijenga LH: Stem cell marker OCT3/4 in tumor biology and germ cell tumor diagnostics: History and future. Crit Rev Oncog. 12:171–203, 2006. 426. Carano KS, Soslow RA: Immunophenotypic analysis of ovarian and testicular mullerian papillary serous tumors. Mod Pathol. 10:414–420, 1997. 427. Wick MR, Swanson PE, Manivel JC: Placental-like alkaline phosphatase reactivity in human tumors: An immunohistochemical study of 520 cases. Hum Pathol. 18:946–954, 1987. 428. Suster S, Moran CA, Dominguez-Malagon H, et al: Germ cell tumors of the mediastinum and testis: A comparative immunohistochemical study of 120 cases. Hum Pathol. 29:737–742, 1998. 429. Cheville JC, Rao S, Iczkowski KA, et al: Cytokeratin expression in seminoma of the human testis. Am J Clin Pathol. 113:583– 588, 2000. 430. Hittmair A, Rogatsch H, Hobisch A, et al: CD30 expression in seminoma. Hum Pathol. 27:1166–1171, 1996. 431. Koshida K, Wahren B: Placental-like alkaline phosphatase in seminoma. Urol Res. 18:87–92, 1990. 432. Balzer BL, Ulbright TM: Spontaneous regression of testicular germ cell tumors: An analysis of 42 cases. Am J Surg Pathol. 30:858–865, 2006. 433. Ulbright TM: The most common, clinically significant misdiagnoses in testicular tumor pathology, and how to avoid them. Adv Anat Pathol. 15:18–27, 2008. 434. Mannuel HD, Mitikiri N, Hussain A: Update on testicular germ cell tumors. Curr Opin Oncol. 23:265–270, 2011. 435. Mannuel HD, Mitikiri N, Khan M, et al: Testicular germ cell tumors: Biology and clinical update. Curr Opin Oncol. 24:266– 271, 2012. 436. Chung PW, Bayley AJ, Sweet J, et al: Spermatocytic seminoma: A review. Eur Urol. 45:495–498, 2004. 437. Decaussin M, Borda A, Bouvier R, et al: Spermatocytic seminoma. A clinicopathological and immunohistochemical study of 7 cases. Ann Pathol. 24:161–166, 2004. 438. Dundr P, Pesl M, Povysil C, et al: Anaplastic variant of spermatocytic seminoma. Pathol Res Pract. 203:621–624, 2007. 439. Looijenga LH, Hersmus R, Gillis AJ, et al: Genomic and expression profiling of human spermatocytic seminomas: Primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. Cancer Res. 66:290–302, 2006. 440. Ulbright TM, Young RH: Seminoma with tubular, microcystic, and related patterns: A study of 28 cases of unusual morphologic variants that often cause confusion with yolk sac tumor. Am J Surg Pathol. 29:500–505, 2005. 441. Rajpert-De Meyts E, Jacobsen GK, Bartkova J, et al: The immunohistochemical expression pattern of Chk2, p53, p19INK4d, MAGE-A4 and other selected antigens provides new evidence for the premeiotic origin of spermatocytic seminoma. Histopathology. 42:217–226, 2003.
References 442. Billings SD, Roth LM, Ulbright TM: Microcystic leydig cell tumors mimicking yolk sac tumor: A report of four cases. Am J Surg Pathol. 23:546–551, 1999. 443. Comperat E, Tissier F, Boye K, et al: Non-leydig sex-cord tumors of the testis. the place of immunohistochemistry in diagnosis and prognosis. A study of twenty cases. Virchows Arch. 444:567–571, 2004. 444. Gilcrease MZ, Delgado R, Albores-Saavedra J: Testicular sertoli cell tumor with a heterologous sarcomatous component: Immunohistochemical assessment of sertoli cell differentiation. Arch Pathol Lab Med. 122:907–911, 1998. 445. Gordon MD, Corless C, Renshaw AA, et al: CD99, keratin, and vimentin staining of sex cord-stromal tumors, normal ovary, and testis. Mod Pathol. 11:769–773, 1998. 446. Groisman GM, Dische MR, Fine EM, et al: Juvenile granulosa cell tumor of the testis: A comparative immunohistochemical study with normal infantile gonads. Pediatr Pathol. 13:389–400, 1993. 447. Young RH, Koelliker DD, Scully RE: Sertoli cell tumors of the testis, not otherwise specified: A clinicopathologic analysis of 60 cases. Am J Surg Pathol. 22:709–721, 1998. 448. Zukerberg LR, Young RH, Scully RE: Sclerosing sertoli cell tumor of the testis. A report of 10 cases. Am J Surg Pathol. 15:829–834, 1991. 449. Ulbright TM, Young RH: Metastatic carcinoma to the testis: A clinicopathologic analysis of 26 nonincidental cases with emphasis on deceptive features. Am J Surg Pathol. 32:1683–1693, 2008. 450. Al-Abbadi MA, Hattab EM, Tarawneh MS, et al: Primary testicular diffuse large B-cell lymphoma belongs to the nongerminal center B-cell-like subgroup: A study of 18 cases. Mod Pathol. 19:1521–1527, 2006. 451. Ferry JA, Harris NL, Young RH, et al: Malignant lymphoma of the testis, epididymis, and spermatic cord. A clinicopathologic study of 69 cases with immunophenotypic analysis. Am J Surg Pathol. 18:376–390, 1994. 452. Ferry JA, Young RH, Scully RE: Testicular and epididymal plasmacytoma: A report of 7 cases, including three that were the initial manifestation of plasma cell myeloma. Am J Surg Pathol. 21:590–598, 1997. 453. Ulbright TM, Orazi A, de Riese W, et al: The correlation of P53 protein expression with proliferative activity and occult metastases in clinical stage I non-seminomatous germ cell tumors of the testis. Mod Pathol. 7:64–68, 1994. 454. Baltaci S, Orhan D, Turkolmez K, et al: P53, bcl-2 and bax immunoreactivity as predictors of response and outcome after chemotherapy for metastatic germ cell testicular tumours. BJU Int. 87:661–666, 2001. 455. Bartkova J, Bartek J, Lukas J, et al: P53 protein alterations in human testicular cancer including pre-invasive intratubular germ-cell neoplasia. Int J Cancer. 49:196–202, 1991. 456. Berney DM, Shamash J, Gaffney J, et al: DNA topoisomerase I and II expression in drug resistant germ cell tumours. Br J Cancer. 87:624–629, 2002. 457. Kersemaekers AM, Mayer F, Molier M, et al: Role of P53 and MDM2 in treatment response of human germ cell tumors. J Clin Oncol. 20:1551–1561, 2002. 458. Sandberg AA, Meloni AM, Suijkerbuijk RF: Reviews of chromosome studies in urological tumors. III. cytogenetics and genes in testicular tumors. J Urol. 155:1531–1556, 1996. 459. Suijkerbuijk RF, Sinke RJ, Meloni AM, et al: Overrepresentation of chromosome 12p sequences and karyotypic evolution in i(12p)-negative testicular germ-cell tumors revealed by fluorescence in situ hybridization. Cancer Genet Cytogenet. 70:85–93, 1993. 460. Cheng L, Zhang S, MacLennan GT, et al: Interphase fluorescence in situ hybridization analysis of chromosome 12p abnormalities
652.e11
is useful for distinguishing epidermoid cysts of the testis from pure mature teratoma. Clin Cancer Res. 12:5668–5672, 2006. 461. Looijenga LH, Zafarana G, Grygalewicz B, et al: Role of gain of 12p in germ cell tumour development. APMIS. 111:161,71; discussion 172–173, 2003. 462. Reuter VE: Origins and molecular biology of testicular germ cell tumors. Mod Pathol. 18(Suppl 2):S51–S60, 2005. 463. Rosenberg C, Van Gurp RJ, Geelen E, et al: Overrepresentation of the short arm of chromosome 12 is related to invasive growth of human testicular seminomas and nonseminomas. Oncogene. 19:5858–5862, 2000. 464. Wehle D, Yonescu R, Long PP, et al: Fluorescence in situ hybridization of 12p in germ cell tumors using a bacterial artificial chromosome clone 12p probe on paraffin-embedded tissue: Clinical test validation. Cancer Genet Cytogenet. 183:99–104, 2008. 465. LeBron C, Pal P, Brait M, et al: Genome-wide analysis of genetic alterations in testicular primary seminoma using high resolution single nucleotide polymorphism arrays. Genomics. 97:341–349, 2011. 466. Smiraglia DJ, Szymanska J, Kraggerud SM, et al: Distinct epigenetic phenotypes in seminomatous and nonseminomatous testicular germ cell tumors. Oncogene. 21:3909–3916, 2002. 467. Netto GJ, Nakai Y, Nakayama M, et al: Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors. Mod Pathol. 21:1337–1344, 2008. 468. Brait M, Maldonado L, Begum S, et al: DNA methylation profiles delineate epigenetic heterogeneity in seminoma and nonseminoma. Br J Cancer. 106:414–423, 2012. 469. Leman ES, Magheli A, Yong KM, et al: Identification of nuclear structural protein alterations associated with seminomas. J Cell Biochem. 108:1274–1279, 2009. 470. Eid H, Geczi L, Magori A, et al: Drug resistance and sensitivity of germ cell testicular tumors: Evaluation of clinical relevance of MDR1/Pgp, p53, and metallothionein (MT) proteins. Anticancer Res. 18:3059–3064, 1998. 471. Eid H, Institoris E, Geczi L, et al: Mdm-2 expression in human testicular germ-cell tumors and its clinical value. Anticancer Res. 19:3485–3490, 1999. 472. Eid H, Van der Looij M, Institoris E, et al: Is p53 expression, detected by immunohistochemistry, an important parameter of response to treatment in testis cancer? Anticancer Res. 17:2663– 2669, 1997. 473. Mayer F, Gillis AJ, Dinjens W, et al: Microsatellite instability of germ cell tumors is associated with resistance to systemic treatment. Cancer Res. 62:2758–2760, 2002. 474. Mayer F, Stoop H, Scheffer GL, et al: Molecular determinants of treatment response in human germ cell tumors. Clin Cancer Res. 9:767–773, 2003. 475. Honecker F, Wermann H, Mayer F, et al: Microsatellite instability, mismatch repair deficiency, and BRAF mutation in treatment-resistant germ cell tumors. J Clin Oncol. 27:2129– 2136, 2009. 476. Dimov ND, Zynger DL, Luan C, et al: Topoisomerase II alpha expression in testicular germ cell tumors. Urology. 69:955–961, 2007. 477. Mazumdar M, Bacik J, Tickoo SK, et al: Cluster analysis of p53 and Ki67 expression, apoptosis, alpha-fetoprotein, and human chorionic gonadotrophin indicates a favorable prognostic subgroup within the embryonal carcinoma germ cell tumor. J Clin Oncol. 21:2679–2688, 2003. 478. Wang X, Zhang S, Maclennan GT, et al: Epidermal growth factor receptor protein expression and gene amplification in the chemorefractory metastatic embryonal carcinoma. Mod Pathol. 22:7–12, 2009.
C H A P T E R 1 8
IMMUNOHISTOLOGY OF THE FEMALE GENITAL TRACT JOSEPH T. RABBAN, TERI A. LONGACRE
Overview 653 Vulva, Vagina, and Cervix 653 Uterus 664 Ovary and Fallopian Tubes 683 Peritoneal Mesothelioma 706 Summary 709
Overview This chapter focuses on the use of immunohistochemistry (IHC) in the setting of diagnostic gynecologic pathology. It is divided into four sections: 1) vulva, vagina, and cervix; 2) uterus; 3) ovary and fallopian tube, and 4) peritoneum. The first part of each section includes a description of the properties and applications of the antibodies that are most useful in diagnostic work. A table of commonly used antibodies is provided for reference (Table 18-1). In the second part of each section, the use of IHC for the resolution of diagnostic problems or for the diagnosis of gynecologic lesions is discussed. Each section also includes a discussion of aspects of molecular pathology as it can be used to supplement, or in some cases substitute for, IHC analysis. The focus of this chapter is on diagnostic IHC, but we wish to emphasize that antibodies must be used and stains must be interpreted in conjunction with careful assessment of routine hematoxylin and eosin (H&E)stained slides and clinicopathologic correlation.
Vulva, Vagina, and Cervix Immunohistochemical Markers Most of the antibodies used in the lower gynecologic tract are also used in other anatomic locations and are described in applicable sections in the text. The most
commonly used antibody in the lower gynecologic tract is p16, therefore we will discuss it in detail in the following section. p16
A tumor suppressor protein that is a cyclin-dependent kinase inhibitor, p16 is essential in regulating the cell cycle, and it inactivates cyclin-dependent kinases that phosphorylate retinoblastoma (Rb); therefore p16 can decelerate the cell cycle. Rb phosphorylation status in turn influences expression of p16. In human papilloma virus (HPV) infection, the HPV oncogenes E6 and E7 can inactivate phosphorylated Rb (pRb) and thus lead to p16 overexpression.1 Therefore p16 overexpression is a surrogate biomarker of HPV infection, the high-risk HPV types in particular, which makes it useful in evaluating HPV-associated squamous and glandular neoplasia of the lower gynecologic tract.2-9 As discussed later in this chapter, the intensity and distribution of p16 is important in interpretation as well as in nuclear versus cytoplasmic localization. HPV-independent mechanisms of p16 overexpression also exist, so p16 expression may be observed in tumors that do not necessarily harbor HPV infection, such as high-grade serous carcinoma.10
Vulva and Vagina VULVAR PAGET DISEASE
Vulvar Paget disease is an intraepidermal adenocarcinoma that may be of primary or metastatic origin. Four diagnostic issues arise when evaluating vulvar Paget disease: 1) determining site of origin, 2) excluding diagnostic mimics, 3) assessing margin status, and 4) assessing for stromal invasion. Unlike Paget disease of the breast, primary vulvar Paget disease is not commonly associated with an underlying adenocarcinoma but is typically a pure intraepidermal tumor. Primary vulvar Paget disease expresses cytokeratin 7 (CK7), gross cystic disease fluid protein (GCDFP; Fig. 18-1), and carcinoembryonic antigen (CEA) but not CDX-2, S-100, human melanoma black 45 (HMB-45), or estrogen or progesterone receptors;11-15 rare cases express CK20,16 653
TABLE 18-1 Commonly Used Antibodies in Gynecologic Pathology Antibody
Vendor
Dilution
Pattern
Alpha-fetoprotein
Dako
1 : 80
Cytoplasmic
Carbohydrate antigen 125
Signet
1 : 500
Cytoplasmic/membranous
Caldesmon
Dako
1 : 200
Cytoplasmic
Calretinin
Zymed
1 : 200
Cytoplasmic/nuclear
Monoclonal carcinoembryonic antigen
Dako
1 : 2
Cytoplasmic/luminal
CD10
Novocastra
1 : 160
Cytoplasmic/membranous
CD30
Dako
1 : 100
Cytoplasmic
CD56
Zymed
1 : 100
Cytoplasmic
CD99
Signet
1 : 200
Membrane
CD117/c-Kit
Dako
1 : 50
Cytoplasmic
CDX-2
BioGenex
1 : 100
Nuclear
Chromogranin
BioGenex
1 : 200
Cytoplasmic
CK7
Dako
1 : 500
Cytoplasmic
CK20
Dako
1 : 100
Cytoplasmic
D2-40
Dako
1 : 50
Cytoplasmic/membranous
Desmin
Cell Marque
Undiluted
Cytoplasmic
Epithelial membrane antigen
Dako
1 : 240
Cytoplasmic/membranous
Estrogen receptor (SP1)
Lab Vision
1 : 50
Nuclear
Gross cystic disease fluid protein 15
Signet
1 : 20
Cytoplasmic
Glypican-3
BioMosaics
Undiluted
Cytoplasmic
Human chorionic gonadotropin
Biomeda
1 : 40
Cytoplasmic
High molecular weight keratin/34βE12
Enzo
1 : 2
Cytoplasmic
Human melanoma black 45
Enzo
1 : 8
Cytoplasmic
Hepatocyte nuclear factor 1β
Santa Cruz
1 : 300
Nuclear
Human placental lactogen
Dako
1 : 20,000
Cytoplasmic
Keratin AE1/AE3
Dako
1 : 100
Cytoplasmic
Inhibin
Serotec
1 : 100
Cytoplasmic
MIB-1
Dako
1 : 1000
Nuclear
MLH1
BD Pharmingen
1 : 100
Nuclear
MSH2
Oncogene
1 : 100
Nuclear
MSH6
BD Transduction
1 : 100
Nuclear
Myogenin
Dako
1 : 200
Nuclear
OCT4
Cell Marque
1 : 2
Nuclear
Pax-8
Protein Tech Group
1 : 100
Nuclear
p16
MTM Lab
Undiluted
Nuclear/cytoplasmic
p53
Novocastra
1 : 300
Nuclear
p57
Lab Vision
1 : 400
Nuclear
p63
Lab Vision
1 : 100
Nuclear
Placental alkaline phosphatase
BioGenex
1 : 2
Cytoplasmic
Progesterone receptor
Dako
1 : 250
Nuclear
SALL4
Sigma-Aldrich
1 : 100
Nuclear
S-100
Dako
1 : 2000
Cytoplasmic
Smooth muscle actin, alpha
Dako
1 : 800
Cytoplasmic
Smooth muscle myosin
Dako
1 : 200
Cytoplasmic
Synaptophysin
Dako
1 : 150
Cytoplasmic
Vimentin
Zymed
1 : 1600
Cytoplasmic
Wilms tumor 1
Dako
1 : 200
Nuclear
Vulva, Vagina, and Cervix
A
B
C
D
655
Figure 18-1 A, Primary vulvar Paget disease (hematoxylin and eosin). Tumor cells express cytokeratin 7 (CK7; B) and gross cystic disease fluid protein (C), but normal epithelium does not. Occult stromal invasion can be highlighted by CK7 (D) when inflammation obscures the epidermal-dermal junction.
and Her2 is typically expressed.17 Questionable cells near surgical margins can be evaluated by CK7, because normal epidermis is CK7 negative. Small foci of stromal invasion may also be highlighted by CK7 (see Fig. 18-1, D); p53 expression may be associated with risk of stromal invasion.18 Secondary vulvar Paget disease most commonly represents spread of primary urinary tract or colorectal adenocarcinoma. The immunophenotype reflects that of the primary tumor: vulvar Paget disease of colorectal origin expresses CK20, CDX-2, and CEA; vulvar Paget disease of urothelial origin may express CK20, uroplakin, and thrombomodulin.13,14,19-21 Rare colorectal cases may express GCDFP, so a panel approach is advised rather than reliance on a single marker. Vulvar melanoma and vulvar high-grade squamous intraepithelial lesions (HSILs) may rarely exhibit features that mimic vulvar Paget disease. Primary Paget disease tumor cells do not express the melanocytic markers S-100 or HMB-45.14,22,23 Pagetoid HSILs do not express GCDFP but may express CK7; better markers to detect these are high-molecular-weight cytokeratin (HMWCK), p16, and p63.24-26
KEY DIAGNOSTIC POINTS Vulvar Paget Disease • Primary vulvar Paget disease is typically positive for CK7 and GCDFP and is negative for CK20 and CDX-2. • Secondary vulvar Paget disease of colorectal or urothelial origin shows a similar immunophenotype as the tumor at the primary site. • A panel approach is advised, because overlap of individual markers may rarely occur. • Pagetoid VIN is distinguished by expression of HMWCK, p16, and p63. • Pagetoid melanoma is distinguished by expression of S-100 and HMB-45.
VULVOVAGINAL MESENCHYMAL LESIONS
Although uncommon, a broad array of mesenchymal lesions may arise in the vulva or vagina. Among these, aggressive angiomyxoma is a mesenchymal neoplasm with a propensity for deep infiltrative growth and local recurrence that should be distinguished from its benign
656
Immunohistology of the Female Genital Tract
A
B
Figure 18-2 A, Vulvar granular cell tumor (hematoxylin and eosin). Tumor cells (inset) express S-100 (B), calretinin, and inhibin (not shown).
mimics, which include fibroepithelial polyp, angiomyofibroblastoma, cellular angiofibroma, myxoid leiomyoma, and nodular fasciitis. The relatively bland morphology of all these entities can make diagnosis problematic. Immunostains are of minimal value, because almost all will express vimentin, estrogen receptor (ER), progesterone receptor (PR), and, to a variable extent, desmin, actin, and CD34.27 Cellular angiofibroma stands out in that it lacks actin and desmin expression.28,29 Gastrointestinal stromal tumors (GISTs) may rarely arise primarily in this location and mimic smooth muscle tumors; as in conventional GIST, CD117 and DOG1 expression are characteristic.30 Proximaltype epithelioid sarcoma of the vulva is distinguished by expression of keratin and epithelial membrane antigen (EMA) and by loss of INI-1.31 Rhabdomyosarcoma of the lower genital tract is distinguished by expression of desmin and myogenin, although regenerating, reactive, and entrapped nonneoplastic skeletal muscle may also express myogenic markers.32,33 Recent study of the cytogenetics of aggressive angiomyxoma has identified that gene rearrangements in HMGA2, an architectural transcription factor encoded on chromosome 12q15, are common. Limited studies suggest that nuclear immunoexpression of HMGA2 is present in approximately 50% of aggressive angiomyxomas and in a minority of vulvar smooth muscle tumors.34-37 This antibody is not widely used in clinical diagnosis. KEY DIAGNOSTIC POINTS Vulvar Mesenchymal Lesions • Classification of vulvar mesenchymal lesions is primarily based on morphologic criteria. • Most entities, including aggressive angiomyxoma, express vimentin, estrogen and progesterone receptors, desmin, actin, and CD34. • Cellular angiofibroma lacks desmin, and actin expression may be focal. • CD117 may help distinguish vaginal GISTs that mimic smooth muscle tumors.
VULVAR GRANULAR CELL TUMOR
Granular cell tumors (GCTs) may occasionally arise in the vulva, and their appearance is similar to tumors at other anatomic sites. They are thought to derive from peripheral nerve sheath and express S-100 protein, inhibin, and calretinin (Fig. 18-2).38,39 The diagnosis is usually straightforward, but occasionally the differential may include melanoma, histiocytes, or even a decidual reaction. VULVAR PAPILLARY SQUAMOUS LESIONS
The main differential diagnosis for papillary squamous lesions of the vulva includes condyloma, fibroepithelial polyp, and squamous papilloma. Vulvar condyloma demonstrates papillomatosis, acanthosis, parakeratosis, and dyskeratosis but often lacks koilocytes, which makes the differential difficult in occasional cases. Molecular immunology Borstel 1 (MIB-1) is useful in this context. In normal vulvar epithelium, fibroepithelial polyp, and squamous papilloma, MIB-1 is confined to parabasal cells; in condyloma, MIIB-1 expression occurs in the middle and upper third of the epithelium (Fig. 18-3).40-43 Vulvar seborrheic keratosis may show increased MIB-1 expression, and focal p16 expression has been reported in rare cases; HPV type 6 has been detected in some vulvar seborrheic keratoses.44 As discussed later in the section on MIB-1 expression in the cervix, caution is advised in interpreting specimens with tangential sectioning or suboptimal orientation, because MIB-1 expression by normal parabasal cells could appear to be located in upper layers of epithelium. VULVAR SQUAMOUS INTRAEPITHELIAL LESION (VULVAR INTRAEPITHELIAL NEOPLASIA)
Similar to cervical squamous lesions associated with high-risk HPV, immunostains can assist in distinguishing vulvar squamous intraepithelial lesion (SIL) and vulvar intraepithelial neoplasia (VIN) from benign mimics as well as identifying the unusual variant of simplex (differentiated) VIN. Normal and atrophic
Vulva, Vagina, and Cervix
A
B
C
D
657
F
E Figure 18-3 In exophytic vulvar lesions, molecular immunology Borstel 1 (MIB-1) is expressed above the basal layer in condyloma (A and B) but is confined to parabasal cells in squamous papilloma (C and D) and in fibroepithelial polyp (E and F).
658
Immunohistology of the Female Genital Tract
A
B
Figure 18-4 A, Simplex (differentiated) vulvar intraepithelial neoplasia consists of nuclear atypia and brisk mitoses confined to the basal zone (hematoxylin and eosin). B, Tumor cells may express p53 and MIB-1 (not shown) but typically do not express p16.
vulvar epithelia contain minimal MIB-1 expression in parabasal cells, whereas the common type of HSIL, the undifferentiated or bowenoid type, shows expression in the middle and upper epithelial layers. Expression of p16 in HSIL is similar, although some cases may be negative.45,46 The expression patterns are similar to those seen in cervical SIL. Simplex (differentiated) VIN is characterized by nuclear atypia and brisk mitoses confined to the basal zone; it is typically not associated with HPV, therefore p16 is usually absent.46,47 Instead, simplex VIN may harbor p53 gene mutations; hence, p53 immunoexpression may be present (Fig. 18-4).48,49 Caution is warranted, however, because benign lesions such as lichen sclerosus may be focally p53 positive, and staining should be interpreted in context with the morphologic findings.50
Cervix MESONEPHRIC REMNANTS
Mesonephric duct remnants may be found in the deep wall of cervix specimens. Sometimes, hyperplasia may be pronounced and may raise concern for minimal deviation–type endocervical adenocarcinoma. Apical expression of CD10 is common in these remnants, as is expression by the intraluminal secretions. However, CD10 is not pathognomonic for mesonephric differentiation, because some endocervical and endometrial adenocarcinomas may also express it. Minimal deviation– type endocervical adenocarcinoma, however, lacks CD10 expression.51 Variable expression of p16 is reported in mesonephric remnants and may lead to misinterpretation as endocervical adenocarcinoma; however, strong, diffuse p16 expression is absent, and MIB-1 is not increased. ER, PR, and CEA are generally negative. Mesonephric adenocarcinoma exhibits a similar phenotype.51-53 CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS
The main diagnostic roles for IHC in evaluation of cervical SILs are distinguishing dysplasia from benign
mimics and evaluating cauterized margins. Grading dysplasia remains an issue to which morphologic criteria should be applied. The markers used most widely are MIB-1 and p16. MIB-1 helps distinguish benign squamous lesions from SIL, but it is less helpful in distinguishing low- and high-grade SILs. Parabasal cells of normal cervical squamous epithelium express MIB-1, as do parabasal cells of immature squamous metaplasia, atrophy, and transitional cell metaplasia (Fig. 18-5). In well-developed HSIL, full-thickness MIB-1 expression occurs. Lesser degrees of dysplasia will also demonstrate MIB-1 expression in cells above the basal layer to varying degrees (Fig. 18-6).54-57 Proper tissue orientation is critical to interpreting MIB-1, because it depends on understanding the relationship between the basal layer and upper layers of epithelium. Tangential sectioning, suboptimal orientation, or artifactual loss of the outer layer of epithelium may lead to difficulty in determining whether an MIB1-positive cell is part of the basal layer or some higher layer. Also, p16 expression is seen in most high-risk HPV– associated squamous lesions2,5,58; absent or occasional focal weak expression is seen in normal, inflamed, and atrophic cervical epithelium and in transitional cell metaplasia. In HSIL, diffuse, strong p16 staining of nuclei and cytoplasm occurs from the upper two thirds to the entire full thickness of the epithelium, although not all cases are positive (Fig. 18-7).2,5,59 In LSIL, p16 expression is variable and is typically limited to the lower half of the epithelium. Positivity of p16 in LSIL appears to be a function of whether high-risk HPV is present, and it has been suggested that it may be a marker of progression to HSIL.2,5,58,59 Immunoexpression of p16 has been studied in comparison to in situ hybridization (ISH) assays for high-risk HPV. Whereas HPV ISH exhibits excellent specificity and may be useful to further evaluate cases of focal p16 positivity, diffuse, strong p16 immunoexpression appears to be a more sensitive marker and, in conjunction with its wider availability and easier interpretation, it is better suited as a first step in evaluating questionable morphology.60
Vulva, Vagina, and Cervix
A
B
C
D
E
F
659
Figure 18-5 Molecular immunology Borstel 1 (MIB-1) expression is confined to the parabasal cells of normal cervix (A and B), postmenopausal atrophy (C and D), and inflamed squamous metaplasia (E and F).
Recent studies suggest potential value for novel markers of cell-cycle regulation, including minichromosome maintenance protein 2 (MCM2) and DNA topoisomerase II alpha (topo-2α), which are involved in early steps of DNA replication and are overexpressed in HPV infection. ProExC is a cocktail of antibodies against MCM2 and topo-2α. Although initial studies show promising sensitivity and specificity for LSIL and HSIL,61,62 further studies are warranted.
KEY DIAGNOSTIC POINTS Cervical Squamous Intraepithelial Lesions • MIB-1 expression in the upper layers of epithelium may distinguish HSILs from benign mimics, as does diffuse p16 expression. • Tangential sectioning or loss of the outer layer of epithelium may make interpretation of MIB-1 difficult, because it is expressed in the parabasal cells of benign cervical epithelium.
660
Immunohistology of the Female Genital Tract
A
B
C
D
Figure 18-6 MIB-1 is expressed variably in the middle and upper layers of cervical low-grade squamous intraepithelial lesions (A and B) and diffusely throughout the full thickness of cervical high-grade squamous intraepithelial lesions (C and D). The superficial layer may be spared.
ENDOCERVICAL ADENOCARCINOMA IN SITU
Two diagnostic issues arise with endocervical adenocarcinoma in situ: distinguishing it from benign mimics and assessing for stromal invasion. IHC can help with the former but not the latter. The constellation of nuclear
enlargement, hyperchromasia, crowding, atypia, mitoses, and cribriform growth define endocervical adenocarcinoma in situ. Tubal metaplasia, microglandular hyperplasia, endometriosis, and inflammatory changes may sometimes harbor one or more of these features. Endocervical adenocarcinoma in situ expresses increased MIB-1 and p16 (diffuse and strong), but not ER, PR, or vimentin (Fig. 18-8). Monoclonal CEA (mCEA) has also been used in this distinction, but it adds little additional information beyond that of p16/HPV in situ, ER/ PR, and vimentin. Tubal metaplasia and endometriosis show cytoplasmic Bcl-2 but no increase in MIB-1 or CEA; p16 is either negative or focal and weak. Microglandular hyperplasia also lacks increased MIB-1, p16, and CEA.3,6,7,63-66
KEY DIAGNOSTIC POINTS Endocervical AIS Figure 18-7 In high-grade squamous intraepithelial lesions, p16 shows diffuse, strong nuclear and cytoplasmic expression throughout the lesion.
• Endocervical AIS is distinguished from endocervical glandular metaplasia, hyperplasia, and endometriosis by increased MIB-1 expression and by strong diffuse p16 expression.
Vulva, Vagina, and Cervix
A
C
INVASIVE ENDOCERVICAL ADENOCARCINOMA
The main diagnostic role for IHC in invasive endocervical adenocarcinoma is distinguishing it from endometrial adenocarcinoma. No single immunostain is accurate, and even a panel of several immunostains is not perfect. Correlation with clinical and radiologic findings is paramount. Generally, endocervical adenocarcinoma expresses p16 but does not express vimentin, ER, or PR, whereas low-grade endometrial endometrioid adenocarcinoma shows the converse profile. In addition, p16 should exhibit a diffuse, strong nuclear and cytoplasmic pattern to qualify as positive for endocervical adenocarcinoma (Fig. 18-9); in fact, p16 can also be expressed in endometrioid adenocarcinoma, sometimes in a weak, patchy pattern but occasionally in a strong, diffuse pattern.67-71 Because of this potential overlap, and because of the importance of staining pattern and intensity, caution is advised in interpreting small samplings. In uterine serous carcinoma, p16 is also positive, although the morphology of serous carcinoma typically allows for distinction from endocervical adenocarcinoma.72 Rarely, endocervical microglandular hyperplasia may mimic endometrial mucinous adenocarcinoma; increased MIB-1 and positive vimentin may distinguish the latter from the former. Both entities have variable ER and PR expression, therefore these stains are not helpful.73
661
B
Figure 18-8 A, Endocervical adenocarcinoma in situ may mimic reactive, inflamed surface epithelium (hematoxylin and eosin); however, it is distinguished by diffuse molecular immunology Borstel 1 (MIB-1) expression (B) and diffuse p16 expression (C). These markers highlight the typical abrupt transition between normal and neoplastic epithelium.
KEY DIAGNOSTIC POINTS Invasive Endocervical Adenocarcinoma • Primary endocervical adenocarcinoma is usually positive for p16 and negative for vimentin and estrogen receptors, whereas primary endometrial adenocarcinoma is usually the converse. • Overlap exists in this profile, therefore clinical and radiologic findings should be the ultimate indicator of origin of adenocarcinoma involving the cervix.
INTESTINAL-TYPE ENDOCERVICAL ADENOCARCINOMA
Intestinal-type endocervical adenocarcinoma and adenocarcinoma in situ (AIS) contain goblet cells and intestinal-type epithelium and may be mistaken for spread of primary intestinal adenocarcinoma. The intestinal marker CDX-2 is expressed in intestinal-type endocervical adenocarcinoma, AIS, and in some nonintestinal types (Fig. 18-10). Therefore CDX-2 does not help define site of origin. Instead, CK7, CK20, and p16 should be used. Whereas primary endocervical adenocarcinoma expresses CK7 and p16 but not CK20, the converse applies to primary colorectal adenocarcinoma involving the cervix.74,75
662
Immunohistology of the Female Genital Tract
A
B
C
D
Figure 18-9 A, Primary endocervical adenocarcinoma may mimic primary endometrial endometrioid adenocarcinoma (hematoxylin and eosin). Monoclonal carcinoembryonic antigen (B) and p16 (C) are diffusely and strongly expressed. Estrogen receptor (D) and vimentin (E) are not expressed, although the stroma may be vimentin positive. Primary endometrial adenocarcinoma typically exhibits the converse profile for these four markers, although overlap may occur in some cases.
MINIMAL DEVIATION ENDOCERVICAL ADENOCARCINOMA
Diagnosis of minimal deviation endocervical adenocarcinoma can be challenging, because the cytologic and architectural features of this tumor can be subtle. Robust forms of lobular or diffuse endocervical glandular hyperplasia or deep endocervical glands may mimic minimal deviation adenocarcinoma. A subtle type of endocervical spread of endometrial adenocarcinoma may also mimic this tumor.
E
Immunostains are of limited value. CEA can be positive in some, but not all, minimal deviation adenocarcinoma (Fig. 18-11), as can MIB-1 and p53; therefore the stains are not informative if negative; and p16 is not helpful, because most cases are not HPV associated.63,76-78 ADENOID CYSTIC CARCINOMA
This tumor is similar to its counterpart in salivary glands in both morphology and immunophenotype, and it may
Vulva, Vagina, and Cervix
A
663
B
Figure 18-10 A, Intestinal-type endocervical adenocarcinoma with prominent goblet cells (hematoxylin and eosin) may resemble primary intestinal adenocarcinoma with spread to the cervix. B, CDX-2 is expressed in the nuclei of this variant of endocervical adenocarcinoma and therefore should not be used to determine site of origin. Cytokeratins 7 and 20 and p16 should be used instead.
arise in the cervix or vulvovaginal soft tissue. It is a dual-cell population tumor that grows in cribriform, tubular, and/or solid patterns. The major cell type is a basaloid polygonal cell with modified myoepithelial features, and it is positive for p63 and smooth muscle actin (SMA). The minor cell type is an epithelial cell that forms tiny ductules that may be inconspicuous. These cells express keratin and CD117. Distinction from basaloid squamous carcinoma is based on the distinct morphology of adenoid cystic carcinoma (ACC), because overlap exists in immunostains: p63 is diffusely expressed in basaloid squamous carcinoma, as opposed to ACC, in which p63 is confined to basaloid cells and not epithelium of the ductules; CD117 can be expressed at low levels in basaloid squamous carcinoma.79-83
A
ADENOID BASAL CARCINOMA
This uncommon tumor of bland basaloid cells without mitoses is arranged in palisaded clusters and nests, and it grows in an infiltrative, scattered pattern in the cervical stroma. Squamous metaplasia can occur. The tumor may coexist with other tumors such as squamous cell carcinoma (SCC) or, rarely, neuroendocrine carcinoma. The basaloid cells express keratin, p63, and p16 (Fig. 18-12).84-86 NEUROENDOCRINE CARCINOMAS
Neuroendocrine carcinoma of the cervix is an aggressive tumor that should be distinguished from small cell
B
Figure 18-11 A, Minimal deviation endocervical adenocarcinoma consists of a deeply invasive, haphazardly infiltrative proliferation of bland endocervical glands that, in a small tissue sampling, may mimic endocervical glandular hyperplasia or mesonephric remnants (hematoxylin and eosin). B, Some cases may express carcinoembryonic antigen, which is not expressed by its benign mimics.
664
Immunohistology of the Female Genital Tract
A
B
C
Figure 18-12 A, Adenoid basal carcinoma exhibiting palisaded uniform basaloid cells growing in packed clusters with focal squamous metaplasia (hematoxylin and eosin). Basaloid cells express p16 (B) and p63 (C).
squamous carcinoma and lymphoma. Although morphologic features are often sufficient, IHC can help if the sample is small or questionable. Most small cell carcinomas will express at least one neuroendocrine marker, such as synaptophysin or chromogranin; CD56 is not specific (Fig. 18-13).87,88 Because p16 tends to be present as well, this may create diagnostic problems in distinguishing small cell carcinoma from small cell SCC.89-91 In such cases, the useful marker is p63, which is negative in small cell carcinoma.92 A similar immunophenotype is reported for large cell neuroendocrine carcinoma. Thyroid transcription factor 1 (TTF-1) can be positive in primary cervical neuroendocrine tumors, therefore it is not necessarily a marker of pulmonary origin.93,94
Uterus Immunohistochemical Markers EPITHELIAL MARKERS: CYTOKERATINS AND EPITHELIAL MEMBRANE ANTIGEN
Expression of cytokeratins AE1/AE3 and CAM5.2 is usually sufficient to confirm epithelial differentiation in tumors of most organ systems. However, in the uterus, cytokeratin expression is not limited to epithelial cells. Both endometrial stromal and smooth muscle cells have shown cytokeratin expression, although much more weakly than in epithelial cells.95-101
Vulva, Vagina, and Cervix
A
B
D
E
665
C
Figure 18-13 Small cell carcinoma of cervix (A, hematoxylin and eosin) shows variable expression of chromogranin (B), synaptophysin (C), keratin (D), and strong p16 (E). Typically, at least one neuroendocrine marker will be positive, but not always; characteristic morphology can be diagnostic in such cases.
666
Immunohistology of the Female Genital Tract
EMA is usually not expressed in these tumors.99-101 Cytokeratin expression can be particularly evident in the sarcomatous portion of carcinosarcomas (malignant mixed müllerian tumors), which frequently coexpresses cytokeratins in addition to mesenchymal markers.102,103 Other commonly used epithelial markers include CK7 and CK20. PAX8
PAX8 is expressed in müllerian tract, thyroid, parathyroid, renal, and thymic carcinomas.104 It is useful in distinguishing müllerian tract carcinomas from metastases, particularly breast, colon, and lung carcinomas. PAX8 is most sensitive for serous carcinomas (95%) but is also strongly expressed in endometrioid (75%), clear cell (80%), and mucinous tumors (60%). Expression is nuclear and is typically strong and diffuse in müllerian tumors; focal, weak expression is not specific, because this degree of staining may be seen in a variety of other neoplasms. VIMENTIN
Vimentin, an intermediate filament, is expressed in mesenchymal tissues, normal proliferative endometrial epithelial cells, and in the majority of endometrial carcinomas.105,106 The coexpression of vimentin in a basolateral membrane pattern and can aid in the differential diagnosis of an endocervical versus an endometrial adenocarcinoma. p53
Overexpression of the p53 tumor suppressor protein is seen in more than 80% of uterine serous carcinomas and in their putative precursor lesion, endometrial intraepithelial carcinoma (EIC).107-109 The most commonly used antibodies against the p53 protein recognize both mutated and wild-type p53, but only mutated p53 gene products result in diffuse and strong overexpression of p53 in the setting of serous endometrial carcinoma. An IHC assay for p53 allows us to recognize the abnormal p53 protein that results from p53 gene mutation, because it accumulates in tumor cell nuclei, likely because of decreased degradation and/or stabilization. The pattern of staining is dramatic: more than 75% of the tumor cells stain with strong intensity, and an abrupt cutoff is apparent with the adjacent uninvolved atrophic endometrium. Complex atypical hyperplasia and International Federation of Gynecology and Obstetrics (FIGO) grade 1 endometrioid adenocarcinomas rarely demonstrate p53 immunoreactivity, and when present, the pattern is weak and focal.110-113 Increasing expression of p53 is seen in endometrioid adenocarcinomas with increasing grade, and some FIGO grade 3 endometrioid adenocarcinomas will stain intensely.110,112-114 Recent work has also been done to define markers that could act as a counterbalance to p53. These include antibodies against antigens associated with endometrioid differentiation: ER, PR,112,115-117 β-catenin,118,119 and PTEN.120-125 ER and PR expression is seen in a wide range of nonuterine tissues and in both benign and malignant
tumors. Of note, ER and PR expression is moderate to strong in endometrioid carcinomas, but it is rarely expressed in clear cell carcinomas.126-128 Serous carcinomas may express ER and, less commonly, PR; these markers are not useful in distinguishing serous from endometrioid carcinoma. ER and PR expression is just as weak in poorly differentiated endometrioid carcinomas, as it is in serous and clear cell carcinomas.115-117,128 β-CATENIN AND PTEN
β-catenin is involved in cell adhesion and, as a component of the Wnt signal transduction pathway, it becomes translocated to the nucleus when mutated or stabilized by another factor. Nuclear β-catenin expression is seen in as many as 50% of endometrioid adenocarcinomas119,125,129-131 and is rarely, if ever, seen in serous carcinomas.119,125 PTEN, the phosphatase and tensin homolog deleted on chromosome 10, is a tumor suppressor gene involved in the genesis of 40% to 75% of endometrioid adenocarcinomas.120-125,132 In contrast to p53, in which expression is upregulated in most cases of p53 mutation, PTEN mutation results in IHC loss compared with normal tissues. PTEN loss has only rarely been documented in serous carcinomas.120-125,132 MUSCLE MARKERS
The muscle actins (muscle-specific actin [MSA] and smooth muscle–specific actin [SMSA]), desmin, and h-caldesmon are useful in identifying smooth muscle cells. Normal endometrial stromal cells express vimentin and muscle actins, but they generally lack expression of cytokeratins and EMA.100 Desmin expression can occur in normal endometrial stromal cells and endometrial stromal neoplasms, but it tends to be significantly decreased relative to CD10, which is typically strong and diffuse.101,133,134 Heavy caldesmon (h-caldesmon) is a more specific marker of smooth muscle differentiation than MSA, SMSA, and desmin and has been used to discriminate between endometrial stromal neoplasms and those showing smooth muscle differentiation.135,136 In addition to other markers, h-caldesmon can also be expressed in portions of endometrial stromal neoplasms that show smooth muscle differentiation. Sex-cord–like elements in uterine mesenchymal tumors show IHC evidence of sex-cord differentiation with inhibin, a peptide hormone normally expressed by ovarian granulosa cells,137 but they also frequently express SMA and sometimes desmin.137-140 Antidesmin antibodies and, more specifically, myogenin and MyoD1, can be used to highlight rhabdomyoblastic differentiation in carcinosarcomas. However, in the absence of malignant heterologous elements, distinction of carcinosarcoma from poorly differentiated carcinoma by using IHC is frequently impossible, because their immunophenotypes overlap.141 CD10
CD10, also known as common acute lymphoblastic leukemia antigen, is now recognized to mark neoplastic and nonneoplastic endometrial stromal cells.139,142,143 Potential uses therefore fall into two categories: distinguishing
Uterus
adenomyosis from invasive endometrial cancer and distinguishing endometrial stromal neoplasms from smooth muscle neoplasms. There are many complexities, however, with the use of antibodies against CD10. These include frequent expression in myometrium that surrounds invading endometrial cancer cells, expression in occasional smooth muscle neoplasms, and loss of expression in endometrial stromal neoplasms with divergent differentiation. WILMS TUMOR 1
The Wilms tumor suppressor gene product, WT1, is expressed in endometrial stroma and endometrial stromal neoplasms as well as in smooth muscle tumors. WT1 immunoreactivity does not discriminate between these two categories of tumors.144
Endometrial Carcinoma ENDOMETRIOID ADENOCARCINOMA
Endometrioid adenocarcinoma is a neoplasm that recapitulates nonneoplastic endometrium in most cases (Table 18-2). That is, the tumor is typically formed of endometrioid tubules, at least focally, and it demonstrates the same range of cytoplasmic changes and metaplasias seen in nonneoplastic endometrium. In general, the immunophenotype is similar to that of its precursor, complex atypical hyperplasia. As with other common adenocarcinomas, endometrioid adenocarcinoma expresses pancytokeratins, EMA, and the glycoprotein-associated markers carbohydrate antigen 125 (CA 125), BerEP4,145 and B72.3,146,147 among others. Expression of CEA, which is uncommon, is almost always limited to apical membranes, although tumors that show extensive mucinous differentiation may express this antigen more diffusely.68-70,105,128,148 Much less striking CEA expression is seen in endometrioid carcinoma when compared with endocervical, pulmonary, and gastrointestinal (GI) carcinomas. Most endometrioid
TABLE 18-2 Key Differential Diagnosis: Primary Uterine Carcinomas
Endometrioid
ER/PR
p53, p16 (Overexpression)
MIB-1 (% Tumor Cells Positive)
+*
R
R*
Serous
S
+
+
Clear cell
R
R
S
*Except in International Federation of Gynecology and Obstetrics (FIGO) grade 3 endometrioid adenocarcinoma, about 50% of which express significant estrogen receptor (ER) and progesterone receptor (PR); these also tend to be more proliferative than grade 1 and 2 carcinomas and may express p53 and p16. +, Almost always positive (or MIB-1 high); S, sometimes positive (or MIB-1 intermediate); R, rarely positive (or MIB-1 low); −, negative. MIB-1, Molecular immunology Borstel 1.
667
adenocarcinomas express LMWCK, and those that show squamous differentiation or morules express HMWCK as well.149 Nearly all endometrioid adenocarcinomas are CK7 positive and CK20 negative.68,150 Occasional mucinous varieties express CDX-2.151,152 Unusually for adenocarcinomas, endometrioid tumors are well known for their frequently strong, predominantly membranous expression of vimentin.68,153,154 Recent advances in our understanding of the molecular pathogenesis of endometrioid adenocarcinoma have led to the study of proteins featured in the relevant pathways (Fig. 18-14). These include ER, PR, p53, β-catenin, p16, PTEN, and the DNA mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2. The preponderance of FIGO grade 1 and 2 endometrioid adenocarcinomas express ER and PR, and approximately 50% of FIGO grade 3 endometrioid adenocarcinomas express these markers as well.112,117,125,127,128,155 Overexpression of p53 (intense expression in greater than 75% of tumor cell nuclei) resulting from p53 mutation and accumulation of mutant p53 protein is encountered in a minority of FIGO grade 2 and 3 adenocarcinomas.107-109,112-114,125,126 That is not to say, however, that p53 expression—as opposed to overexpression, defined previously—is uncommon in endometrioid carcinomas; in fact, it is very common to find weak expression in less than 50% of endometrioid carcinoma tumor cell nuclei. As a consequence of CTNNB1 mutation, β-catenin expression in tumor cell nuclei and cytoplasm, as opposed to cell membranes alone, is found in perhaps 33% of FIGO grade 1 and 2 adenocarcinomas, especially those showing squamous or morular metaplasia.119,125 Occasionally, p16 is expressed in endometrioid carcinomas, and this appears related to grade; a minority of grade 1 and 2 adenocarcinomas express p16 in scattered tumor cells, whereas more diffuse and intense staining is seen in occasional grade 3 adenocarcinomas.128,156 A diffuse, strong “every cell” expression pattern is exceptional in endometrioid adenocarcinomas; PTEN is frequently mutated, and expression of this gene is sometimes silenced via hypermethylation of its promoter.120-125,132 Detection of these abnormalities with IHC is challenging.125,157 Although imperfect, the 6H2.1 antibody appears to allow recognition of PTEN protein loss; correct interpretation of PTEN immunostains involves the identification of an intact positive internal control, the cytoplasm and sometimes nuclei of endometrial stroma and nonneoplastic endometrial glands, and expression loss in at least 90% of tumor cells. DNA MMR proteins are found to be deficient in tumor cell nuclei by IHC in up to 33% of endometrioid adenocarcinomas;158-161 this results from MLH1 promoter hypermethylation in most cases or in mutation of MLH1, MSH2, MSH6, or PMS2 in the remaining cases. Interpreting DNA MMR protein deficiency by IHC requires complete expression loss in the setting of a valid positive internal control. Valid internal controls include nonneoplastic endometrial stroma and glands or infiltrating lymphocytes with reproducibly stained nuclei. In most instances of DNA MMR protein deficiency, loss of expression occurs in couplets (MLH1 with PMS2 and MSH2 with MSH6). A more detailed discussion of ER
668
Immunohistology of the Female Genital Tract
A
B
C
D
Figure 18-14 Markers of emerging importance in distinguishing uterine endometrioid carcinoma and uterine serous carcinoma include p53, β-catenin, and PTEN. Although serous carcinomas characteristically overexpress p53 (A), endometrioid adenocarcinomas, especially when gland forming, frequently express estrogen receptors (B), commonly show at least focal nuclear and cytoplasmic staining with anti–βcatenin (C), and lose expression of PTEN (D).
and PR, p53, and DNA MMR can be found later in this chapter.
KEY DIAGNOSTIC POINTS Endometrioid Carcinomas • Endometrioid carcinomas typically express CK7, CA 125, estrogen receptors, progesterone receptors, and vimentin, and they are usually negative for CK20. • Overexpression of p53 and p16 is only occasionally seen. • Nuclear β-catenin expression and loss of PTEN and DNA MMR proteins are seen in significant minorities.
ENDOMETRIAL SEROUS CARCINOMA
It is easy to recognize endometrial serous carcinoma when it is tumor forming and present in its characteristic papillary form with bizarre tumor cell nuclei. However, endometrial serous carcinoma can be difficult
to diagnose correctly when 1) glandular, clear cell, or solid forms predominate; 2) the tumor is admixed with an endometrioid or clear cell neoplasm; 3) tumor is intraepithelial and apparently confined to an endometrial polyp; or 4) uncertainty exists regarding the possibility of ovarian, tubal, or peritoneal primaries. To further complicate matters, the morphologic and immunophenotypic threshold at which serous and endometrioid carcinomas can be meaningfully and reproducibly separated is currently being debated. Despite important immunophenotypic differences between serous, clear cell, and endometrioid carcinomas of the endometrium, discussed subsequently, these tumors share notable similarities. As with endometrioid carcinomas, endometrial serous carcinomas commonly express pancytokeratins, EMA, CA 125, BerEP4, B72.3, CK7, and vimentin, and they are usually negative for CK20 and lack diffuse, strong cytoplasmic expression of CEA. The immunophenotype of endometrial serous carcinoma resembles that of its putative precursor, intraepithelial serous carcinoma (endometrial intraepithelial carcinoma).107,162-165
Uterus
It is not surprising that the expression of proteins discussed in reference to molecular pathways essential to endometrioid carcinogenesis differs significantly in serous carcinoma (Figs. 18-15 and 18-16; see Fig. 18-14 also). Approximately 90% of endometrial serous carcinomas show p53 overexpression (intense expression in greater than 75% of tumor cell nuclei) as a result of p53 mutation and the consequent accumulation of mutant protein.107-109,113,114 The majority of remaining tumors, most of which show absolutely no p53 expression, harbor p53 mutations that result in a truncated p53 protein or a protein with conformational changes that
669
cannot be detected by using commercially available antibodies.108 Proliferative activity, approximated with an MIB-1 labeling index, is extremely high, with rates that mimic p53 overexpression patterns (i.e., >75% of tumor cell nuclei).112 Although typical serous carcinoma were once considered to lack diffuse ER and PR expression,112,115-117,125 the newer hormone receptor antibodies have demonstrated significant expression of ER and, to a lesser extent, PR in uterine serous carcinoma. In addition, carcinomas with hybrid endometrioid/serous features and admixtures of endometrioid and serous components may express considerable amounts of ER.166 PR is
A
B
C
D
E
F
Figure 18-15 MIB-1 and p53 may be useful for distinguishing endometrial surface metaplastic and degenerative changes from endometrial intraepithelial carcinoma (A and C), which contrasts with metaplastic-degenerative change (D and F) by virtue of uniform and intense nuclear expression for p53 (B vs. E) and MIB-1 (C vs. F).
670
Immunohistology of the Female Genital Tract
A
B
C
D
E
F
Figure 18-16 Immunohistochemical staining for p53 can be used when the differential diagnosis includes uterine serous carcinoma (USC) and uterine endometrioid carcinoma (UEC). USC shows diffuse and intense nuclear immunoreactivity for p53 (A and B), as does the USC precursor, endometrial intraepithelial carcinoma (C and D). Not infrequently, USC can demonstrate a glandular architectural pattern (E). Diffuse and intense p53 immunoreactivity in glandular USC (F) can provide support for this entity when simple atypical hyperplasia and endometrioid adenocarcinoma are considerations.
expressed less frequently than ER, and p16 is almost always highly expressed in an every-cell pattern.128,156 In contrast to most endocervical carcinomas, this does not imply HPV infection; rather, it may reflect disturbances in the cell cycle that favor hyperproliferative activity. Abnormalities that involve the CTNNB1 (β-catenin) and PTEN pathways are very rare in endometrial serous carcinomas; this means that nuclear β-catenin expression is almost never encountered.
Finally, it should be noted that although many serous adenocarcinomas of endometrium resemble ovarian serous carcinomas, there are some important differences. The most important of these is infrequent WT1 expression in endometrial serous carcinomas (seen in at most 20% to 30% of such cases) and the very common diffuse nuclear expression of WT1 in ovarian, tubal, and primary peritoneal examples (at least 70% to 80% of such cases).167-169
Uterus
KEY DIAGNOSTIC POINTS Endometrial Serous Carcinoma • Serous carcinomas express CK7, CA 125, and vimentin, whereas they are usually negative for CK20, just like endometrioid carcinomas. • Unlike endometrioid carcinomas, endometrial serous carcinomas typically overexpress p53 and p16 and show extremely high proliferative indices with MIB-1; most examples have low-level expression of estrogen receptors and low-level or absent progesterone receptors.
CLEAR CELL CARCINOMA
Clear cell carcinoma is increasingly strictly defined, which means that endometrioid and serous carcinomas composed of cells that show cytoplasmic clearing should not be diagnosed as clear cell carcinomas.170 As with endometrioid and serous carcinomas, clear cell carcinomas usually express pancytokeratins, EMA, CA 125, BerEP4, B72.3, CK7, and vimentin, whereas they are usually negative for CK20 and WT1. Clear cell carcinomas are typically ER and PR negative and show rates of p53, p16, and MIB-1 expression that are intermediate between endometrioid and serous carcinomas.126-128 Although it is conceivable that some clear cell carcinomas might show abnormalities in CTNNB1 (β-catenin) and PTEN pathways,171,172
KEY DIAGNOSTIC POINTS Clear Cell Carcinomas • Clear cell carcinomas express CK7, CA 125, and vimentin, and they are usually negative for CK20, just like endometrioid and serous carcinomas. • Overexpression of p53 and p16 is uncommon; unlike typical endometrioid carcinomas, estrogen and progesterone receptor expression is low or absent.
671
insufficient numbers of well-characterized cases have been studied, and it is thought that abnormalities are largely lacking. Hepatocyte nuclear factor 1β (HNF-1β) is expressed in the majority of endometrial clear cell carcinomas, but it is also expressed in secretory and gestational endometria, endometrial carcinoma with secretory change, and in endometriosis.104,173-178 OTHER HISTOLOGIC SUBTYPES OF ENDOMETRIAL CARCINOMA AND SECONDARY CARCINOMAS, INCLUDING METASTASES
Endometrial carcinomas with mucinous, squamous, neuroendocrine, and undifferentiated features have been described (Table 18-3), but information about the immunophenotype of squamous cell and small cell carcinomas (excluding case reports and small series) is limited. Data that concern the immunophenotype of mucinous carcinoma center mainly on its distinction from microglandular hyperplasia and endocervical adenocarcinoma, which were discussed earlier in this chapter. NEUROENDOCRINE CARCINOMAS
Extrapolating data from the cervical and pulmonary literature, assorted case reports, and reviews,179-181 it appears that small cell and large cell neuroendocrine carcinomas that involve endometrium express chromogranin, synaptophysin, neuron-specific enolase (NSE), and CD56 in a significant number of cases. The extent to which these endometrial neuroendocrine carcinomas can express TTF-1 and p16 has not been studied extensively.181-183 Conventional endometrioid carcinomas can contain significant subpopulations of neuroendocrine cells, which can be a pitfall when using IHC alone to diagnose small cell and large cell neuroendocrine carcinomas. Significant neuroendocrine marker expression (e.g., greater than 20% of tumor cells) should be present before an undifferentiated neoplasm is classified as definitively neuroendocrine, especially when it
TABLE 18-3 Key Differential Diagnosis: Adenocarcinomas Involving the Uterus ER/PR
WT1
CK20
Pax-8
TTF-1
Endometrioid of endometrium
+†
−
−
+
−‡
Serous of endometrium
R
R
−
+
−‡
Serous of ovary*
S
+
−
+
−‡
Endocervix
−
−
−
+
−‡
Breast
S
−‡
−
−
−
Colorectal
−
−
+
−
−
Pulmonary
−
−
−
−
+
‡
‡
‡
*Includes ovarian, peritoneal, and tubal serous carcinomas. † Except in International Federation of Gynecology and Obstetrics (FIGO) grade 3 endometrioid adenocarcinoma, about 50% of which express significant ER and PR. ‡ Rare examples have been reported to be positive. +, Almost always positive; S, sometimes positive; R, rarely positive; −, negative. CK, Cytokeratin; ER/PR, estrogen receptor/progesterone receptor; TTF-1, thyroid transcription factor 1; WT1, Wilms tumor 1.
672
Immunohistology of the Female Genital Tract
does not exhibit compelling features of neuroendocrine differentiation on an H&E-stained slide. UNDIFFERENTIATED CARCINOMAS
The immunophenotype of undifferentiated and so-called dedifferentiated endometrial carcinomas has been explored in recent publications.184,185 It should be noted, however, that these tumors probably constitute only one clinical and morphologic manifestation of undifferentiation. Other varieties undoubtedly exist. Compared with typical endometrioid adenocarcinomas, undifferentiated carcinomas tend to show relative loss of cytokeratin, ER, and PR expression when juxtaposed with the typical endometrioid carcinoma. EMA expression, however, may be retained, at least partially, in the undifferentiated endometrial carcinoma. A disproportionate number of undifferentiated and dedifferentiated endometrial carcinomas have been reported to show abnormal expression of DNA MMR proteins.186 MIXED EPITHELIAL CARCINOMA
Mixed epithelial carcinoma (i.e., mixed endometrioid and serous carcinoma) can be diagnosed when at least two histologically distinctive elements are present, and each constitutes at least 10% of the tumor. The elements should be obvious, separable, and characteristic to diagnose a mixed tumor. When performed, IHC stains should demonstrate that the immunophenotypes of each component are distinctive as well. Mixed epithelial carcinomas should not be diagnosed when the overall morphology is a hybrid of features generally encountered in different endometrial cancer subtypes.187,188 METASTATIC CARCINOMAS
Data regarding the sources of secondary or metastatic carcinomas to the endometrium are limited, but it is estimated that lobular breast cancers, high-grade serous ovarian and tubal carcinomas, HPV-associated endocervical adenocarcinomas, and typical colorectal adenocarcinomas probably constitute at least 90% of such cases.
Endometrial Carcinoma Staging
therefore absent CD10 staining does not entirely exclude the presence of endometrial stroma and adenomyosis. LYMPHOVASCULAR INVASION
A number of studies have correlated lymphovascular invasion (LVI) with lymph node metastasis, but LVI is not an independent indicator of prognosis because of the extent to which it associates with other features of high-risk endometrial cancer. Although assessment of LVI is not required for stage assignment, a surgical pathology report that lacks this information is generally considered incomplete. Vascular endothelial markers such as CD34, CD31, and ERG and the lymphatic endothelial marker podoplanin (D2-40) have been used to verify the presence of a vascular space that contains a tumor embolus, but the contribution of these tests with respect to their association with lymph node metastases and prognosis has not been extensively studied in endometrial cancer.191-194 IHC for these endothelial markers should therefore be considered essentially untested for diagnostic purposes in endometrial cancer. LYMPH NODE METASTASIS AND SENTINEL LYMPH NODE EVALUATION
Cytokeratin immunostains are occasionally used to detect microscopic deposits of occult carcinoma cells in lymph nodes. Carcinoma that demonstrates the microcystic, elongated, and fragmented (MELF) pattern of myometrial invasion is frequently associated with lymphovascular invasion195 and deposits of carcinoma in lymph nodes that resemble histiocytes—this is an obvious application of the use of keratin stains to confirm epithelial differentiation and the presence of rare, single tumor cells in lymph node sinuses.196 Keratin stains have also been studied in the setting of sentinel lymph node evaluation, but this is currently experimental, and the significance, if any, of isolated tumor cells in lymph nodes in endometrial carcinoma is unknown. Lymph nodes involved by isolated tumor cells should probably not be considered evidence of lymph node involvement for staging purposes.197
MYOMETRIAL INVASION
Synchronous Endometrial and Ovarian, Tubal, and Peritoneal Carcinomas
Although it has been proposed that CD10 expression can help to distinguish invasive endometrial cancer from adenocarcinomas that colonize adenomyosis, this has not proved to be useful in diagnostic practice.189,190 Many invasive adenocarcinomas are surrounded by a rim of tissue that expresses CD10, such that the appearance mimics the endometrial stroma that one expects in adenomyosis. In addition, endometrial stroma that supports endometrial cancer may undergo metaplasia to a smooth muscle or fibroblastic phenotype, similar to the stroma that is characteristic of endometrial polyps. This metaplastic stroma frequently expresses CD10 only weakly or focally,
IHC evaluation is unnecessary for the two categories of endometrial and ovarian tumors that can be recognized as highly likely to be synchronous. The first is a noninvasive or minimally invasive well-differentiated endometrioid carcinoma of endometrium with coincident complex atypical hyperplasia and a well-differentiated endometrioid carcinoma of ovary with coincident endometriosis or endometrioid adenofibromatous tumor but without ovarian surface involvement. The second concerns endometrial and ovarian tumors of obviously different grades and histologic types. At the other end of the spectrum are the obviously metastatic tumors: deeply invasive, high-grade endometrial carcinomas with
Uterus
multiple ovarian tumor nodules, including ovarian surface deposits; the occasional huge ovarian, tubal, or peritoneal carcinoma with multifocal, small “drop metastases” along a cycling endometrium; and an endometrial serous carcinoma that arises in an endometrial polyp with intraepithelial serous carcinoma along with a serous carcinoma that involves ovary, fallopian tube, and peritoneum. It is not debatable that the former two scenarios represent metastasis from one organ to another, but whether the latter situation is an example of metastasis from endometrium to ovary is up for discussion. Both evidence of monoclonality198 and IHC substantiation of endometrial serous differentiation support the idea that the latter situation is one in which an endometrial serous carcinoma metastasized to extrauterine organs.199 In a paper published by MD Anderson,199 the authors studied WT1 IHC in two groups of patients, one with peritoneal carcinomatosis and no endometrial disease and another with peritoneal carcinomatosis and serous carcinomas in endometrial polyps. Peritoneal tumors in the first group were almost always WT1 positive (supporting ovarian, tubal, or peritoneal serous carcinoma), but the endometrial and peritoneal tumors in the second group were almost always WT1 negative (supporting endometrial serous carcinoma). Therefore given the appropriate context, WT1 immunostains can be used to determine the likelihood that a given serous carcinoma is endometrial or extrauterine in origin. However, because as many as 20% to 30% of endometrial serous carcinomas have been reported to be WT1 positive and 20% to 30% of ovarian, tubal, and peritoneal carcinomas have been reported to be WT1 negative, it is imprudent to claim that this test offers unqualified support for one situation over another.167-169,200,201
Uterine Mesenchymal Tumors LEIOMYOMA AND LEIOMYOSARCOMA
Uterine smooth muscle tumors (Tables 18-4 and 18-5), particularly those composed of spindled cells, characteristically express SMA, MSA, desmin, h-caldesmon, and vimentin.135,136 Leiomyosarcomas may also express WT1,202 Bcl-2,203-205 ER, PR,206-209 and CD10.139 Less frequently assayed uterine smooth muscle–associated TABLE 18-4 Key Differential Diagnosis: Primary Uterine Mesenchymal Tumors CD10
Desmin/hCaldesmon
Cytokeratin
Smooth muscle, spindled
R
+
S
Smooth muscle, epithelioid
R
S
S
Endometrial stromal*
+
−
R
*Includes the mesenchymal component of müllerian adenosarcoma without stromal overgrowth. +, Almost always positive; S, sometimes positive; R, rarely positive; −, negative.
673
TABLE 18-5 Key Differential Diagnosis: Epithelioid Uterine Tumors Cytokeratin
Desmin
Inhibin
S-100
Carcinoma
+
−
−
−
Epithelioid smooth muscle
S
S
−
R
Trophoblastic
+
−
S
−
Melanoma
−
R
R
+
+, Almost always positive; S, sometimes positive; R, rarely positive; −, negative.
markers include histone deacetylase 8 (HDAC8)210 and oxytocin receptors.211 Note that cytokeratin expression can be found in up to 25% to 30% of cases, particularly epithelioid varieties,139,212 although it is usually patchy in distribution, whereas up to 25% to 30% of epithelioid and myxoid leiomyosarcomas fail to demonstrate appreciable expression of smooth muscle–associated markers.139 Although leiomyosarcomas express more p53, MIB-1, and p16 than leiomyomas and less Bcl-2, ER, and PR, this IHC panel is not clinically useful, because the results have shown a broad gradient of expression for each of these markers in leiomyomas, atypical smooth muscle tumors (symplastic or bizarre leiomyomas), and leiomyosarcomas instead of sharp cutoff points that facilitate quantitation.204,207,213-217 The mitosis marker antiphosphohistone H3 (PHH3) is emerging as an attractive alternative to MIB-1.218 This marker is detectable specifically during mitotic chromatin condensation and should therefore be preferable to MIB-1 for estimating proliferative activity. It is also useful for separating bizarre nuclear forms and apoptotic bodies from true mitotic figures.217,218 KEY DIAGNOSTIC POINTS Leiomyosarcomas • Uterine leiomyosarcoma is composed of spindle cells that typically express smooth muscle actin and desmin, although desmin expression is sometimes lacking. It may express CD10. • Uterine leiomyosarcomas frequently express cytokeratins in a patchy distribution. • Uterine epithelioid leiomyosarcomas frequently express cytokeratins but may lack desmin expression. • Uterine myxoid leiomyosarcomas frequently fail to express desmin. • Assays for p53, MIB-1, Bcl-2, p16, and estrogen and progesterone receptors are not currently used for diagnostic purposes in this setting.
ENDOMETRIAL STROMAL NODULE AND LOW-GRADE ENDOMETRIAL STROMAL SARCOMA
Endometrial stromal neoplasms, both nodules and sarcomas, almost always express CD10, ER, PR, and
674
Immunohistology of the Female Genital Tract
WT1 (Fig. 18-17).139,142-144,219,220 Many also express β-catenin.221-223 They frequently express SMA,100,101,139,220 cytokeratin,99,220 or androgen receptors in a patchy distribution.224 These tumors only rarely express desmin100,136,225,226 and CD34 (unpublished observation) in varieties in which the constituent cells resemble nonneoplastic proliferative endometrial stroma. Diffuse desmin and h-caldesmon expression supports smooth
muscle differentiation and disqualifies categorization as an endometrial stromal tumor in the absence of strong CD10 expression, except in rare cases. Several endometrial stromal tumor variants have been described (Fig. 18-18). These include the smooth muscle variant, also referred to as mixed endometrial stromal and smooth muscle tumor or stromomyoma134,226; fibromyxoid variant133,226; sex cord variant; and variants
A
B
C
D
E
Figure 18-17 Endometrial stromal tumors (nodules and sarcomas) and cellular leiomyomas can show significant morphologic similarity. Although desmin reactivity can frequently distinguish these entities, h-caldesmon antibody appears to be more specific for smooth muscle differentiation. A shows an endometrial stromal sarcoma that is not immunoreactive for h-caldesmon (B). Endometrial stromal sarcomas nearly always express CD10 (C), estrogen receptor (D), and progesterone receptor (PR). Several histologic mimics of metastatic endometrial stromal sarcoma, including solitary fibrous tumor (SFT), can also express PR and CD10 (E). Although endometrial stromal sarcomas tend to express CD10 strongly and diffusely, many express it in a patchy pattern, which overlaps with that seen in SFT. C to E, From Bhargava R, Shia J, Hummer AJ, et al: Distinction of endometrial stromal sarcomas from “hemangiopericytomatous” tumors using a panel of immunohistochemical stains. Mod Pathol 2005;18:40-47.
Uterus
A
675
B
Figure 18-18 A, Immunohistochemistry can be used to evaluate endometrial stromal neoplasms with smooth muscle differentiation. Areas that resemble endometrial stroma (left) contrast with areas that resemble smooth muscle (right). CD10 marks endometrial stromal cells, whereas muscle markers, such as h-caldesmon, mark zones that show smooth muscle differentiation. B, Endometrial stromal neoplasms are generally diagnosed when conventional-appearing endometrial stroma is present; the diagnosis should not be abandoned solely because of divergent differentiation, such as the smooth muscle differentiation illustrated here.
that include endometrioid glands or epithelioid cells.227,228 The key to understanding the immunophenotype of these tumors is that variant or metaplastic elements often lose the phenotype of endometrial stroma and acquire the phenotype of the corresponding metaplastic element.226 Therefore the smooth muscle in a smooth muscle variant of endometrial stromal sarcoma expresses muscle markers (see Fig. 18-18, B) and is frequently CD10 negative.139,226 The endometrioid glands in a low-grade endometrial stromal sarcoma, glandular variant, will express epithelial markers such as EMA. Similarly, the sex cord component of an endometrial stromal nodule, sex cord variant, might express inhibin and lose CD10 expression.137 Hybrid forms also exist that further complicate interpretation. For example, some stromal neoplasms with sex cord features contain elements that coexpress muscle markers and cytokeratins.139 With the exception of the fibroblastic variant, in which CD10 expression can be muted, all other variants should have at least remnants of a proliferation that resemble nonneoplastic proliferative-phase endometrial stroma and retain CD10, ER, and PR expression. A negative CD34 result, particularly in a tumor with fibroblastic features, can be useful in the discrimination of metastatic stromal sarcoma and solitary fibrous tumor, which is CD34 positive.220 KEY DIAGNOSTIC POINTS Endometrial Stromal Tumors • Endometrial stromal tumors are typically positive for CD10 and estrogen and progesterone receptors and are negative or weakly positive for desmin, h-caldesmon, and CD34. • If confronted with a possible endometrial stromal tumor variant, focus on components that resemble proliferative phase endometrial stroma. • Expression of CD10 without h-caldesmon and/or desmin in these areas supports an endometrial stromal component.
CARCINOSARCOMA
The diagnosis of carcinosarcoma (malignant mixed müllerian tumor) requires clearly defined and separable carcinomatous and sarcomatous components. If they are not clearly defined on H&E examination, a diagnosis of carcinosarcoma is difficult or impossible to substantiate. Often, IHC study of an apparently undifferentiated neoplasm, whose differential diagnosis includes carcinoma and sarcoma, reveals a confusing immunophenotype coexpression of epithelial and mesenchymal markers or a complementary expression of both in areas that are morphologically similar.102,103 If geographic zones are present that express cytokeratins exclusively and separate, distinct zones express mesenchymal markers exclusively, it is reasonable to consider a diagnosis of carcinosarcoma, but other patterns of immunoreactivity are probably not informative. The presence of heterologous elements, particularly rhabdomyoblasts, may herald a more aggressive clinical course in surgically staged patients with FIGO stage I carcinosarcoma, but heterologous elements have not been demonstrated to confer worse prognosis in highstage disease.229 A myogenin or MyoD1 immunostain is therefore recommended for establishing skeletal muscle differentiation when it is suspected. Rare case reports exist of carcinosarcomas that harbor yolk sac tumor230 or neuroectodermal elements, including primitive peripheral neuroectodermal tumor.231,232 When it has been studied, the immunophenotype of these components has been similar to that described in tumors outside the context of carcinosarcoma. MÜLLERIAN ADENOSARCOMA
The immunophenotype of this tumor closely parallels that of endometrial stromal neoplasms when stromal overgrowth is not present. The mesenchymal component of müllerian adenosarcomas without stromal
676
Immunohistology of the Female Genital Tract
overgrowth typically expresses ER, PR, androgen receptors, CD10, and WT1, whereas significant minorities also express SMA and even pancytokeratins (Fig. 18-19).206,233-235 Cases that demonstrate stromal overgrowth, particularly when it is associated with highgrade sarcoma, generally lose strong and diffuse ER, PR, CD10, and WT1 expression.235 Tumors that contain heterologous elements exhibit an immunophenotype similar to eutopic tumors, therefore the mesenchymal component of an adenosarcoma with rhabdomyoblastic differentiation would be expected to express desmin, myogenin, and MyoD1. As with carcinosarcoma, recent work suggests that the presence of rhabdomyoblastic differentiation may indicate a poor prognosis.236 The proliferative index, estimated with an MIB-1 immunostain, increases with mitotic index and the presence of sarcomatous stromal overgrowth.235 OTHER MESENCHYMAL TUMORS Undifferentiated Sarcoma
As is the case with undifferentiated carcinomas, undifferentiated sarcomas are an extraordinarily heterogeneous collection of different tumors. Undifferentiated endometrial sarcoma, many examples of which are considered high-grade endometrial stromal sarcoma, is the best known member of this group. Despite this, the criteria for diagnosing this tumor and its immunophenotype are not well understood. Some examples might represent dedifferentiation from a low-grade endometrial stromal sarcoma.223 IHC has been studied in a small series of such cases. CD10 and PR expression were noted to be partially or completely lost when compared with the differentiated components in most cases. Recently, several subtypes of undifferentiated sarcoma have been delineated in the uterine corpus. Should a pleomorphic tumor with diffuse SMA expression be classified as leiomyosarcoma? Should a high-grade spindle cell tumor without diffuse desmin expression be classified as undifferentiated sarcoma? Unfortunately, the criteria that separate this tumor from morphologically similar ones and the answers to these questions are unclear. Regardless, some practical points are worth discussing. Undifferentiated carcinoma (primary or metastatic), leiomyosarcoma, adenosarcoma with stromal overgrowth, melanoma, lymphoma, and leukemia should be considered before a diagnosis of undifferentiated uterine sarcoma is established. An IHC panel that includes antibodies against cytokeratin, EMA, S-100, leukocyte common antigen (LCA), CD43, CD30, and a muscle marker should be helpful with respect to excluding carcinoma, melanoma, lymphoma, and leukemia. Diffuse, strong keratin expression supports carcinoma, and diffuse muscle-marker expression without keratin immunoreactivity supports a sarcoma with a myogenous phenotype. Focal but strong EMA expression in a tumor that closely resembles an undifferentiated carcinoma would also support that entity.184,185 Many undifferentiated carcinomas are composed of a diffuse, sheetlike proliferation of small to intermediate-sized round cells that resemble lymphoma,
plasmacytoma, or rhabdoid tumor; they do not show glandular or squamous differentiation and do not contain pleomorphic spindled cells. If clinical correlation is unsatisfactory, and the immunophenotype is not contributory, a diagnosis of undifferentiated malignant neoplasm should be considered. It is worthwhile to discuss with the contributing gynecologist the extent of IHC workup needed for surgical planning and the consideration of adjuvant therapy. It is usually more efficient to scrutinize the tumor in a very wellsampled hysterectomy specimen than to spend time and resources with IHC on a scant endometrial biopsy specimen. An important exception to this rule is the need to exclude metastasis and lymphoma/leukemia before hysterectomy, if at all possible. Perivascular Epithelioid Cell Tumors
Perivascular epithelioid cell (PEC) tumors, also known as PEComas, have been described to occasionally arise in the uterus237-239 and belong to a group of tumors with histologic appearances that overlap with those of epithelioid smooth muscle tumors, especially those with clear cytoplasm. PEComas are related to other tuberous sclerosis–associated proliferations (e.g., lymphangioleiomyomatosis and angiomyolipoma) and are composed of specialized or hybrid muscle cells that coexpress muscle markers and some of those associated with melanocytic differentiation, namely, HMB-45 and melan-A. Uterine smooth muscle neoplasms can occasionally express HMB-45 and/or melan-A but only in a very restricted distribution.240,241 PEComas, which are frequently but not always desmin negative, tend to show much stronger expression of these melanoma-associated markers. It is unknown whether important clinical differences exist between uterine PEComas and conventional uterine epithelioid smooth muscle tumors. Only rare patients with uterine PEComas have been found to have the tuberous sclerosis complex.237 Uterine Tumor Resembling Ovarian Sex Cord Tumor
As the name implies, uterine tumor resembling ovarian sex cord tumor (UTROSCT) contains elements reminiscent of sertoliform tubules or aggregates of granulosa cells.242 From a morphologic perspective, the differential diagnosis usually concerns an epithelioid smooth muscle tumor, an endometrial stromal tumor with sex cord differentiation, or, rarely, an adenocarcinoma. Tumors that show any appreciable endometrial stromal features are assigned to that category (i.e., endometrial stromal tumor with sex cord–like differentiation) and are not regarded as UTROSCTs, which are polyphenotypic tumors that frequently coexpress cytokeratins, muscle markers, and inhibin.137-140,210 Gastrointestinal Stromal Tumor
Although only one primary uterine GIST has been reported,243 rectal examples may occasionally be mistaken for fibroids. Spindle cell examples would then suggest leiomyoma or perhaps even leiomyosarcoma, and epithelioid varieties could mimic an epithelioid smooth muscle tumor, carcinoma, or even a PEComa or UTROSCT. The successful treatment of GISTs and
Uterus
A
B
C
D
E
F
677
Figure 18-19 The immunophenotype of müllerian adenosarcoma with and without stromal overgrowth differs. Expression of progesterone receptor (A and B) and CD10 (C and D) is significantly more diffuse in adenosarcoma without stromal overgrowth (A to C) than those with stromal overgrowth (B to D). Note that epithelial expression of estrogen receptor is retained in this adenosarcoma with stromal overgrowth (B). Most adenosarcomas without stromal overgrowth show a proliferative index with MIB-1 of less than 5% overall, but the proliferative index is also generally elevated in periglandular cuffs (E). The proliferative index in adenosarcomas with stromal overgrowth almost always exceeds 5% (F). From Soslow RA, Ali A, Oliva E: Mullerian adenosarcomas: an immunophenotypic analysis of 35 cases. Am J Surg Pathol 2008;32:1013-1021.
678
Immunohistology of the Female Genital Tract
chronic myelogenous leukemia with imatinib mesylate has resulted in a great deal of interest in CD117 expression in neoplasms, for which good adjuvant therapy does not exist. In several studies, uterine smooth muscle and endometrial stromal neoplasms were negative or rarely immunoreactive for CD117,139,244,245 although others have shown CD117 positivity in leiomyosarcomas and carcinosarcomas.245-247 Importantly, in one study, IHC expression of CD117 was found in leiomyosarcomas and carcinosarcomas but without activating mutations of the CD117 gene.246 We therefore believe that it is wasteful to analyze uterine smooth muscle tumors and endometrial stromal neoplasms with CD117 antibodies for determining candidacy for treatment with imatinib. However, IHC stains for CD117, along with DOG1, are indicated when the differential diagnosis includes both a gynecologic smooth muscle neoplasm and a GIST.217 Inflammatory Myofibroblastic Tumor
Inflammatory myofibroblastic tumor is a very rare, possibly benign uterine tumor characterized by a cytologically bland and largely mitotically inactive proliferation of spindle cells in a myxoid matrix.248 The main differential diagnostic considerations include myxoid leiomyoma and leiomyosarcoma and endometrial stromal neoplasm with myxoid stroma. All inflammatory myofibroblastic tumors reported in the largest series were ALK positive, which is similar to what has been described to occur in other sites.248 All uterine mesenchymal neoplasms studied were ALK negative, including smooth muscle and endometrial stromal tumors and carcinosarcomas.
Gestational Trophoblastic Disease A number of different trophoblastic lesions occur in the uterus. Some of these are benign, nonneoplastic proliferations, such as placental site reactions and placental site nodules. Hydatidiform mole usually has a benign clinical evolution after therapy, but it can give rise to recurrent or progressive gestational trophoblastic disease. Malignant trophoblastic tumors include choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor. The various types of trophoblastic cells have distinctive immunophenotypes, so it is possible to use IHC to assist in the diagnosis of trophoblastic lesions, which include hydatidiform mole (partial, complete, and invasive), choriocarcinoma, placental site trophoblastic tumor, epithelioid trophoblastic tumor, exaggerated placental site, and placental site nodule. With the exception of p57, IHC is rarely used for the diagnosis of hydatidiform moles. Trophoblasts that include cytotrophoblasts, intermediate trophoblasts, and syncytiotrophoblasts stain strongly and diffusely with cytokeratins (AE1/AE3),249 inhibin,250,251 CD10,52,252 CK18,253,254 and 3β-hydroxyD5-steroid dehydrogenase (3βHSD).253,255 Mel-CAM, also known as CD146, a membrane glycoprotein of the immunoglobulin gene superfamily involved in cell-to-cell interaction,256 is a good general marker of
intermediate trophoblasts, as is human leukocyte antigen G (HLA-G).257 Syncytiotrophoblasts stain strongly with human chorionic gonadotropin (hCG), as do some intermediate trophoblasts of the implantation-site type.252 Human placental lactogen (hPL) marks intermediate trophoblasts of the implantation-site type, and it also stains syncytiotrophoblasts.258 Placental alkaline phosphatase (PLAP) is expressed in intermediate cells and also in syncytiotrophoblastic cells,259 but the intermediate trophoblasts marked here are of the chorionic type.258,260 Chorionic-type intermediate trophoblasts also express p63,248 and cytotrophoblasts express β-catenin.261-263 The trophogram is a diagram developed by Shih and Kurman254,255 that provides diagnostic help for trophoblastic lesions by using a decision tree format (Fig. 18-20). All trophoblastic lesions express CK18 and HLA-G diffusely. hCG expression in numerous syncytiotrophoblasts intimately admixed with mononuclear trophoblasts favors choriocarcinoma. For lesions without numerous syncytiotrophoblasts, hPL expression without p63 favors a lesion composed of implantation-site intermediate trophoblasts (exaggerated placental site versus placental site trophoblastic tumor [PSTT]); p63 expression with variable hPL favors a lesion composed of chorionic-type intermediate trophoblast (placental site nodule vs. epithelioid trophoblastic tumor [ETT]). In general, MIB-1 labeling of less than 10% to 15% favors exaggerated placental site reaction over PSTT, and MIB-1 labeling of less than 10% to 15% favors placental site nodule over ETT, but exceptions exist, as does overlap in MIB-1 expression values in these entities. Diagnoses should be rendered on the basis of the constellation of morphology (including immunohistology) and the clinical setting. COMPLETE HYDATIDIFORM MOLE
The morphologic criteria for distinguishing complete hydatidiform moles from mimickers are robust, particularly when the tissue is derived from a second trimester conceptus. Detection of abnormal gestations in the first trimester has led to diagnostic difficulties, however, because increased villous size and trophoblastic proliferation are less pronounced than in tissue derived from more advanced gestations. Complete moles diagnosed in the first trimester have been referred to as “early” complete hydatidiform moles. IHC evaluation with antibodies against the p57 protein, a maternally transcribed gene product, has emerged as a useful adjunct to morphologic, flow cytometric, and cytogenetic study of molar tissue. Whereas p57 protein is expressed in intermediate trophoblasts of complete moles, partial moles, and nonmolar abortuses, only complete hydatidiform moles lack p57 expression in cytotrophoblasts and villous stromal cells.264-268 Subtle but reproducible morphologic differences between early complete moles and other entities in the differential have been reported.269,270 Using ancillary diagnostic techniques that include cytogenetics, assays for DNA ploidy, and molecular
Uterus
679
Possible trophoblastic lesions CK18 HLA-G Trophoblastic lesions
p63 hPL Lesions of implantation site intermediate trophoblast MIB-1 1% Exaggerated placental site MIB-1 1% Placental site trophoblastic tumor
p63 or hCG (highlighting syncytiotrophoblast)
p63 hPL or
Choriocarcinoma
Lesions of chorionic-type intermediate trophoblast MIB-1 10% Placental site nodule MIB-1 10% Epithelioid trophoblastic tumor
Figure 18-20 An algorithm using a panel of immunohistochemical markers is useful for the differential diagnosis of trophoblastic lesions. The algorithm begins with immunostaining with cytokeratin 18 (CK18) and human leukocyte antigen G (HLA-G) antibodies. If they are both diffusely positive, the lesion is trophoblastic. If p63 is selectively stained in mononucleate trophoblasts, and human chorionic gonadotropin (hCG) is selectively stained in syncytiotrophoblasts, the lesion is a choriocarcinoma. If p63 is negative and human placental lactogen (hPL) is diffusely positive, the lesion is either an exaggerated placental site or is a placental site trophoblastic tumor. These lesions can often be distinguished based on the MIB-1 labeling index. If p63 is diffusely positive and only focally positive for hPL, the lesion is either a placental site neoplasm or an endothelial trophoblastic tumor. These lesions can often be distinguished based on the MIB-1 labeling index (+++, diffusely positive; +, focally positive; −, negative). From Shih IM, Kurman RJ: p63 expression is useful in the distinction of epithelioid trophoblastic and placental site trophoblastic tumors by profiling trophoblastic subpopulations. Am J Surg Pathol 2004;28:1177-1183.
techniques (see the “Genomic Applications” section), it has become evident that many early complete hydatidiform moles have historically been misdiagnosed as nonmolar abortuses or partial moles. This misdiagnosis has led to the mistaken impression that the development of choriocarcinoma from a partial mole is a common event. In fact, choriocarcinoma almost never follows a partial mole when it is diagnosed correctly; early complete moles, in contrast, remain a common source from which choriocarcinoma evolves. PLACENTAL SITE NODULE
Placental site nodules are composed of mitotically inactive intermediate trophoblastic cells of the chorionic type within a nodular, hyalinized stroma. Lesional cells express pantrophoblastic markers as well as the chorionic intermediate trophoblast-associated markers PLAP and p63. The MIB-1 index in a placental site nodule is usually less than 10% to 15%.271 EXAGGERATED PLACENTAL SITE
This lesion is actually an exuberant, nonneoplastic physiologic finding that frequently accompanies molar pregnancies and is composed mostly of implantation-site intermediate trophoblasts. The constituent cells express hPL and HLA-G.257 The MIB-1 index in an exaggerated placental site reaction is generally less than 10% to 15%.272
PLACENTAL SITE TROPHOBLASTIC TUMOR
Placental site trophoblastic tumor (PSTT) is composed of neoplastic implantation-site intermediate trophoblasts that infiltrate myometrium, and hPL and HLA-G are typically expressed. PSTTs have an MIB-1 index of 15% (±7%).272 PSTTs can also contain scattered hCG-expressing syncytiotrophoblasts, but they are rare and haphazardly situated, not intimately admixed with mononuclear trophoblasts as is typical of choriocarcinoma. Biopsy or curettage material may suggest the presence of PSTT or a placental site nodule. Both the immunophenotype and proliferative index of these lesions differ, with preferential expression of hPL and HLA-G in PSTT as opposed to expression of PLAP and p63 in placental site nodule.254,255 EPITHELIOID TROPHOBLASTIC TUMOR
Epithelioid trophoblastic tumor (ETT) is composed of chorionic site intermediate trophoblasts that tend to arise in the cervix, where they have a rounded contour and a pushing pattern of stromal infiltration.273 PLAP and p63 are expressed, but hPL and HLA-G are typically not expressed.254,255 Because of its frequently cervical location and its epithelioid, eosinophilic appearance, this tumor can be confused with squamous carcinoma. ETT shares expression of p63 with SCC, but ETT expresses inhibin, CD10,250,273 CK18,254 and 3βHSD.254
680
Immunohistology of the Female Genital Tract
ETTs lack the p16 expression typical of HPV-associated squamous neoplasms.274
encountered in occasional pleomorphic carcinomas also express hCG.
CHORIOCARCINOMA
Genomic Applications
It was previously thought that choriocarcinomas were biphasic neoplasms composed of mononuclear cytotrophoblasts and multinucleate syncytiotrophoblasts. Although it is certainly still the case that these tumors have a biphasic appearance with H&E stains, they are actually triphasic, using markers that recognize cytotrophoblasts, intermediate trophoblasts, and syncytiotrophoblasts.263 Cytotrophoblasts, which show nuclear labeling with antibodies against β-catenin, represent a small minority of the mononuclear cells; most of the mononuclear cells in choriocarcinomas are actually intermediate trophoblasts.263 The main differential diagnostic entities to consider with a tumor that resembles gestational choriocarcinoma include germinal or nongestational choriocarcinoma, complete hydatidiform mole with sparse villi, ETT and PSTT, and carcinomas that contain trophoblastic elements. Germinal choriocarcinoma without admixture of other germ cell elements is so rare in women that it really is not a viable diagnostic consideration; however, nongestational choriocarcinomas do occur in the uterus in older women. The trophoblastic proliferation associated with a complete mole may be histologically indistinguishable from choriocarcinoma, but it is unclear whether IHC differences exist. Therefore with the exception of the rare choriocarcinomas that arise in nonmolar placentas and those that coexist with a nonmolar twin, choriocarcinoma cannot be diagnosed confidently when villi are present. Choriocarcinomas and tumors composed of intermediate trophoblasts may resemble one another when syncytiotrophoblasts are few; this is particularly problematic when presented with tissue from choriocarcinomas previously exposed to chemotherapeutic agents.275 Serum hCG levels are likely to be very helpful, because choriocarcinomas, even those relatively deficient in syncytiotrophoblasts, are frequently associated with higher serum levels than is the case with intermediate trophoblastic tumors. As expected, an hCG immunostain would mark syncytiotrophoblasts, which are almost always more numerous in choriocarcinoma compared with PSTTs and ETTs.258 An hCG immunostain would also be expected to highlight the characteristic morphologic relationship of syncytiotrophoblasts and mononuclear trophoblasts in choriocarcinoma, in which the multinucleated cells envelop the mononuclear cells. An MIB-1 immunostain is likely to be even more helpful when syncytiotrophoblasts are very few in number; the proliferative rate is usually 15% to 25% in tumors derived from intermediate trophoblasts, whereas it frequently exceeds 70% in choriocarcinoma.272 Based on current data, study with IHC is not useful for the separation of gestational choriocarcinoma from a carcinoma that contains trophoblastic elements, which can be seen in cervical squamous carcinoma and transitional cell carcinoma of the bladder, in addition to others (see Table 18-4).276,277 Syncytiotrophoblasts
DNA MISMATCH REPAIR PROTEINS
Women with hereditary nonpolyposis colon cancer syndrome (Lynch syndrome) have an increased risk for various types of cancer. The most frequent cancers in this patient population are endometrial adenocarcinoma and colorectal adenocarcinoma. Lynch syndrome is caused by mutation of one of the MMR genes: MLH1, PMS2, MSH2, or MSH6. Definitive testing requires molecular analyses, but screening for possible mutations can be performed by using IHC, because antibodies are available that detect the presence of the various MMR proteins. If a mutation has occurred, the corresponding MMR protein is not produced, and staining for that protein is absent.161 Staining is nuclear and is somewhat variable from area to area on the slide; a positive result is complete absence of nuclear staining in all tumor cells on the slide with a positive internal control (positive staining in the nuclei in normal tissues, no staining in tumor cell nuclei). Generally, loss of MLH1 results in loss of PMS2, and loss of MSH2 results in loss of MSH6. On the other hand, a mutation in PMS2 or MSH6 will result in loss of the corresponding protein only. Interpretation is further complicated by the fact that loss of MLH1 can be due not only to mutation of the gene, but also, more commonly, to hypermethylation of the MLH1 gene promoter. IHC staining for the MMR proteins is a screening test, and women with a positive result are usually referred for genetic counseling and possible molecular testing to identify a mutation of one of the MMR genes or hypermethylation of the MLH1 gene promoter. IHC testing for MMR proteins is of value not only for selecting patients for further testing but also for deciding which gene needs to be tested first. Testing can commence with the gene for which staining is lost, and it may not be necessary to test for mutations in all four genes. DNA MMR proteins are found to be absent or deficient in tumor cell nuclei by IHC in up to one third of endometrioid adenocarcinomas158-161; this results from MLH1 promoter hypermethylation in most cases and from mutation of MLH1, MSH2, MSH6, or PMS2 in the remaining cases (Fig. 18-21). Only complete expression loss in the setting of a valid positive internal control is considered interpretable. Valid internal controls include nonneoplastic endometrial stroma and glands and/or infiltrating lymphocytes with reproducibly stained nuclei. Care should be taken to ensure that the lesion being assessed is adenocarcinoma, not hyperplasia.161 It is also extremely important that the IHC methodology and interpretation of stains be performed with the strictest guidelines in mind, particularly with respect to MLH1 and MSH6 stains, because these tend to be more problematic.161 Common pitfalls in interpreting these stains include 1) patchy or weak expression of MSH6; 2) retention of MMR protein expression in numerous intratumoral lymphocytes,
Uterus
A
B
C
D
681
Figure 18-21 DNA mismatch repair protein expression in endometrial carcinoma. An endometrioid adenocarcinoma (A, hematoxylin and eosin) shows negative staining for PMS2 (B), focal and weak positive nuclear staining for MLH1 (C), and positive nuclear staining for MSH2 (D). (B) shows positive nuclear labeling in stromal cells that serves as a positive internal control. From Modica I, Soslow RA, Black D, et al: Utility of immunohistochemistry in predicting microsatellite instability in endometrial carcinoma. Am J Surg Pathol 2007;31:744-751.
mistakenly interpreted as tumor cell nuclei, when the tumor cell nuclei are actually MMR protein deficient; 3) poor or inadequate fixation with loss of internal positive control; or 4) no internal control tissue is present for evaluation. In an effort to define a target endometrial cancer population in which the DNA MMR immunostains might be informative, some institutions test for DNA MMR abnormalities in endometrial carcinomas that occur in women younger than 50 years and in older women whose tumors exhibit morphologic features that have been reported to covary with deficient MMR protein expression.187 Such morphologic features, although not specific and currently considered of debatable significance, include dense peritumoral lymphocytes and tumor-infiltrating lymphocytes (TILs; >40 TILs per 10 HPF) and biphasic tumors with the appearance of a collision tumor, such as the dedifferentiated carcinoma described earlier in the chapter.185,187,278-281 Although many Lynch syndrome–associated tumors are endometrioid, a proportion of endometrial clear cell
carcinomas, serous carcinomas, and carcinosarcomas may also belong to this group.173,186 Moreover, a significant proportion of women with endometrial cancer and Lynch syndrome have endometrial cancer develop after 50 years of age. In one study that compared various combinations of age and/or family history in conjunction with other potential screening criteria for Lynch syndrome in women with endometrial cancer, triage of endometrial cancer patients with at least one firstdegree relative with a Lynch syndrome–associated cancer was found to have the best incremental costeffectiveness ratio.282 However, not all women with Lynch syndrome have affected first-degree relatives, and a recent recommendation has been made to offer MMR deficiency testing to all patients newly diagnosed with endometrial cancer, regardless of age and personal or family history, similar to that recently proposed for colorectal cancer screening (Fig. 18-22).283,284 Both screening procedures can be accomplished in a cost-effective model that incorporates the twoMMR protein antibody panel. If reflex testing is to be performed, it is recommended that all involved
682
Immunohistology of the Female Genital Tract
IHC testing for loss of MMR protein expression for all endometrial carcinomas
MMR intact per IHC but clinical suspicion of LS
Loss of MMR per IHC
Order LS microsatellite instability by PCR
Instability at Instability 1 microsatellite ≥2/5 of marker microsatellite markers
High
No instability present
Indeterminate
Consider germline testing of MMR genes
Low
Abnormal staining for MLH1 & PMS2
Abnormal staining for MSH2 & MSH6
Abnormal staining for MSH6
Abnormal staining for PMS2
Test for MLH1 promoter methylation
Genetic mutation testing for LS: recommend LS MSH2 sequencing and deletion/duplication as first test
Genetic mutation testing for LS: recommend LS MSH6 sequencing and deletion/duplication as first test
Genetic mutation testing for LS: recommend LS PMS2 (and MLH1 if PMS2 negative) sequencing and deletion/duplication as first test
Methylation present
Likely sporadic endometrial carcinoma*
Methylation absent
Genetic mutation testing for LS: recommend LS MSH1 sequencing and deletion/duplication as first test
*If strong clinical suspicion for LS, consider MLH1 promoter methylation analysis of non-neoplastic tissue/peripheral blood to evaluated for germline epigenetic MLH1 promoter methylation. Figure 18-22 Mismatch repair (MMR) protein immunohistochemistry can be incorporated with microsatellite instability testing by polymerase chain reaction (PCR) and with clinical history to identify which patients with endometrial carcinoma are at high risk for Lynch syndrome (LS) and need formal genetic evaluation and MMR germline analysis. One possible algorithm is illustrated here. IHC, Immunohistochemistry.
pathologists, gynecologic oncologists, and geneticists agree to the medical necessity of performing the test and arrange a chain of command that guarantees that all targeted patient material is tested and that patients are referred for the appropriate counseling.285
Theranostic Applications ENDOMETRIAL CARCINOMA p53, Estrogen Receptor, and Progesterone Receptor
Although stage and grade are still considered the most important prognostic indices in endometrial cancer, much has been written about using IHC to predict prognosis. Of possible relevance is IHC overexpression of p53. Several studies have documented that overexpression of p53 is a negative prognostic indicator in endometrial cancer.200,286-291 It is possible that ER and PR expression is also of therapeutic and even prognostic
relevance here, as it has recently been shown that tumors that overexpress p53 while coexpressing ER and PR are less aggressive than those without ER and PR expression.166 This observation was understood to mean that occasional high-grade endometrial carcinomas represent tumors that have progressed or transformed from ERand PR-positive low-grade endometrioid carcinomas that have acquired p53 mutations. Clinicians commonly request ER and PR testing for metastatic or recurrent endometrial carcinoma when therapy with hormonal agents, particularly high-dose progestins, is considered. HER2
A large percentage of uterine serous carcinomas overexpress HER2,292-296 but there is an imperfect correlation with ERBB2 (formerly HER2/neu) gene amplification. Several studies have reported a relationship between HER2 overexpression and poor clinical outcomes. The Gynecologic Oncology Group (GOG) attempted to study the efficacy of trastuzumab (GOG
Ovary and Fallopian Tubes
181B), but the results of this have not yet been published. UTERINE SARCOMA Estrogen Receptor, Progesterone Receptor, p53, MIB-1, and p16
As independent variables, expression of ER, PR, p53, and MIB-1 has a prognostic significance in leiomyosarcoma204,207,209,213,214,216; however, these markers lose significance in multivariate models, in which stage remains the most important prognostic factor. In several studies, uterine smooth muscle and endometrial stromal neoplasms were negative or rarely immunoreactive for CD117,139,244,245 although others have shown CD117 positivity in leiomyosarcomas and carcinosarcomas.246,247 Importantly, IHC expression of CD117 in leiomyosarcomas and carcinosarcomas is not associated with activating mutations of the KIT gene.246 Currently, there is no clinical indication to analyze uterine smooth muscle tumors and endometrial stromal neoplasms with CD117 for determining candidacy for treatment with imatinib. Finally, a number of recent publications have called attention to the significance of p16 expression in atypical uterine smooth muscle tumors, although the mechanisms that underlie p16 overexpression in uterine sarcomas has not been elucidated.297-300 Among uterine leiomyomas, smooth muscle tumors of uncertain malignant potential (STUMPs), and leiomyosarcomas, p16 expression is found mostly in leiomyosarcomas and in a subset of STUMPs, some of which behave in a malignant fashion. The implication is that STUMPS that overexpress p16 might be at higher risk of relapse compared with those that are p16 negative, but larger studies with clinical follow-up have failed to substantiate this. Retrospective data suggest that progestational agents are a beneficial adjuvant therapy, so clinicians frequently request ER and PR testing for low-grade sarcomas such as endometrial stromal sarcomas and adenosarcomas. More than 90% of these tumors express ER and PR when they are histologically low grade and when highgrade stromal overgrowth is absent, as in the case of adenosarcoma.235
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications to Diagnosis JAZF1-SUZ12 TRANSLOCATION
The translocation between chromosome 7p15 and 17q21 gives rise to a fusion gene that contains contributions from two genes with prominent zinc finger motifs: JAZF1 and SUZ12 (formerly JJAZ1).301 This translocation has been documented in approximately 50% of low-grade endometrial stromal sarcomas and endometrial stromal nodules.302-309 Endometrial stromal tumors with typical morphologic features and the smooth muscle variant are more frequently composed of cells
683
that harbor this abnormality compared with tumors that show fibroblastic differentiation.301 Reverse transcription polymerase chain reaction (RT-PCR) to detect the fusion gene product304,306,307 and fluorescence in situ hybridization (FISH) for the gene rearrangement308,309 are assays used to identify this aberration. The translocation t(7;17) is apparently specific for tumors in the endometrial stromal category, because this has not yet been described in other tumor types.
Gestational Trophoblastic Disease Studies for DNA ploidy that include computer-assisted image analysis of Feulgen stains and cytogenetics that include FISH have been used to distinguish between nonmolar abortuses and partial hydatidiform mole and between partial and complete hydatidiform moles. In contrast to partial moles, which are almost always triploid, nonmolar abortuses and complete moles are usually diploid. Some complete moles are tetraploid,310 and some nonmolar abortuses are triploid.311 In the case of a triploid conceptus, distinguishing between diandric triploidy and digynic triploidy is important, because partial moles are specifically diandric.312 That is, partial moles contain two sets of paternal chromosomes and one set of maternal chromosomes. Molecular genotyping is perhaps the most accurate technique to distinguish partial moles, because it can distinguish androgenetic diploidy, diandric triploidy, and biparental diploidy to diagnose complete moles, partial moles, and nonmolar gestations, respectively.313
Ovary and Fallopian Tubes Ovarian neoplasms can be divided into four main categories: 1) primary epithelial tumors, 2) sex cord– stromal tumors, 3) germ cell tumors, and 4) other tumor types. The latter includes a wide variety of primary and metastatic tumors and includes mesenchymal neoplasms, lymphoma and leukemia, and metastatic epithelial tumors. Tumors of the fallopian tube are primarily epithelial and resemble their ovarian counterparts. A detailed classification of ovarian and tubal tumors is presented in the World Health Organization (WHO) fascicle Pathology and Genetics of Tumours of the Breast and Female Genital Organs.314 Benign and borderline tumors of the fallopian tube are relatively less common than their ovarian counterparts; most fallopian tube tumors are carcinomas. Although the same types of carcinoma that occur in the ovary also occur in the fallopian tube, certain types that are relatively common in the ovary, such as clear cell carcinoma and mucinous carcinoma, rarely arise in the fallopian tube. The most common histologic type of tubal carcinoma is serous carcinoma, followed by endometrioid carcinoma and undifferentiated carcinoma.315-318 In recent years, studies of risk-reducing salpingooophorectomy specimens from women with BRCA mutations have shown that incidentally discovered intraepithelial and invasive carcinomas, almost invariably of the high-grade serous type, are found mainly in
684
Immunohistology of the Female Genital Tract
the fallopian tubes rather than in the ovaries.319-321 Detection of small, grossly invisible carcinomas in these patients requires sectioning of all tissue. Intraepithelial carcinoma is characterized by marked nuclear atypia, nuclear stratification and disorder, and mitotic activity (see Fig. 18-50, A). Small invasive carcinomas form masses of malignant cells in the tubal mucosa. The neoplasms, which are preferentially located in the fimbriae of the tubes,322 typically show diffuse strong nuclear staining for p53, MIB-1, and WT1 (see Fig. 18-50, B). A possible precursor of intraepithelial carcinoma, the so-called p53 signature lesion, is p53 positive but lacks the atypia and proliferative activity present in intraepithelial carcinoma; this entity is of theoretic and scientific interest and is not a clinical diagnosis.323,324 The discovery of the frequent presence of intraepithelial and invasive carcinomas, usually of the high-grade serous type, in fallopian tubes of women with high-grade serous carcinoma of the ovaries or peritoneum has led to the proposal that many carcinomas that involve these sites represent metastases from a carcinoma of the fallopian tube rather than primary neoplasms.325 Multifocal tumorigenesis has also been proposed.326 Ovarian and tubal neoplasms can usually be correctly diagnosed by routine H&E-stained sections. However, each of the main categories of tumors has distinctive IHC features, and IHC can be useful to establish or confirm a diagnosis in problematic cases. IHC may be an especially important aid in the classification of poorly differentiated neoplasms and in the diagnosis and classification of metastatic or systemic tumors that involve the ovary.
Immunohistochemical Markers A relatively small number of antibodies are sufficient for the diagnosis of most ovarian and tubal tumors. These core antibodies are listed in the following paragraphs. Additional antibodies are sometimes helpful and are discussed where appropriate in subsequent sections. CYTOKERATIN
Keratins are intermediate filament proteins that contribute to the cytoskeleton of epithelial cells. Because they are present in all epithelial cells, immunostains for cytokeratins are useful as a screening test to identify a neoplasm as being of the epithelial type. Human cytokeratins (CKs) have been classified according to their molecular weights and isoelectric pH values in the catalog published by Moll and colleagues.327 Twenty epithelial cytokeratin polypeptides have been identified. Some of these have specific tissue distributions that can be exploited for the differential diagnosis of tumors. For screening purposes, a wide-spectrum antibody that recognizes many different cytokeratins is most valuable. We use a cocktail of AE1/AE3 and CAM5.2. Cytokeratin 7
CK7 is a type II basic LMWCK that is found in simple epithelia in a variety of organs, including all epithelia in
the female genital tract.327,328 Epithelial tumors of the ovary and fallopian tube exhibit cytoplasmic and/or membranous staining for CK7.329-331 This characteristic staining pattern of female genital tract tumors can be used, usually in combination with staining for other keratins such as CK20, to differentiate primary female genital tract adenocarcinomas from those arising in other organs.150,332 A panel of immunostains must be evaluated, because some primary ovarian neoplasms fail to stain for CK7, and a proportion of metastatic carcinomas in the ovary are CK7 positive.333 Cytokeratin 20
CK20 is a type I acidic LMWCK initially described in 1992.334 It is found in normal tissues of the stomach, intestine, urothelium, and in Merkel cells and in most adenocarcinomas of the large and small intestines, in some mucinous tumors of the ovary, and in Merkel cell carcinomas. It is frequently present in urothelial carcinoma (UCa) and in adenocarcinoma of the stomach, pancreas, and bile ducts.332,335 CK20 is a useful marker for primary mucinous tumors of the ovary and for various types of metastases found in the ovaries.336-338 Most primary nonmucinous epithelial tumors are CK20 negative. ANTIADENOCARCINOMA ANTIBODIES
Antibodies that are reactive against adenocarcinoma but nonreactive with mesothelial cells are used in a panel for the differential diagnosis between adenocarcinoma and mesothelioma. Antibodies that have been used for this purpose include CD15, BerEP4, mCEA, and MOC-31. CD15 (LeuM1) is used mainly in hematopathology, because it is reactive in neutrophils, histiocytes, immunoblasts, and classic Reed-Sternberg cells, but it also stains various adenocarcinomas. Positive staining is reported in approximately one third to two thirds of ovarian serous carcinomas and in a higher percentage of endometrioid and clear cell adenocarcinomas.339,340 Staining can be either granular and cytoplasmic or membranous. BerEP4 is a monoclonal antibody directed against glycoproteins on epithelial cells, and it shows diffuse, strong membrane staining in nearly all serous carcinomas of the ovary and peritoneum.339,341 In ovarian cancers, mCEA is rarely positive, with the exception of mucinous adenocarcinoma, which is typically CEA positive.339,341-348 Areas of squamous differentiation in endometrioid adenocarcinoma can also show staining for CEA. Polyclonal CEA (pCEA) appears to be less specific than mCEA and stains a somewhat greater percentage of ovarian carcinomas. B72.3 is a monoclonal antibody against a tumor-associated glycoprotein (TAG-72).349 It is usually positive in carcinomas of the ovary and exhibits a granular cytoplasmic staining pattern.339,341,350 This stain can be difficult to interpret, because there is often background staining of mucin and other secretions. MOC-31 is a monoclonal antibody against a glycoprotein that shows strong membrane staining in most cases of ovarian cancer.339 All of these antibodies are negative or only rarely immunoreactive in mesothelioma. In general, BerEP4, MOC-31, and B72.3 are more sensitive
Ovary and Fallopian Tubes
for detection of ovarian surface epithelial tumors than are LeuM1 and CEA. PAX8
PAX8, a member of the paired box (PAX) family of transcription factor genes, is expressed in the müllerian tract.104 It is useful in distinguishing müllerian tract carcinomas from malignant mesotheliomas and metastases, particularly breast, colon, and lung carcinomas. In addition to the müllerian tract, Pax-8 is also a highly sensitive marker for thyroid, parathyroid, renal, and thymic tumors. Although Pax-8 expression has also been reported in pancreatic neuroendocrine tumors, appendiceal and rectal carcinoids, and B-cell lymphomas, the observed Pax-8 positivity in these tumors is due to a cross-reactivity of the polyclonal antibody used with the N-terminal region of PAX6 and PAX5, respectively; it is not seen with monoclonal Pax-8 antibody. Pax-8 is most sensitive for serous carcinomas (95%) but is also strongly expressed in endometrioid (75%), clear cell (80%), and mucinous tumors (60%). Expression is nuclear and typically strong and diffuse in müllerian tumors; focal weak expression is not specific, because this degree of staining may be seen in a variety of other neoplasms. Carbohydrate Antigen 125
Carbohydrate antigen 125 (CA 125) is a high-molecularweight (HMW) glycoprotein recognized by the monoclonal antibody OC125, which has intracellular, transmembrane, and extracellular domains.351-353 The OC125 and M11 binding sites are located in the extracellular domain.351 CA 125 is commonly expressed by primary nonmucinous epithelial ovarian cancers, but it can also be expressed by various other gynecologic cancers that include tumors of the cervix, endometrium, and fallopian tube and by certain nongynecologic cancers such as those of pancreas, breast, colon, lung, and thyroid.354-356 Normal endometrium expresses CA 125 and can be used as a positive control.357 Mesothelioma can also be immunoreactive for CA 125.350 Evaluation for the expression of CA 125 (OC125) is of limited value, because female genital surface epithelial proliferations, metastatic carcinomas from extragenital sites, and mesothelial proliferations can all express CA 125.354,358 Inhibin
Inhibin is a dimeric 32-kD glycoprotein hormone that participates in the regulation of the pituitary-gonadal feedback system.359-362 Inhibins secreted in the ovary consist of an α-subunit linked to one of two β-subunits. In inhibin A, an α-subunit is linked to a β-A subunit; in inhibin B, an α-subunit is linked to a β-B subunit. The monoclonal antibody in general use for IHC recognizes inhibin A. Inhibin is a sensitive and relatively specific marker of sex cord–stromal tumors of the ovary, and its main use in gynecologic pathology is in the differential diagnosis of such tumors.363-368 Luteinized stromal cells sometimes present around carcinomas can express inhibin,363,365,369 which can lead to the erroneous interpretation that a carcinoma expresses inhibin. In general,
685
inhibin expression in carcinomas is encountered uncommonly, and when it is seen, the pattern is only exceptionally strong and diffuse.363,365,369-371 Adrenal cortical neoplasms also commonly express inhibin.372-377 Calretinin
Calretinin is a 29-kD calcium-binding protein initially detected in the central nervous system.378 It belongs to the same family of EF-hand proteins as the S-100 proteins.379,380 Subsequent studies revealed that calretinin is present in benign mesothelial cells and mesothelioma, and it is now the most widely used marker for mesothelioma. Staining is both cytoplasmic and nuclear, with nuclear staining required for specificity for mesothelioma. Calretinin is also present in mast cells, schwannomas, granular cell tumors, adrenal cortical tumors, and in sex cord–stromal tumors of the ovary, which is of particular interest to gynecologic pathologists. Calretinin stains a broader range of sex cord–stromal tumors than inhibin, and it is a more sensitive but less specific marker of such tumors.368,381-383 It is typically used in an IHC panel that also includes inhibin, steroidogenic factor 1 (SF-1), and FOXL2. FOXL2
FOXL2 is a forkhead transcription factor that may have a role in ovarian development and function. The FOXL2 antibody appears to be a relatively sensitive and highly specific marker for sex cord–stromal tumors. FOXL2 staining is present in almost all sex cord–stromal tumors with a FOXL2 mutation and also in a majority of sex cord–stromal tumors without a mutation. Together with inhibin, calretinin, and SF-1, FOXL2 can be used in a panel for positive identification of sex cord–stromal tumors.384 Steroidogenic Factor 1
Steroidogenic factor 1 (SF-1) is a major protein that regulates the complex cascade of steroidogenesis. It is a nuclear transcription factor involved in gonadal and adrenal development and is expressed in adrenal cortical tumors and sex cord–stromal tumors of the ovary. In combination with inhibin, calretinin, and FOXL2, it can be used in a panel for identification of sex cord– stromal tumors.385,386 Wilms Tumor 1
The Wilms tumor gene, WT1, is located on chromosome 11 at 11p13. It functions in the development of the genitourinary tract and is thought to have a tumor suppressor function.387 The WT1 gene product is a DNA binding protein localized in the nucleus. Positive nuclear staining is observed in Wilms tumor,388 desmoplastic small round cell tumor,389-391 and mesothelioma.392,393 WT1 is expressed in the ovarian surface epithelium, inclusion cysts, and normal tubal epithelium.394 Expression is seen in serous carcinoma of the ovary, fallopian tube, and peritoneum,393 but expression in serous carcinoma of the endometrium tends to be limited. Both polyclonal and monoclonal antibodies to WT1 are available. The sensitivity for staining of serous carcinoma is high, and nuclear staining is seen in more
686
Immunohistology of the Female Genital Tract
than 90% of cases reported in some studies.168,392 Other ovarian tumors reported to express WT1 include small cell carcinoma of the hypercalcemic type and some sex cord–stromal tumors.368,395,396 Placental Alkaline Phosphatase
Placental alkaline phosphatase (PLAP) is a marker for malignant GCTs, especially dysgerminoma and embryonal carcinoma (ECa). However, positive staining is also seen in some epithelial tumors, especially serous carcinomas.397,398 PLAP is a useful marker for dysgerminoma and neoplasms that contain related cells,399 such as gonadoblastoma.400 Absence of PLAP expression is unusual in dysgerminoma, but expression of PLAP does not prove that a tumor is a dysgerminoma, because nondysgerminomatous GCTs and some carcinomas also express PLAP. CD117
The CD117 protein is a transmembrane tyrosine kinase growth factor receptor that is the product of KIT gene expression. It is present in a variety of normal human cell types, including breast epithelium, germ cells, melanocytes, immature myeloid cells, and mast cells.401,402 Staining for CD117 occurs in a variety of tumor types, although strong staining is present mainly in mast cell disease and in GISTs, for which CD117 is the preferred marker.401,403-405 A minority of serous ovarian carcinomas stain strongly for CD117.401 In ovarian pathology, CD117 is most useful as a marker of dysgerminoma, which shows diffuse strong membrane staining in nearly all cases.402,406,407 CD117 does not stain ECa and is thus a more specific marker of dysgerminoma than is PLAP. Because metastatic melanoma can occasionally mimic dysgerminoma, it is worth noting that melanoma is occasionally CD117 positive, although usually with a cytoplasmic staining pattern. OCT4
OCT4, now known as POU5F1, is a nuclear transcription factor necessary for maintenance of the pluripotentiality of stem cells and primordial germ cells.408 Positive staining is observed in the nuclei of such pluripotential GCTs as dysgerminoma and ECa and for in situ germ cell neoplasias, such as intratubular germ cell neoplasia in the testis and gonadoblastoma in dysgenetic gonads.409,410 Other types of GCTs are negative. OCT4 has proven to be an excellent immunostain, because staining is generally strong and diffuse, and interpretation is usually straightforward even in small samples and in those with crush artifact or necrosis. In one study, staining for OCT4 was noted in the germ cells in dysgenetic gonads in children up to at least the age of 14 months.411 Therefore positive staining of germ cells in a dysgenetic gonad from a young patient should not be taken as an indication of germ cell neoplasia, unless a tumor with clear cut features of a gonadoblastoma, germinoma, or ECa is present. NANOG is another stem cell marker that stains pluripotential GCTs such as dysgerminoma in a manner similar to OCT4,412 however, NANOG has not been as widely used in diagnostic pathology as OCT4.
Alpha-Fetoprotein
Alpha-fetoprotein (AFP) is an oncofetal glycoprotein expressed in yolk sac tumor and its variants, including hepatoid and endometrioid yolk sac tumor.413-419 Other ovarian tumors that are frequently AFP positive are the rare ovarian hepatoid carcinoma,420-422 metastatic hepatocellular carcinoma,421,423 and SertoliLeydig cell tumors with heterologous hepatocytic differentiation.424-426 Among ovarian GCTs, AFP expression is almost entirely confined to yolk sac tumors,415,427 although focal expression can be seen in ECa and in hepatic or enteric tissues in teratomas.428-432 Rarely, AFP-positive yolk sac tumors have been reported to arise from somatic adenocarcinomas such as endometrioid adenocarcinoma.230,433 For the most part, however, AFP expression in an ovarian tumor is indicative of a yolk sac tumor given the appropriate morphologic context. However, focal strong AFP expression may be occasionally encountered in surface epithelial tumors, particularly in clear cell carcinoma. Glypican-3
The oncofetal protein glypican-3 is highly expressed by yolk sac tumors, typically at higher levels than AFP.434,435 Endometrioid carcinomas and approximately 15% to 20% of clear cell carcinomas may express glypican-3, but the degree of expression is typically focal and/or weak and is not nearly as strong as that seen in yolk sac tumors. Glypican-3 is also expressed by a variety of hepatic tumors, including hepatocellular carcinoma and hepatoblastoma, and in some SCCs of the lung. Other tumors that demonstrate hepatoid or yolk sac differentiation may also express glypican-3. SALL4
SALL4, a zinc finger transcription factor, is a highly sensitive marker for yolk sac tumor, dysgerminoma, ECa, choriocarcinoma, and teratoma. SALL4 is a nuclear protein and is also expressed in adrenal cortical lesions. Less than 15% of ovarian clear cell carcinomas express SALL4, and the incorporation of this antibody in a panel of markers that includes CK7, AFP, and glypican-3 is useful in distinguishing yolk sac tumor from clear cell carcinoma in problematic cases.436 HUMAN CHORIONIC GONADOTROPIN
Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by syncytiotrophoblastic cells. Like other glycoprotein hormones, it consists of an α-chain linked to a β-chain by disulfide bonds. The α-chain is identical to that in follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH). The β-chain is unique, and it is to this chain that antibodies used for IHC are directed. In GCTs, hCG expression is limited to syncytiotrophoblastic cells and some intermediate trophoblastic cells. Primary ovarian tumors that contain syncytiotrophoblastic cells, including choriocarcinoma and some dysgerminomas and ECas, express hCG.432,437-439 Rare, poorly differentiated carcinomas show choriocarcinomatous
Ovary and Fallopian Tubes
differentiation, and the syncytiotrophoblastic cells in such tumors express hCG.440 In addition, occasional carcinomas that lack syncytiotrophoblastic cells have also been reported to express hCG.441,442 S-100 PROTEIN
S-100 protein is a multigenic family of small acidic EF-hand calcium-binding proteins initially discovered in brain extract.443-445 Melanoma is nearly always strongly S-100 positive, so staining for S-100 is a practical way to screen for primary or metastatic ovarian melanoma.446 S-100 can also be identified in a variety of other neoplasms, including some carcinomas,447,448 peripheral nerve sheath tumors (PNSTs), tumors that contain myoepithelial cells, and in the dendritic cells that frequently accompany neoplasms. Sex cord–stromal tumors—including granulosa cell tumors,449 Sertoli cell tumors,450 and tumors in the fibroma-thecoma group—occasionally show positive staining for S-100. A diagnosis of melanoma can be confirmed by positive staining with antibodies against HMB-45 or melan-A (A103) when the diagnosis remains unclear.451-453 HMB-45 and melan-A are more specific for melanoma than is S-100, but they are less sensitive.454 HMB-45 is also expressed in lymphangioleiomyomatosis, angiomyolipoma, and PEComa,238,455-461 and melan-A can be expressed in luteinized cells, Leydig cells, and ectopic adrenal tissue.371,462,463 CD45
CD45, also known as leukocyte common antigen (LCA), is a family of transmembrane protein tyrosine phosphatases. It is expressed on the surface of all hematopoietic cells except erythroid and megakaryocytic cells. Commercially available monoclonal antibodies effectively mark lymphoid cells, both benign and malignant, and are therefore useful for screening tumors to determine whether they might be hematopoietic neoplasms.464 Staining is usually membranous. Plasma cell neoplasms tend to stain weakly or not to stain at all, and staining of myeloid leukemic cells is variable. If a neoplasm is suspected of being of the hematopoietic type, numerous additional markers are available to further characterize it, as discussed in detail in the chapters on lymphoma (Chapters 5 and 6). MARKERS OF NEUROENDOCRINE DIFFERENTIATION
Markers of neuroendocrine differentiation are helpful in the diagnosis of primary and metastatic neuroendocrine carcinomas and primitive neuroectodermal tumors (PNETs) and for identification of primary and secondary carcinoid tumors. The antibodies used for this purpose are discussed in detail in the chapter on endocrine neoplasms (Chapter 10). Chromogranin and synaptophysin are the most specific markers of neuroendocrine differentiation in general use. NSE and CD56 (neural cell adhesion molecule; NCAM) are less specific but are more sensitive and should be used for screening purposes only.
687
Epithelial Tumors Epithelial tumors are by far the most common ovarian and fallopian tube neoplasms. They account for 60% of all ovarian tumors and approximately 95% of all malignant tumors. Keratin antibodies are helpful in the diagnosis of epithelial tumors. Broad-spectrum antikeratins, such as AE1/AE3, can be used to confirm the epithelial nature of a tumor. Although the epithelial nature of low- to intermediate-grade carcinomas is usually obvious, ovarian cancers are often poorly differentiated, and some are difficult to recognize as carcinomas. Positive staining for keratin and EMA suggests that a tumor is a carcinoma. Antibodies against specific keratins have become indispensable in the evaluation of ovarian tumors. Immunoreactivity for CK7 is characteristic of epithelial tumors of the female genital tract, including those of the ovary and fallopian tube. Nearly 100% of primary epithelial tumors of the ovary and fallopian tube are CK7 positive (Fig. 18-23, A),150,329-331,338 so lack of staining for CK7 suggests the possibility of a metastasis. Antibodies to CK20 are also of assistance in the evaluation of ovarian and tubal tumors. Except for mucinous tumors of intestinal type, stains for CK20 are generally negative in primary epithelial tumors (see Fig. 18-23, B). Primary ovarian epithelial tumors are generally CK7 positive and CK20 negative, so the CK7negative/CK20-positive immunophenotype suggests a metastatic tumor in the ovary, especially one from the intestine or appendix. Pax-8 is also extremely useful in confirming müllerian origin, although this marker will not differentiate müllerian carcinoma from renal, thyroid, parathyroid, or thymic carcinomas. KEY DIAGNOSTIC POINTS Epithelial Tumors • All common primary epithelial tumors of the ovary express CK7. • If an epithelial tumor is CK7 negative, consider a metastasis or one of the rare types of primary epithelial tumors. • Serous carcinoma and transitional cell carcinoma are commonly positive for WT1. • Most primary ovarian tumors are negative for CK20 and CDX-2. The exception is the intestinal type of mucinous ovarian tumor, which is positive for CK7 and variably positive for CK20 and CDX-2.
SEROUS TUMORS Most Useful Antibodies: Cytokeratin 7, Wilms Tumor 1
As with other primary epithelial tumors of the ovary and fallopian tube, serous tumors exhibit positive staining for CK7 and negative staining for CK20 (Table 18-6).329,331 Keratin stains can be used to highlight foci of microinvasion in borderline serous tumors.465 Staining of the cell membranes with CA 125 is typically positive in serous carcinoma (Fig. 18-24).466 Stains for estrogen and progesterone receptors are positive in up
688
Immunohistology of the Female Genital Tract
B
A
Figure 18-23 Cytokeratin (CK) subsets in primary epithelial tumors. A, Primary epithelial tumors usually exhibit diffuse, strong staining for CK7, as with this endometrioid adenocarcinoma. B, Except for mucinous tumors, primary epithelial tumors do not stain for CK20.
to 50% of serous carcinomas,466 and strong positive nuclear staining for p53 is found in 30% to 50% of serous carcinomas.168,466-468 Small foci of intraepithelial carcinoma on the surface of the ovary or in the fallopian tube can sometimes be highlighted with this stain. Benign and borderline serous tumors, including micropapillary borderline tumors, are p53 negative. Lowgrade serous carcinoma, which appears to evolve via a different pathway than high-grade serous carcinoma, is significantly less likely than high-grade serous carcinoma to express p53.469 Positive nuclear staining for the Wilms tumor gene product WT1 is generally observed in serous carcinoma of the ovary, fallopian tube, and peritoneum, although the extent and intensity of staining is variable (Fig. 18-25).168,347,392,470-472 Staining is also observed in borderline serous tumors.469,471 Serous carcinoma of the endometrium tends to lack staining for WT1167 or to stain only weakly and focally,168 so absence of staining for WT1 and strong staining for p53 suggests that a metastatic serous carcinoma is more likely to be of endometrial than ovarian origin. Of note, sex cord– stromal tumors of the ovary are also reported to exhibit nuclear staining for WT1.368
PAX8 is a transcription factor involved in the development of the müllerian system, and it is expressed as positive nuclear staining in a significant percentage of serous carcinomas.473,474 Staining for the Pax-8 protein is not specific for serous carcinoma, because other types of epithelial tumors also show positive staining. Pax-2 expression is also seen in müllerian carcinomas, but this marker is not generally recommended for use in clinical diagnosis, because of poor nuclear localization and significant background staining when compared with Pax-8. MUCINOUS TUMORS Most Useful Antibodies: Cytokeratin 7, Cytokeratin 20, Pax-8
IHC can play an important role in the diagnosis and classification of mucinous tumors of the ovary. Two
TABLE 18-6 Primary vs. Metastatic Adenocarcinoma CK7
CK20
CDX-2
Pax-8
DPC4
Primary mucinous
+
+
S
+
+
Primary endometrioid
+
−
−
+
+
Metastatic colorectal
−
+
+
−
+
Metastatic pancreas
S
S
−
−
−
+, Almost always positive; S, sometimes positive; R, rarely positive; −, negative. CK, Cytokeratin.
Figure 18-24 Many nonmucinous ovarian epithelial tumors, particularly serous tumors, exhibit positive staining for carbohydrate antigen 125. This microscopic serous carcinoma, found in a woman with a family history of ovarian cancer, illustrates the typical strong membranous staining pattern. Although often positive, this is not a specific marker for müllerian differentiation.
Ovary and Fallopian Tubes
Figure 18-25 Positive staining for Wilms tumor 1 in serous carcinoma. Strong staining of most tumor cell nuclei is a characteristic finding in serous carcinoma of the ovary.
types of primary mucinous tumors occur in the ovary. Tumors composed of cells with an intestinal phenotype are most common, but a minority of mucinous tumors have a müllerian endocervical-like or seromucinous phenotype. These two types of mucinous tumors have different immunophenotypes, and immunostains can be used to differentiate between the two types. Pax-8 expression is seen in both types, although it is less frequent than in serous carcinomas. Pax-8 expression is not seen in mucinous tumors associated with ovarian teratomas. Immunostains for cytokeratin or EMA can help identify foci of microinvasion. Intestinal-type mucinous tumors are diffusely and strongly positive for CK7 (Fig. 18-26, A),338,475,476 and the majority are also immunoreactive for CK20.150,475 Staining for CK20 tends to be patchy, and the extent and intensity of staining is variable in primary mucinous tumors of the ovary (see Fig. 18-26, B).476 Some pathologists advocate using a high threshold for a positive result, claiming that it will improve the reproducibility of results (i.e., >25% or >50% of tumor cells stained); if this practice is adopted, only 40% to 50% of ovarian
A
689
mucinous carcinomas will be designated as CK20 positive.331,347 CDX genes encode homeobox nuclear transcription factors involved in the proliferation and differentiation of intestinal epithelial cells. Diffuse, strong nuclear staining for CDX-2 is present in normal intestine and in most colorectal adenocarcinomas and their ovarian metastases.477-479 Diffuse, strong positive staining for CDX-2 is observed in only a minority of primary mucinous tumors of the ovary.333,475,480-482 Nonmucinous ovarian tumors are CDX-2 negative. Strong positive staining for CK7, coupled with variable or negative staining for CK20 and CDX-2, typically differentiates a primary mucinous tumor from metastatic colorectal adenocarcinoma because the latter typically is CK7 negative and shows diffuse, strong positive staining for CK20 and CDX-2 (see Table 18-6).150,329,331,332,348 Occasional mucinous tumors that arise in the ovary in association with a benign cystic teratoma have a lower intestinal tract phenotype (CK7−, CK20+, CDX-2+).483 These tumors are often associated with pseudomyxoma ovarii. If a metastasis from a lower intestinal tract carcinoma can be excluded, such tumors are acceptable as primary ovarian mucinous neoplasms with an aberrant immunophenotype, although all such tumors will also be negative for Pax-8. It is important to remember that occasional primary and metastatic tumors show aberrant cytokeratin staining patterns. In particular, rectal adenocarcinomas can be CK7 positive, and occasional primary mucinous tumors of the ovary are CK7 negative.476,484 Immunostains for pCEA and mCEA show staining of luminal mucin, apical cell borders, and cytoplasm of the tumor cells.347,485 Immunostains for villin intensely stain the apical border of the tumor cells in most cases.477 Chromogranin and synaptophysin stains reveal scattered basal endocrine cells among the columnar tumor cells. The epithelium in mucinous tumors is inhibin negative, but pericystic stromal cells are often partly or completely luteinized and are strongly positive for inhibin. Intestinal mucinous tumors are usually negative for estrogen and progesterone receptors.486
B
Figure 18-26 Cytokeratin (CK) subsets in primary intestinal-type mucinous tumors. A, As with other primary epithelial tumors, intestinaltype mucinous tumors generally show strong positive staining for CK7. B, In contrast to other primary epithelial tumors, intestinal-type mucinous tumors are generally CK20 positive. Staining for CK20 tends to be patchy, as seen here, and can be less intense than is observed in metastatic colorectal adenocarcinoma, which is typically CK7 negative and CK20 positive.
690
Immunohistology of the Female Genital Tract
A
B
Figure 18-27 Cytokeratin (CK) subsets in primary endocervical-type mucinous tumors (seromucinous tumors). This type of mucinous tumor has a typical müllerian immunophenotype. A, Endocervical-type mucinous tumors show diffuse strong staining for CK7. B, In contrast to intestinal-type mucinous tumors, endocervical-type mucinous tumors are CK20 negative.
Several additional immunostains are useful for the evaluation of mucinous tumors of the ovary. Stains for the mucin gene product MUC5A and for the pancreas cancer tumor suppressor gene product SMAD4 (formerly DPC4) are generally positive in mucinous carcinoma of the ovary.338 Mutation of SMAD4 occurs in approximately 50% of pancreatic adenocarcinoma and results in loss of staining for the protein, thus lack of staining for the gene product suggests that a mucinous tumor in the ovary may be metastatic from the pancreas. Pseudomyxoma peritonei can occur in patients with mucinous tumors involving ovary. The ovarian tumors have a varied appearance that ranges from that of benign cystadenoma to adenocarcinoma, although most resemble borderline mucinous tumors of the intestinal type. Clinicopathologic and molecular studies suggest that in most instances, pseudomyxoma peritonei is secondary to a tumor of the GI tract, especially one of the appendix, and the ovarian tumors represent secondary involvement of the ovaries by a metastatic neoplasm. IHC stains tend to support this impression, because most tumors lack staining for CK7 and show positive staining for CK20.476,487,488 Rarely, such tumors may arise in association with an ovarian teratoma; these latter tumors will express a similar phenotype to that of the appendiceal tumors and can only be diagnosed with confidence by exclusion of an appendiceal primary and by identification of other teratomatous elements in the ovarian tumor. Mural nodules are occasionally detected in the walls of mucinous tumors. The mucinous tumors can be benign or borderline, or they can be carcinoma. Three types of mural nodules have been described: 1) anaplastic carcinoma,489,490 2) sarcoma,491 and 3) sarcoma like reactive spindle cell proliferations.492,493 Mixtures of the three types may also occur. Anaplastic carcinoma is an obviously malignant proliferation that expresses cytokeratin, usually in a strong and diffuse pattern, and which often coexpresses vimentin.494,495 Sarcomatous mural nodules are malignant spindle or epithelioid cell proliferations that express vimentin but not cytokeratin.496 Sarcoma like nodules have a varied morphology,
but usually cytologic atypia and mitotic activity are less than in malignant nodules. Some of these benign proliferations show limited, usually weak and focal expression of cytokeratin in addition to vimentin.493,497 Seromucinous (endocervical-like) tumors exhibit a different immunophenotype than intestinal-type mucinous tumors. They are CK7 positive (Fig. 18-27, A), but in contrast with intestinal-type mucinous tumors, they are CK20 negative (see Fig. 18-27, B) and CDX-2 negative regardless of what threshold is selected for a positive result.475 Seromucinous tumors tend to be CEA negative, except that the eosinophilic undifferentiated cells found in borderline seromucinous tumors can exhibit strong staining for CEA.485 Seromucinous tumors frequently exhibit positive nuclear staining for estrogen and progesterone receptors and show positive cytoplasmic staining for vimentin.486,498 These tumors are Pax-8 positive. ENDOMETRIOID TUMORS Most Useful Antibodies: Cytokeratins 7 and 20, CDX-2, Cytokeratin Cocktail, Epithelial Membrane Antigen, Inhibin, Pax-8
Benign, borderline, and malignant endometrioid tumors occur in the ovary. Benign and borderline endometrioid tumors are mainly adenofibromatous neoplasms, although rare borderline endometrioid tumors are papillary or glandular. All endometrioid tumors have a similar immunophenotype. Endometrioid carcinoma is CK7 positive, and it is negative or at most weakly and focally positive for CK20 (see Fig. 18-23).479 Most endometrioid tumors express Pax-8. Approximately 33% of endometrioid carcinomas show basal membranous and/ or cytoplasmic staining for vimentin.105 Among the epithelial tumors, endometrioid neoplasms are most likely (38% in one series) to show nuclear and cytoplasmic staining for β-catenin,179 and a majority of these have β-catenin mutations.499 Carcinomas with β-catenin mutations tend to be low grade and low stage, exhibit morular or squamous differentiation, and have a
Ovary and Fallopian Tubes
Figure 18-28 Endometrioid carcinoma, including its sertoliform variant, shows diffuse strong staining for epithelial membrane antigen (EMA). Sertoli cell and Sertoli-Leydig cell tumors are EMA negative.
favorable prognosis. Borderline endometrioid tumors, particularly those with morules, appear to be particularly likely to show nuclear staining for β-catenin.500 Staining is present in the nuclei and cytoplasm of a variable percentage of glandular tumor cells and tends to be particularly prominent in the nuclei of cells in morules or foci of squamous differentiation.501 Most epithelial tumors, including endometrioid tumors, show membrane staining with β-catenin. More common in endometrioid tumors than in other types of epithelial neoplasms, only nuclear staining is associated with β-catenin mutation, and nuclear staining can be difficult to identify in cases with intense membrane staining. Morules and foci of squamous differentiation also show nuclear staining for CDX-2 and cytoplasmic staining for CD10; staining is more likely to be present and more intense in morules.501 Some endometrioid carcinomas exhibit growth patterns that mimic sex cord–stromal tumors such as Sertoli cell tumors, Sertoli-Leydig cell tumors, or granulosa cell tumors. Sertoliform variants of endometrioid carcinoma are the most common of these. IHC stains help to differentiate these variants of endometrioid carcinoma from sex cord–stromal tumors, because endometrioid carcinoma is EMA positive (Fig. 18-28) and does not stain for inhibin or calretinin (Table 18-7).502,503
691
In contrast, sex cord–stromal tumors may stain for cytokeratin, but they are typically negative for EMA, and they show cytoplasmic staining for inhibin and cytoplasmic and nuclear staining for calretinin.370 Metastatic colorectal adenocarcinoma can mimic endometrioid carcinoma, of which diffuse strong staining for CK7 is characteristic. On the other hand, metastatic colorectal adenocarcinoma tends to show diffuse strong cytoplasmic staining for CK20 and nuclear staining for CDX-2, both of which are negative in endometrioid carcinoma, except as noted earlier in the chapter, in morules and foci of squamous differentiation (see Table 18-6).329,479 Pax-8 is expressed in endometrioid carcinoma but is rarely seen in colorectal carcinoma, and it tends to be focal and/or weak in expression when present. Cytoplasmic staining is negative for CEA in glandular areas of endometrioid carcinoma, although apical membrane staining and strong staining can be found in areas of squamous differentiation.105 Metastatic cervical adenocarcinoma can also mimic endometrioid adenocarcinoma. The cervical primary adenocarcinoma is sometimes only minimally invasive, and the possibility of ovarian metastasis is often not suspected. Metastatic cervical adenocarcinoma exhibits histologic features that suggest the correct diagnosis: the tumor is glandular, and the tumor cells are columnar with hyperchromatic fusiform nuclei, pale cytoplasm, and mitotic figures in the upper poles of the columnar tumor cells. Immunostains are usually necessary to confirm the diagnosis. Positive immunostaining for p16 and a positive molecular test for HPV (ISH or PCR) support a diagnosis of metastatic cervical adenocarcinoma.504 Staining for p16 must be strong and diffuse (nearly all tumor cells positive) to be indicative of metastatic cervical adenocarcinoma, because primary ovarian carcinomas, including endometrioid adenocarcinomas, can show lesser degrees of staining.505,506 Carcinosarcoma, adenosarcoma, and endometrioid stromal sarcoma are classified as types of endometrioid tumor of the ovary.314 In carcinosarcoma and adenosarcoma, IHC testing for cytokeratin or EMA can help the pathologist identify an epithelial component in a predominantly mesenchymal neoplasm, but in most cases, IHC is not useful in establishing this diagnosis.507,508 However, immunostains for myogenin or MyoD1 are useful to confirm the presence of rhabdomyoblasts. Endometrioid stromal sarcoma exhibits a distinctive positive staining reaction for CD10,142 whereas sex cord–stromal tumors, with which it may
TABLE 18-7 Endometrioid Carcinoma Sex Cord–Stromal Tumors Cytokeratin
EMA
Inhibin
Calretinin
SF-1
FOXL2
Endometrioid carcinoma
+
+
−
−
−
−
Granulosa cell tumor
S
−
+
+
+
+
Sertoli-Leydig cell tumor
+
−
+
+
+
+
Sertoli cell tumor
+
−
+
+
+
+
+, Almost always positive; S, sometimes positive; −, negative. EMA, Epithelial membrane antigen; SF-1, steroidogenic factor 1.
692
Immunohistology of the Female Genital Tract
A
B
Figure 18-29 Clear cell carcinoma. A, Cytokeratin 7 (CK7) is diffusely and strongly positive in clear cell carcinoma. B, Although its primary use is in hematopathology, CD15 stains some adenocarcinomas, including clear cell carcinoma. Yolk sac tumor, an important differential diagnostic consideration, is typically CK7 and CD15 negative.
be confused, are typically positive for inhibin and calretinin. Endometrioid stromal sarcomas that contain foci of epithelioid or sex cord–like differentiation pose particular diagnostic problems, because these can express inhibin or calretinin.509 In addition to diffuse staining for CD10, endometrioid stromal sarcomas tend to show strong positive staining for estrogen and progesterone receptors. CLEAR CELL CARCINOMA Most Useful Antibodies: Cytokeratins 7 and 20, Hepatocyte Nuclear Factor 1β, Wilms Tumor 1, Epithelial Membrane Antigen, Glypican-3, Pax-8
Clear cell carcinoma shows the typical cytokeratin pattern of immunoreactivity seen in other primary epithelial tumors of the ovary. The tumor cells stain for cytokeratin, Pax-8, CK7 (Fig. 18-29, A), HMWCK, and EMA.127,510 They also stain for CD15 (see Fig. 18-29, B) but usually not for CK20.127 More than 90% of clear cell carcinomas exhibit minimal or no staining for estrogen and progesterone receptors.468,510 Recently, hepatocyte nuclear factor 1β (HNF-1β) has emerged as a sensitive and specific marker for ovarian clear cell carcinoma, as long as yolk sac tumor is excluded.174,176,468 Diffuse strong nuclear staining for HNF-1β is observed in more than 80% of clear cell carcinomas (Fig. 18-30) and in the majority of yolk sac tumors. A panel that includes HNF-1β, WT1, and estrogen receptor (ER) has proved effective in resolving the common differential diagnosis of clear cell carcinoma versus serous carcinoma in the ovary, although this has not proven to be as useful in the endometrium. HNF-1β is likely to be positive in ovarian clear cell carcinoma, whereas WT1 and ER are likely to be positive in ovarian serous carcinoma. The osteopontin gene is a target of HNF-1β protein, and clear cell carcinomas that express HNF-1β also tend to show cytoplasmic and membrane staining for osteopontin.511 Diffuse positive nuclear staining for p53 has been reported by some authors127 but not by others.512,513 Clear cell carcinoma frequently contains
waxy eosinophilic hyaline material in the stroma between glands or in papillae. This material, which is not observed in other types of epithelial tumors, is thought to be basement membrane material, because it stains positively for laminin and type IV collagen.514,515 In the past, clear cell carcinoma and yolk sac tumor were thought to comprise a single tumor type (mesonephroma). Yolk sac tumor still poses a differential diagnostic problem in some cases, because there is a degree of overlap in the histologic appearance of these tumors. Knowledge of the patient’s age, the clinical setting, and the serum AFP level can assist with the differential diagnosis. Clear cell carcinoma generally does not stain for AFP, but yolk sac tumor is usually at least focally positive (Table 18-8).340 Glypican-3 shows strong diffuse cytoplasmic and membrane staining in most yolk sac tumors, but it only rarely stains clear cell carcinoma.419,436 Clear cell carcinoma stains for CD15 and EMA, and these are typically negative or weakly positive in yolk sac tumor.417 CK7 staining has been reported to be absent in yolk sac tumor of the ovary,340 although it may
Figure 18-30 Clear cell carcinoma. Strong positive nuclear staining for hepatocyte nuclear factor 1β (HNF-1β) is characteristic of clear cell carcinoma. Serous and endometrioid carcinoma are usually negative for this marker.
Ovary and Fallopian Tubes
693
TABLE 18-8 Clear Cell Carcinoma vs. Yolk Sac Tumor CK
CK7
EMA
SALL4
Glypican-3
AFP
Clear cell carcinoma
+
+
+
−
−*
−*
Yolk sac tumor
+
−*
−
+
+
+
*May exhibit focal expression. +, Almost always positive; −, negative. AFP, α-fetoprotein; CK, cytokeratin; EMA, epithelial membrane antigen.
be rarely seen in ovarian yolk sac tumors.418 Clear cell carcinoma is almost always CK7 positive, so lack of staining for CK7 favors a diagnosis of yolk sac tumor.516 The clear cell variant of renal cell carcinoma (RCC) can mimic a primary clear cell carcinoma of the ovary. Primary clear cell carcinoma is CK7 positive, whereas metastatic clear cell RCC is rarely CK7 positive.510 Another useful stain for differentiating between these tumors is CD10,52 which is negative in primary clear cell carcinoma of the ovary but shows positive staining, often with membrane accentuation, in metastatic clear cell RCC. Additional stains that are more likely to be positive in clear cell carcinoma include estrogen and progesterone receptors.517 On the other hand, positive staining for the RCC antigen (RCC) favors metastatic RCC.510 The most useful panel includes CK7, CD10, and RCC.510 Diffuse strong positive staining for CK7 and absence of staining for CD10 and RCC favor primary clear cell carcinoma of the ovary. Both tumors express HNF-1β and Pax-8. BRENNER TUMORS Most Useful Antibodies: Cytokeratins 7 and 20, Wilms Tumor 1
Recent studies show similarities between the immunophenotypes of Brenner tumor epithelium and urothelium, suggesting that the transitional cell appearance of the epithelium in Brenner tumors represents true urothelial metaplasia. All authors report that Brenner tumors are CK7 positive and that many of them stain for CEA.395,518-520 Although not all authors have observed the same staining patterns, most recent reports indicate that Brenner tumors stain in a manner similar to that of urothelium: positive for CK7, uroplakin III,521 and thrombomodulin (Fig. 18-31), and in some studies, for CK20.519,521 Some authors have been unable to detect immunostaining for such markers of urothelial cells as CK20 and thrombomodulin.518,522 Positive nuclear staining for p63 is found in urothelium and in UCa of the bladder, and benign and borderline Brenner tumors show diffuse strong nuclear staining for p63, which supports a urothelial phenotype.523-525 Although the number of cases studied is limited, p63 appears to be a good marker for Brenner tumors, because they are p63 positive, whereas other types of ovarian tumors are negative for p63. Borderline and malignant Brenner tumors express urothelial markers less often than do benign Brenner tumors.395
OTHER EPITHELIAL TUMORS Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, CD56, Synaptophysin, Chromogranin, Wilms Tumor 1
The immunophenotype of primary undifferentiated/ poorly differentiated carcinoma is not well defined. Our experience is that high-grade carcinomas stain for general epithelial markers such as cytokeratin and EMA. These stains can help differentiate poorly differentiated carcinomas from carcinosarcoma and various poorly differentiated nonepithelial neoplasms, such as lymphoma. Three types of small cell carcinoma occur in the ovary: small cell carcinoma of the neuroendocrine type can arise in the ovary as a pure tumor, but most often it is admixed with a more common type of primary epithelial neoplasia, such as mucinous or endometrioid carcinoma; small cell carcinoma of the lung can metastasize to the ovary. Small cell carcinoma of neuroendocrine type, whether primary or metastatic, is typically immunoreactive for such general neuroendocrine markers as NSE (Fig. 18-32) and CD56, but these markers are nonspecific. Some examples stain for synaptophysin and/or chromogranin. These tumors sometimes show limited staining for cytokeratin, which can exhibit a dotted or cytoplasmic rim pattern of staining that is highly suggestive of small cell carcinoma.
Figure 18-31 The epithelial elements in Brenner tumors are cytokeratin 7 positive. They also stain for markers of urothelial differentiation such as uroplakin, p63, and thrombomodulin, in which there is cytoplasmic and membrane staining, as shown here.
694
Immunohistology of the Female Genital Tract
cytokeratin, myogenin, FLI-1, and WT1 (with a polyclonal antibody that detects the c-terminal portion of the protein) for desmoplastic small round cell tumor (Fig. 18-34); CD99 and FLI-1 for ES/PNET; and myogenin and MyoD1 for rhabdomyosarcoma.
Sex Cord–Stromal Tumors
Figure 18-32 Small cell carcinoma, neuroendocrine type. This tumor arose in association with an endometrioid adenocarcinoma. The tumor cells are strongly positive for neuron-specific enolase.
Extrapulmonary small cell carcinomas can be immunoreactive for TTF-1, so positive staining does not necessarily indicate that a small cell carcinoma in the ovary is metastatic from the lung. Most rhabdomyosarcomas, PNETs, and sex cord–stromal tumors, including granulosa cell tumors, show positive staining for CD56,526,527 so staining for this marker is not, by itself, indicative of small cell carcinoma. The third type of small cell carcinoma, small cell carcinoma of the hypercalcemic type, also occurs in the ovary. This is a highly malignant neoplasm that occurs mainly in young women, and approximately two thirds of patients have hypercalcemia. The nature of this tumor has been controversial, but recent studies suggest that it may represent an epithelial tumor. IHC stains reveal an epithelial phenotype, because most tumors show staining for EMA and a significant proportion are cytokeratin positive.396,528-530 Recent studies have revealed at least focal staining for WT1, CD10, and p53 in a significant percentage of cases.182,399 Other stains that are nonspecific but frequently positive include vimentin, NSE and chromogranin, and CD99.528,538 Occasional tumors stain for parathyroid hormone–related protein or parathyroid hormone,529 but the cause of the hypercalcemia that accompanies small cell carcinoma has yet to be elucidated. Absence of staining for inhibin, generally weak staining for calretinin, and frequent positive staining for EMA help to differentiate small cell carcinoma from juvenile granulosa cell tumor and suggest that small cell carcinoma is not a sex cord–stromal neoplasm.366,383,396,528 Other primary and metastatic small round cell tumors that enter into the differential diagnosis of ovarian small cell carcinoma include lymphoma, melanoma,446,531,532 desmoplastic small round cell tumor,533-535 Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET),536 and primary and metastatic round cell sarcomas that include embryonal and alveolar rhabdomyosarcomas.537-539 Antibodies that are useful for the differential diagnosis of these tumors include CD45 and related markers for lymphoma (Fig. 18-33); S-100, HMB-45, and related markers for melanoma;
Tumors derived from the sex cords or ovarian mesenchyme compose 5% to 12% of all ovarian neoplasms.540,541 Benign tumors in the fibroma-thecoma group are relatively common. Other sex cord–stromal tumors and mesenchymal tumors are rare. The most common malignant sex cord–stromal tumor is granulosa cell tumor, which comprises 1% of 2% of all malignant ovarian tumors. Two types of granulosa cell tumors are found: the adult type occurs mainly in postmenopausal women and is associated with mutations in FOXL2, and the juvenile type occurs mainly in children and young adults and is not associated with the FOXL2 mutation.542 Other sex cord–stromal tumors that commonly cause diagnostic problems and require IHC testing include Sertoli-Leydig cell tumors, Sertoli cell tumors, sex cord tumors with annular tubules, Leydig cell tumors, and unclassified steroid cell tumors. Several antibodies have proved to be invaluable for the diagnosis of sex cord–stromal tumors. These include inhibin, calretinin, and FOXL2, which are positive in most sex cord–stromal tumors; in addition, EMA is almost always negative in these tumors, a finding that excludes various types of carcinoma from the differential diagnosis. Inhibin is the most specific marker for sex cord–stromal tumors.336,364,366,367,369,370,463,528,543 FOXL2 and calretinin are more sensitive, staining more tumors and tumor types, but calretinin is less specific, because it also stains mesotheliomas and about 20% of epithelial neoplasms.382,383,463 Staining for inhibin tends to be patchy and of variable intensity. The sensitivity and specificity for FOXL2 in identifying sex cord–stromal tumors is 80% and greater than 90%, respectively. Although FOXL2 stains most sex cord–stromal tumors,
Figure 18-33 Lymphoma of the ovary. This diffusely infiltrative tumor proved to be a large cell lymphoma of the B-cell type; an immunostain for CD20, a B-cell marker, shows strong staining of the membranes of every tumor cell.
Ovary and Fallopian Tubes
A
C
protein expression is not associated with an underlying gene mutation except in the adult granulosa cell tumors (greater than 90%), occasional Sertoli-Leydig cell tumors (10%), thecomas, and sex cord–stromal tumors otherwise unclassified.384 Ovarian stromal cells and most types of sex cord–stromal tumors show positive staining for CD56 and WT1.527 Staining for CD56 may be membranous, cytoplasmic, or both, and staining for WT1 is nuclear. In many sex cord–stromal tumors,
KEY DIAGNOSTIC POINTS Sex Cord–Stromal Tumors • Sex cord–stromal tumors can be either positive or negative for cytokeratin. • Sex cord–stromal tumors are almost always EMA negative; if positive, the expression is focal and/or weak. Diffuse positive staining for EMA suggests an epithelial tumor, either primary or metastatic, mimicking a sex cord–stromal tumor. • Inhibin and FOXL2 are relatively specific markers for sex cord–stromal tumors. • Calretinin and SF-1 are more sensitive but less specific markers for sex cord–stromal tumors than are inhibin and FOXL2. • Other markers for sex cord–stromal tumors include CD56 and WT1, both of which are nonspecific.
695
B
Figure 18-34 Desmoplastic small round cell tumor (DSRCT) involving the ovary. A, The growth pattern is nested, and desmoplastic fibrous stroma is evident between the tumor cell nests. B, The tumor cells show strong cytoplasmic staining for cytokeratin and also tend to stain for epithelial membrane antigen and neuron-specific enolase (not shown). C, A perinuclear dotted pattern of staining for desmin is characteristic of DSRCT. Courtesy Anjali Saqi, MD.
CD99 is positive, and the cells stain in a membranous pattern. Leydig cell and steroid cell tumors tend to stain strongly for melan-A (formerly MART-1). CD10 is expressed in many types of sex cord–stromal tumors, but expression is usually focal and weak.544 CD10 is not particularly useful in the diagnosis of sex cord– stromal tumors. Finally, steroidogenic factor 1 (SF-1) is reported to stain most types of sex cord–stromal tumors.385 Therefore the standard panel for the diagnosis of sex cord–stromal tumors should incorporate inhibin, calretinin, and FOXL2; the other markers mentioned above are useful on occasion, depending on the differential diagnosis under consideration. SF-1 is positive in a high percentage of cases, but with weaker and more limited staining than is observed with the other markers.385
Fibroma, Thecoma, and Related Tumors MOST USEFUL ANTIBODIES: INHIBIN, CALRETININ
A fibroma is a benign stromal tumor in which spindleshaped stromal cells grow in abundant collagenous stroma. IHC is rarely performed on fibromas, because their appearance on H&E-stained slides is usually distinctive. Fibromas and related tumors, such as cellular fibromas and fibrosarcomas, stain only infrequently for inhibin, but most are FOXL2 and calretinin positive.366,368,383,384 They show patchy and weak to
696
Immunohistology of the Female Genital Tract
moderate staining for CD56 and WT1.545 Staining for CD56 tends to be cytoplasmic, rather than membranous, as is typical in most other types of sex cord– stromal cells.527 Thecoma is a benign spindle cell stromal tumor that differs from a fibroma in that it is often hormonally active, usually secreting estrogen. Morphologic differences are apparent as well: the tumor cells in a thecoma tend to be plump with clear or vacuolated cytoplasm, and less collagen is present in the background stroma than is present in a fibroma. A thecoma is usually positive for both inhibin and calretinin,364-366,368,383 and strong positive staining for inhibin favors classifying a stromal tumor as a thecoma rather than as a fibroma. Thecomas express FOXL2.384 Stains for myoid markers, such as SMA, are often positive as well.545,546 A sclerosing stromal tumor is a benign, hormonally inactive stromal tumor of the ovary.547,548 The histologic appearance is variegated with cellular spindle cell zones that alternate with paucicellular fibrous zones. Scattered throughout are branched, dilated blood vessels with a hemangiopericytoma-like appearance. The tumor cells adjacent to the vessels are often polygonal and vaguely myoid in appearance. The tumor cells are vimentin positive, and they often stain for SMA, with the SMA stain concentrated in the plump cells in perivascular regions of the tumor.549-551 Immunostains for inhibin, calretinin, and FOXL2 are positive in more than 50% of sclerosing stromal tumors.366,383,384,549 Several authors have correlated the high vascularity observed in these tumors with staining for vascular endothelial growth factor (VEGF) in tumor cells.548,552 Stains for vascular markers such as CD31, CD34, and ERG highlight the prominent branched vessels.549,553 GRANULOSA CELL TUMORS Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Inhibin, Calretinin, FOXL2
Of the two types of granulosa cell tumors of the ovary, the adult type granulosa cell tumor is most common; it is an indolent neoplasm of low malignant potential, which most patients survive. However, granulosa cell tumors can spread beyond the ovary, recur, and cause death.554,555 Recurrences tend to be late and in some
cases are detected more than 20 years after primary therapy.556 Microscopically, the tumor cells are small and uniform with pale or euchromatic nuclei and scanty cytoplasm. The mitotic rate is typically very low. The tumor can often be recognized by the distinctive arrangement of tumor cells in insular, microfollicular, trabecular, and diffuse patterns. More than 90% of adult granulosa cell tumors harbor a somatic mutation in FOXL2. The FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W) and appears to be highly specific for adult granulosa cell tumors. IHC staining for FOXL2, inhibin, and calretinin is helpful in establishing a diagnosis of granulosa cell tumor (Table 18-9).364,366,370,371,382-384,528,557 FOXL2, inhibin, and calretinin are typically strongly positive, with diffuse staining for FOXL2 and calretinin and either diffuse or patchy staining for inhibin (Fig. 18-35, A).363 These stains are not specific, because other types of sex cord–stromal tumors also show positive staining, and occasional carcinomas show weak staining for calretinin and inhibin. Immunostains for LMWCKs are positive, often in a patchy pattern, in occasional tumors (see Fig. 18-35, B),449,558,559 but granulosa cell tumors are usually EMA negative.449 Other immunostains that are often positive include CD56, WT1, SF-1, SMA, S-100, and CD99.368,370,385,449,527,560-562,568 Stains for hormone receptors are frequently positive. Staining for progesterone receptors (PRs) tends to be stronger and more extensive than staining for estrogen receptors (ERs), but neither is positive in some tumors.563 The tumor most often mistaken for a granulosa cell tumor is a poorly differentiated carcinoma, either primary in the ovary or metastatic to it. Carcinomas exhibit features not found in granulosa cell tumors such as bilaterality, high nuclear grade, and a high mitotic index. In addition, IHC can help to establish the correct diagnosis; because carcinomas are usually strongly and diffusely positive for keratin and EMA, they often stain for subsets of keratin such as CK7 or CK20, and they are negative for FOXL2, inhibin, and calretinin. Carcinoid tumors can also mimic granulosa cell tumors, particularly when they grow in insular, microglandular, or diffuse patterns. Clues to the correct diagnosis include the presence of other teratomatous elements and tumor cells with coarse, clumped nuclear chromatin and granular cytoplasm. The immunophenotype of carcinoid
TABLE 18-9 Differential Diagnosis of Granulosa Cell Tumor FOXL2
CR
CK
EMA
LCA
CD99
CGR/SYN
Granulosa cell tumor
+
+
S
−
−
+
−
Carcinoma
−
−
+
+
−
−
−
Carcinoid
−
−
+
+
−
−
+
Lymphoma
−
−
−
−
+
S
−
Small cell carcinoma
−
S
S
S
−
S
S
+, Almost always positive; S, sometimes positive; −, negative. CGR/SYN, Chromogranin and synaptophysin; CK, cytokeratin; CR, calretinin; EMA, epithelial membrane antigen; LCA, leukocyte common antigen.
Ovary and Fallopian Tubes
A
697
B
Figure 18-35 Adult-type granulosa cell tumor. A, Immunostains for inhibin are positive in granulosa cell tumors, but staining is variable in intensity and distribution. As shown here, some cells show strong cytoplasmic staining, whereas in other cells, staining is weak or absent. B, Staining for cytokeratin is variable in granulosa cell tumors. Patchy, moderate cytoplasmic staining appears in this example, but many granulosa cell tumors are cytokeratin negative.
tumors differs from that of granulosa cell tumors, because carcinoids tend to be strongly and diffusely positive for keratin and negative for FOXL2, inhibin, and calretinin, and they stain for markers of neuro endocrine differentiation, such as synaptophysin or chromogranin. The second type of granulosa cell tumor that occurs in the ovary is the juvenile granulosa cell tumor.564,565 It occurs mainly in children and young women but can occur at any age, including after menopause in women.566 Juvenile granulosa cell tumor has a favorable prognosis when confined to the ovary, but its histologic appearance can be alarming. The tumor cells are large, with atypical nuclei that can contain prominent nucleoli. The cytoplasm is abundant and is often luteinized, and mitotic figures tend to be frequent. The tumor cells grow in macrofollicular or diffuse patterns. Although this variant does not harbor the FOXL2 somatic mutation, the immunophenotype is similar to that of adult type granulosa cell tumor. The tumor cells stain for FOXL2, inhibin, and calretinin365,366,370 and usually show strong membrane staining for CD99 (Fig. 18-36).370 SF-1, WT1, and CD56 also are also positive,527 and staining for cytokeratin is present in some tumors. IHC can help differentiate juvenile granulosa cell tumor from small cell carcinoma of the hypercalcemic type (see Table 18-9). Juvenile granulosa cell tumor is positive for FOXL2 and inhibin, whereas small cell carcinoma does not usually stain for these two markers.366,528 Small cell carcinoma stains more intensely for cytokeratin, and a majority of small cell carcinomas are EMA positive.396,528 Most sex cord–stromal tumors are EMA negative, but juvenile granulosa cell tumor can be immunoreactive for EMA,368,567 although staining is usually weak and focal. Staining for calretinin can be present in small cell carcinoma, but it is usually weaker than in juvenile granulosa cell tumor.396 Conflicting results are found in the literature regarding staining of small cell carcinoma for CD99: some authors report no staining, and others report staining in about half of the cases.396,528 Although small cell carcinoma can be CD99
negative, nearly all juvenile granulosa cell tumors are CD99 positive. SERTOLI-LEYDIG CELL TUMOR Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Inhibin, FOXL2, Calretinin
Sertoli-Leydig cell tumors occur mainly in young women, and approximately half are virilizing.568 In welldifferentiated variants, Sertoli cells line well-formed tubules set in a fibrous stroma that contains clusters of polygonal Leydig cells.569,570 Immature stromal and Sertoli cells are not present. The more common intermediate and poorly differentiated Sertoli-Leydig cell tumors contain variably mature Sertoli cells that grow in trabeculae or nests or lining round or retiform tubules.569,571-573 The stroma is cellular and immature, and Leydig cells, present either singly or in clusters, are present in most tumors. Most patients come to medical attention with tumors confined to the ovary and have a favorable prognosis.
Figure 18-36 Juvenile granulosa cell tumor. Strong membrane staining for CD99 is present in nearly all juvenile granulosa cell tumors.
698
Immunohistology of the Female Genital Tract
A
B
Figure 18-37 Sertoli-Leydig cell tumor. The Sertoli cell cords and tubules can be difficult to identify. A, Staining for cytokeratin can be helpful; positive cytoplasmic staining in Sertoli cells growing in cords or tubules, as shown here, is characteristic. B, Sertoli cell cords and tubules are epithelial membrane antigen (EMA) negative. The lack of staining for EMA in Sertoli cords and tubules contrasts with positive staining for both cytokeratin and EMA in sertoliform variants of endometrioid carcinoma (shown in Fig. 18-28).
IHC stains are important in the diagnosis of SertoliLeydig cell tumors, because the various cell types can be difficult to recognize, and because other tumor types, especially the sertoliform variant of endometrioid carcinoma, share some histologic features with SertoliLeydig cell tumors. Sertoli cells stain for cytokeratin, which highlights tubules and delineates cords, nests, and sheets of immature Sertoli cells, but they are EMA negative (Fig. 18-37).368,370,574,575 The stromal cells and Leydig cells are CK and EMA negative; inhibin and calretinin are usually positive in Sertoli-Leydig cell tumors, although staining can be patchy and is strongest in the Leydig cells.359,363-366,381,383,463,528,562 Approximately 50% are positive for FOXL2.384 Sertoli cells tend to show strong membrane staining with CD99,561 and they can also exhibit strong nuclear staining for WT1, a finding seen in various types of adenocarcinoma that can enter into the differential diagnosis of Sertoli-Leydig cell tumor.463 Staining for ERs and/or PRs is present in some Sertoli-Leydig cell tumors,563 and staining for ERs is likely to be stronger and more diffuse than staining for PRs. Heterologous elements are present in about 20% of Sertoli-Leydig cell tumors, and immunostains can help with their identification and classification. GI-type epithelium is the most common heterologous element,576 and it stains for cytokeratin and EMA but is negative for inhibin. Cells that are immunoreactive for chromogranin, serotonin, and various peptides such as corticotropin, somatostatin, and calcitonin are often present in the heterologous enteric epithelium.577 Rarely, SertoliLeydig cell tumors exhibit foci of heterologous hepatoid differentiation and secrete AFP (Fig. 18-38).424,426 The hepatoid cells stain for LMWCKs (CK 8/18), with antihepatocyte antibody, and for AFP but not for inhibin. Heterologous carcinoid differentiation is chromogranin and/or synaptophysin positive. Foci of heterologous rhabdomyoblastic differentiation, a poor prognostic finding, stain for myogenin.578 The uncommon sertoliform variant of endometrioid carcinoma is generally positive for CK and EMA and lacks staining for FOXL2, inhibin, and calretinin.
Positive staining for FOXL2, inhibin, and calretinin and lack of staining for EMA helps to differentiate between a Sertoli-Leydig cell tumor and the sertoliform variant of endometrioid carcinoma (see Table 18-7).502,574,575,579 Rare Sertoli-Leydig cell tumors with pseudoendometrioid tubules almost invariably contain at least focal areas of more typical Sertoli-Leydig cell tumor morphology, and the pseudoendometrioid tubules show positive staining for FOXL2, inhibin, and calretinin and lack staining for EMA.384,580 SERTOLI CELL TUMOR Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Inhibin, Calretinin
Sertoli cell tumors are rare benign neoplasms in which Sertoli cells line tubules or grow in trabeculae.446,581 They lack the primitive stroma and Leydig cells present in Sertoli-Leydig cell tumors. Oxyphilic and lipid-rich variants have been described,582 and Sertoli cell tumors
Figure 18-38 Sertoli-Leydig cell tumor. This tumor shows focal heterologous hepatic differentiation in which the hepatocytic cells show strong cytoplasmic staining for α-fetoprotein; they also stained with antihepatocyte antibody.
Ovary and Fallopian Tubes
can contain areas of diffuse growth. IHC can help to confirm the diagnosis and to differentiate a Sertoli cell tumor from such differential diagnostic considerations as sertoliform endometrioid carcinoma and metastatic carcinoma. Sertoli cell tumors exhibit positive staining for vimentin and cytokeratin, but they do not stain for EMA.450 They are positive for inhibin and calretinin, and most also stain for CD99.383,450 Sertoliform endometrioid carcinomas stain not only for CK but also for EMA, and they are usually negative for inhibin and calretinin.502,503,574,579,583,584 SEX CORD TUMOR WITH ANNULAR TUBULES Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Inhibin, Calretinin
The sex cord tumor with annular tubules (SCTAT) is an unclassified sex cord–stromal tumor that occurs in two clinical settings. Approximately 33% of patients with SCTAT have Peutz-Jeghers syndrome and small, often microscopic tumors that are usually multifocal and bilateral. The remaining 66% of patients do not have the Peutz-Jeghers syndrome, rather they have larger, unilateral tumors that can be hormonally active and can recur or metastasize. Regardless of the clinical setting, the tumors are composed of columnar cells with basal nuclei that line closed annular tubules, which often surround cores of eosinophilic hyaline material. The SCTAT has an immunophenotype similar to that of other sex cord–stromal tumors and stains for vimentin, cytokeratin, inhibin, and calretinin365,366,368,585 but not for EMA. The hyaline material in the eosinophilic cores and stroma has been shown to be basement membrane material by ultrastructural study. It stains, at least in some cases, for laminin and type IV collagen. LEYDIG CELL TUMOR Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Inhibin, Calretinin
Leydig cell tumors are benign ovarian tumors that frequently secrete sufficient testosterone to cause symptoms that lead to their discovery at a small size. They are composed of polygonal Leydig cells with vesicular nuclei, often conspicuous nucleoli, and abundant eosinophilic or pale, vacuolated cytoplasm. The cytoplasm characteristically contains eosinophilic hyaline globules or rod-shaped inclusions known as crystalloids of Reinke, and their presence is diagnostic of a Leydig cell tumor. Most Leydig cell tumors develop in the hilum of the ovary and are sometimes called hilus cell tumors. Nonhilar Leydig cell tumors and stromal Leydig cell tumors are difficult to diagnose, because crystalloids of Reinke are required for their positive identification, and these can only be found in approximately 50% of cases. IHC stains are usually not required for the diagnosis of a Leydig cell tumor, and they do not help to differentiate such tumors from other sex cord–stromal tumors and tumorlike conditions that enter into the differential diagnosis, such as luteinized thecoma, unclassified
699
steroid cell tumors, and luteoma of pregnancy. Leydig cells stain for inhibin, calretinin, and melan-A, and they are usually negative for keratin and EMA.381 STEROID CELL TUMOR Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Inhibin, Calretinin
Steroid cell tumors tend to be large unilateral neoplasms. Many are hormonally active and secrete testosterone or other hormones. Microscopically, they are composed of large polygonal cells with abundant cytoplasm. Some cells have clear, vacuolated cytoplasm and resemble adrenal cortical cells. Others have dense eosinophilic cytoplasm and resemble Leydig cells, except for the absence of crystalloids of Reinke. Both cell types can be present in a steroid cell tumor, but one or the other cell type can predominate. Large size, marked nuclear atypia, and a high mitotic index correlate with malignant behavior. Steroid cell tumors are typically immunoreactive for inhibin, calretinin, and melan-A.363,366,385,463,586 Only approximately 10% are positive for FOXL2.384 Steroid cell tumors tend to show positive staining for SF-1, staining for CD99 is weak and variable, and most tumors lack staining for WT1. Most steroid-cell tumors are vimentin positive, and 40% to 50% stain for cytokeratin.587 Stains for EMA are negative.
Germ Cell Tumors GCTs can be grouped into three main categories. The first includes the common benign cystic teratoma, which accounts for most of the GCTs observed in general practice, and a less common solid mature variant. These tumors tend to be grossly and microscopically distinctive, and they contain skin and skin appendages, as well as benign tissues derived from other germ cell layers, and glial tissue is often conspicuous. IHC is rarely required for the diagnosis of simple benign teratomas. The second group includes variants of benign cystic teratoma in which one line of differentiation predominates or completely overgrows the teratoma, resulting in a monodermal teratoma. Tumors in this category include struma ovarii, carcinoid tumors, and somatic tumors such as appendiceal-like mucinous tumors, SCC, adenocarcinoma, melanoma, and other malignant non– germ cell neoplasms that have arisen in the benign cystic teratoma. IHC may be useful in the diagnosis of tumors in this category. The third main group comprises the malignant GCTs: dysgerminoma, immature teratoma, yolk sac tumor, ECa, choriocarcinoma, polyembryoma, and mixed GCTs that contain two or more of the pure types. These rare tumors tend to occur in young patients. Treatment is by conservative surgery, often followed by chemotherapy. Accurate diagnosis is essential to ensure proper management, and immunostains are often used to confirm the diagnosis. The stains most often used for the diagnosis of malignant GCTs include CD117, PLAP, OCT4, D2-40, AFP, glypican-3, SALL4, CD30, hCG, broad-spectrum antikeratins such as AE1/AE3, and EMA.
700
Immunohistology of the Female Genital Tract
A
B
Figure 18-39 Dysgerminoma shows strong cytoplasmic and membrane staining for placental alkaline phosphatase (A) and strong membrane staining for CD117 (B).
KEY DIAGNOSTIC POINTS Germ Cell Tumors • PLAP can be positive in most types of malignant germ cell tumors, but it can also stain epithelial neoplasms. • OCT4, CD117, and D2-40 are the most useful markers for dysgerminoma. • OCT4 and CD30 are the most useful markers for embryonal carcinoma. • Alpha-fetoprotein, SALL4, and glypican-3 are the most useful markers for yolk sac tumor. • Cytokeratin AE1/AE3 stains many malignant germ cell tumors, and the pattern of staining may help with classification. • EMA is negative in most malignant germ cell tumors.
DYSGERMINOMA Most Useful Antibodies: Cytokeratin, Epithelial Membrane Antigen, Placental Alkaline Phosphatase, CD117, OCT4, D2-40, Human Chorionic Gonadotropin
Dysgerminoma is the ovarian analog of testicular seminoma. It consists of large polygonal tumor cells with round vesicular nuclei that have conspicuous nucleoli. The cells have abundant clear or eosinophilic cytoplasm, and the cell membranes tend to be well defined. The tumor cells grow in nests and sheets divided by fibrous trabeculae. Lymphocytes are present in variable numbers in the fibrous trabeculae and among the tumor cells, and sarcoidlike granulomas are commonly present. Dysgerminoma shows cytoplasmic and membrane staining for PLAP and membrane staining for CD117 (Fig. 18-39).399,407,412 Staining for CD117 is especially helpful, because other tumors in the differential diagnosis, such as ECa and yolk sac tumor, do not show the membranous pattern of CD117 staining characteristic of dysgerminoma (Table 18-10).340 Similar to seminoma and ECa of the testis, dysgerminoma and ECa exhibit strong nuclear staining for the stem cell–related proteins OCT4 and NANOG.369,409 OCT4 is more widely used and is an excellent marker for dysgerminoma, and it shows
strong positive nuclear staining in almost every case (Fig. 18-40). Most other types of GCTs, such as yolk sac tumor and choriocarcinoma, lack staining for OCT4. As previously noted, ECa is positive. Clear cell carcinoma, which enters the differential diagnosis of dysgerminoma in some cases, shows focal staining (<10% of tumor cells) in a minority of cases.409 D2-40 (podoplanin) is widely used to mark lymphovascular endothelial cells and mesothelioma; it shows diffuse strong positive cytoplasmic and membrane staining in dysgerminoma, but this marker is not generally used in a primary panel for this diagnosis. ECa is usually negative for D2-40 or shows only limited staining.588 Dysgerminoma can show patchy positive staining for cytokeratin, often in a rimlike or dotlike cytoplasmic pattern.589 Diffuse strong staining such as that seen in other GCTs and in epithelial tumors is very rare. The tumor cells are negative for EMA and CD30 as well as for S-100 protein, lymphoid markers, and neuroendocrine markers. Approximately 5% of dysgerminomas contain syncytiotrophoblastic giant cells that show strong cytoplasmic staining for cytokeratin and hCG (Fig. 18-41).439
Figure 18-40 Dysgerminoma. Strong nuclear staining for OCT4, as shown here, is characteristic of dysgerminoma. In the ovary, the only other tumor types that stain for this marker are embryonal carcinoma and the germ cells in gonadoblastoma.
Ovary and Fallopian Tubes
701
TABLE 18-10 Differential Diagnosis of Dysgerminoma PLAP
CD117
OCT4
CK
CD30
S-100
LCA
MPO
Dysgerminoma
+
+
+
−
−
−
−
−
Embryonal carcinoma
+
−
+
+
+
−
−
−
Yolk sac tumor
+
−
−
+
−
−
−
−
Lymphoma
−
−
−
−
−
−
+
−
Granulocytic sarcoma
−
+
−
−
−
−
+
+
Melanoma
−
−
−
−
−
+
−
−
CD117 shows membrane staining; rare dysgerminomas show weak staining with AE1/AE3, embryonal carcinoma shows membrane staining and yolk sac tumor cytoplasmic staining. Melanoma may show CD117 staining. +, Almost always positive; −, negative. CK, Cytokeratin; LCA, leukocyte common antigen; MPO, myeloperoxidase; PLAP, placental alkaline phosphatase.
Yolk sac tumor is an uncommon malignant GCT that grows in a confusing variety of histologic patterns, so IHC staining is helpful to establish the diagnosis. The most useful stain is for AFP, because positive staining is characteristic of yolk sac tumor (Fig. 18-42).413 Staining for AFP is variable and often patchy, and diffuse strong staining is not seen in every tumor.340 Nevertheless, more than 75% of yolk sac tumors show positive cytoplasmic staining for AFP. Secretory material in gland lumens and the hyaline globules often seen in yolk sac tumors can also stain for AFP. Positive staining for AFP is particularly helpful in the identification of some of the rare variants of yolk sac tumor, such as the endometrioid and glandular variants. Hepatoid yolk sac tumor is AFP positive and also shows positive cytoplasmic staining with antihepatocyte antibody.421 This can help with the diagnosis of this rare variant of yolk sac tumor, but positive staining does not differentiate hepatoid
yolk sac tumor from hepatoid carcinoma of the ovary or from metastatic hepatocellular carcinoma, because these also show positive staining. Glypican-3 shows strong positive cytoplasmic staining in more than 95% of yolk sac tumors, and it has emerged as an additional positive confirmatory stain for the diagnosis.419 Glypican-3 rarely stains clear cell carcinoma, but similar to AFP, it is positive in tumors with hepatoid features, which include hepatoid yolk sac tumor, hepatoid carcinoma, and metastatic hepatocellular carcinoma.419 Yolk sac tumor is positive for broad-spectrum cytokeratins, and it shows diffuse cytoplasmic staining with AE1/AE3 (Fig. 18-43), as opposed to the membrane staining seen in ECa. This difference can be exploited for differential diagnostic purposes (see Table 18-10). Yolk sac tumor usually does not stain for EMA or CD15,419,427 and it is reported to be CK7 negative.340 When only small foci of yolk sac tumor are present, they often fail to stain for AFP, although they usually mark with cytokeratin and/or glypican-3. Immunostains for PLAP are often positive, but they are nonspecific, because other types of GCTs and some epithelial tumors are also positive.427 The extracellular hyaline material in
Figure 18-41 Dysgerminoma. The patient had an elevated serum β-human chorionic gonadotropin (hCG), and the tumor contained many syncytiotrophoblastic giant cells. These show diffuse strong cytoplasmic staining for hCG (left), whereas the dysgerminoma cells are hCG negative (right).
Figure 18-42 Yolk sac tumor, showing positive staining for α-fetoprotein (AFP). Weak to moderate patchy staining, as shown here, is characteristic. Diffuse strong staining for AFP is unusual.
YOLK SAC TUMOR Most Useful Antibodies: Cytokeratins AE1/AE3 and 7, Epithelial Membrane Antigen, Alpha-Fetoprotein, Glypican-3, SALL4
702
Immunohistology of the Female Genital Tract
staining of ECa cells, staining of cells showing early functional differentiation toward yolk sac tumor, or a difficult-to-recognize component of yolk sac tumor. CHORIOCARCINOMA Most Useful Antibodies: Cytokeratin AE1/AE3, Human Chorionic Gonadotropin
Figure 18-43 Mixed germ cell tumor. An immunostain for cytokeratin AE1/AE3 helps delineate the various elements. Foci of yolk sac tumor (top) show strong cytoplasmic staining for keratin. The surrounding dysgerminoma is keratin negative.
yolk sac tumors is laminin positive, and immunostains for hCG are negative except for positive cytoplasmic staining in the STGCs occasionally present in yolk sac tumors. EMBRYONAL CARCINOMA Most Useful Antibodies: Cytokeratin AE1/AE3, Epithelial Membrane Antigen, CD30, OCT4
Although it is a relatively common form of germ cell neoplasia in the testis, embryonal carcinoma (ECa) is rare in the ovary. It occurs most often as a constituent of a mixed GCT, mixed with yolk sac tumor or other tumor types, but it can occur in pure form. Immunostains are helpful to confirm the diagnosis and delineate areas of ECa from yolk sac tumor, which can be difficult in routine H&E-stained sections. ECa is keratin positive, and we find staining with cytokeratin AE1/AE3 useful, because it shows staining of tumor cell membranes with little if any staining in tumor cell cytoplasm. This contrasts with the cytoplasmic pattern of staining seen in yolk sac tumor. The two most characteristic IHC features of ECa are diffuse strong nuclear staining for OCT4 and diffuse strong membrane staining for CD30 (Fig. 18-44). ECa and dysgerminoma are the only two types of GCTs that stain for OCT4, and ECa is the only one that stains for CD30. Diffuse positive staining for cytokeratin and CD30 and absence of staining for CD117 differentiate ECa from dysgerminoma. Immunostains for PLAP are frequently positive, and embryonal carcinoma does not stain for EMA. Based on limited testing of ovarian tumors and staining of testicular tumors, ECa shows no staining, or at most limited staining, of the tumor cells for D2-40,588 which are strongly positive in dysgerminoma. Syncytiotrophoblastic giant cells are commonly present in ECa and show strong homogeneous cytoplasmic staining for keratin, and they are also positive for hCG. Patchy staining for AFP is noted in some ECa. It is unclear whether this represents
Choriocarcinoma rarely occurs in pure form in the ovary but rather is usually present as a constituent of a mixed GCT. Choriocarcinoma is characterized by an admixture of cytotrophoblastic, intermediate trophoblastic, and syncytiotrophoblastic cells. Staining for hCG is positive in the cytoplasm of the syncytiotrophoblastic giant cells but not in cytotrophoblastic or intermediate trophoblastic cells. Stains for hCG can be somewhat difficult to interpret because of high background staining, most likely caused by hCG in the serum. Inhibin can also be used as a marker for syncytiotrophoblastic cells and has the advantage of low background staining.250,251,590,591 It is worth noting that CD10 stains trophoblastic cells,592 all of which are positive for cytokeratin and negative for EMA. The syncytiotrophoblastic giant cells have abundant cytoplasm that shows diffuse strong staining for cytokeratin, thereby making them easy to identify even at low magnification. Although choriocarcinoma is most often present as a component of a mixed GCT or in pure form, rare ovarian carcinomas with choriocarcinomatous differentiation have been reported.444,593 TERATOMAS Most Useful Antibodies: Depends on Specific Type of Teratoma
IHC usually has a limited role in the evaluation of mature and immature teratomas. Glial tissue can be abundant, and its identity can be confirmed by staining
Figure 18-44 Embryonal carcinoma (ECa). An immunostain for CD30 shows strong membrane staining characteristic of ECa. Staining for cytokeratin AE1/AE3 shows a similar membrane pattern in ECa compared with a cytoplasmic pattern of staining in yolk sac tumor (see Fig. 18-43).
Ovary and Fallopian Tubes
for glial fibrillary acidic protein (GFAP) if necessary. Staining for the proliferation marker Ki-67 (MIB-1) can be of assistance in the differential diagnosis between mature and immature teratoma. The ependymal rosettes that can be found in mature teratomas show low proliferative activity, whereas the neuroepithelial tubules that occur in immature teratomas show high proliferative activity. Recently, glial cell line–derived neurotrophic factor receptor α-1 (GFR-α1) has been shown to exhibit strong membranous staining in immature neuroectodermal cells in immature teratoma, and it has been proposed that it might be helpful in identifying areas of immaturity.594 Increased serum levels of AFP are detected in some patients who have an immature teratoma. IHC staining in such cases shows AFP staining in endodermal glandlike vesicles, immature enteric epithelium, and hepatic cells.428,595 IHC has a greater role in the diagnosis of monodermal teratoma. Some examples of struma ovarii are largely cystic and contain few follicles,596 grow in unusual patterns or are composed of cells with clear or eosinophilic cytoplasm,597 or resemble thyroid adenoma or carcinoma.598 In such cases it may be necessary to identify thyroglobulin in the tumor cell cytoplasm and colloid or TTF-1 in the tumor cell nuclei (Fig. 18-45) to confirm the diagnosis.596,597 Various types of carcinoid tumors, which may or may not be associated with other teratomatous elements, occur in the ovary. The types of carcinoids that occur in the ovary include insular, trabecular, strumal, and mucinous carcinoids. Carcinoid tumors can mimic other ovarian tumors, and IHC stains help to establish the correct diagnosis. Insular and trabecular carcinoids can resemble sex cord– stromal tumors, such as granulosa cell tumors, Sertoli cell tumors, or Sertoli-Leydig cell tumors. However, in carcinoids, diffuse, strong positive staining for cytokeratin and for the neuroendocrine markers chromogranin and synaptophysin (Fig. 18-46) plus a lack of staining for inhibin and calretinin can establish the correct diagnosis. A variety of peptide hormones, such as serotonin, can also be identified in carcinoids.599
703
Figure 18-46 Carcinoid tumors in the ovary can be found with other tissues as a component of a teratoma. They can also be primary in the ovary but not associated with other teratomatous tissues, in which case they are viewed as monodermal teratomas. Finally, they can be metastatic from the appendix, small intestine, or other sites. Regardless of their origin, they show cytoplasmic staining for chromogranin, as shown here, and for synaptophysin.
Strumal carcinoids show partial thyroid differentiation and can be mistaken for cellular or malignant struma ovarii. Positive staining for thyroglobulin in parts of the tumor that show thyroid differentiation and positive staining for chromogranin and synaptophysin in the carcinoid component serve to confirm the diagnosis.600-602 Some strumal carcinoids exhibit positive staining for calcitonin, and most have the interesting characteristic of staining for prostatic acid phosphatase (PSAP), although they do not stain for prostate-specific antigen (PSA).600,601 In addition, strumal carcinoids associated with a severe constipation syndrome show positive staining for protein YY.603 Mucinous carcinoids tend to exhibit positive staining for chromogranin, synaptophysin, and/or serotonin, and their immunophenotype can help differentiate them from various types of primary and metastatic mucinous neoplasms.604 We have observed that primary insular and mucinous carcinoids express CDX-2, so staining for CDX-2 cannot be used to determine whether a carcinoid in the ovary is primary or metastatic.605 Primary ovarian carcinoids other than strumal carcinoids lack staining for TTF-1, and they do not stain for CK7.605 Only mucinous carcinoids show staining for CK20. GONADOBLASTOMA
Figure 18-45 Struma ovarii is a form of monodermal teratoma in which thyroid tissue predominates. The thyroid cells show strong nuclear staining for thyroid transcription factor 1, as shown here. The tumor cell cytoplasm and the colloid also stain positively for thyroglobulin.
Gonadoblastoma contains a mixture of primitive germ cells and sex cord cells, typically arranged around cores of hyaline material. In some cases an invasive malignant GCT, usually a germinoma, arises in the gonadoblastoma. IHC stains reveal that the germ cells stain for PLAP, CD117, and OCT4 and that the sex cord cells stain for vimentin, cytokeratin, and inhibin.366,409,543,606,607 The hyaline material intermixed with the tumor cells will stain for laminin, which indicates that it is basement membrane material.607
704
Immunohistology of the Female Genital Tract
A
B
Figure 18-47 Colorectal adenocarcinoma is the tumor that most often metastasizes to the ovary. Metastatic colorectal adenocarcinoma is typically cytokeratin (CK) 7 negative (A) but shows diffuse strong staining for CK20 (B).
METASTATIC TUMORS
Metastases to the ovary are common and are usually of the epithelial type, and they have been found to account for as many as 10% of malignant ovarian tumors in some series. The most common primary sites of tumors that metastasize to the ovary are the GI tract, particularly the large intestine, stomach, and appendix; the breast; the female genital tract, including the endometrium and cervix; and the pancreas, although tumors of any type and from any site can on occasion give rise to ovarian metastases.608 The differentiation of a primary surface epithelial tumor of the ovary from an extragenital primary tumor can be difficult, especially when the surgeon or pathologist, or both, do not know the patient’s clinical history. Morphologic clues are useful, but in many instances, the histopathologic appearance is misleading; IHC can be particularly valuable in such cases and is most helpful in identifying metastases from nongenital sites. Differentiation between a primary ovarian tumor and a metastasis from another genital primary site, such as the endometrium, is more difficult, and IHC staining may not be helpful. Among common epithelial tumors, serous neoplasms are usually readily recognized as primary neoplasms and are only infrequently mimicked by metastases. On the other hand, metastatic adenocarcinomas commonly mimic both mucinous and endometrioid tumors. In a recent report, 77% of mucinous ovarian cancers were metastatic.609 Primary mucinous and endometrioid tumors tend to be unilateral, and primary mucinous tumors are usually larger than 10 cm in diameter.609 Small mucinous tumors and bilateral tumors thought to be of mucinous or endometrioid types should be evaluated for the possibility that they represent metastases.610 This rule of thumb applies not only to obviously invasive tumors (carcinoma) but also to apparently borderline tumors. Metastatic mucinous adenocarcinoma from the appendix, colon, pancreas, and gallbladder can on occasion show remarkable morphologic overlap with borderline mucinous and endometrioid tumors. Evaluation of tumors that might represent metastases from these sites should include antibodies against CK7,
CK20, and CDX-2 (see Table 18-6). Primary ovarian carcinomas are almost invariably diffusely and strongly positive for CK7; metastatic colorectal carcinomas are usually entirely negative, although there are exceptions (Fig. 18-47, A),476 or they show only minimal staining.611 Tumors from the rectum and appendix are somewhat more likely to show staining for CK7,338,612 and biliary tract tumors are almost invariably CK7 positive.613,614 Metastatic colorectal tumors are usually diffusely and strongly positive for CK20 and CDX-2 (Fig. 18-48; see also Fig. 18-47, B).611 Carcinomas of the biliary tract and pancreas stain variably for CK20. Endometrioid carcinoma does not stain for CK20 or CDX-2. Primary mucinous carcinoma is CK20 positive, and it can stain for CDX-2, but the staining for both of these markers is typically weaker and more focal than that observed in metastatic colorectal carcinoma.476,482 Thus immunophenotypes positive for CK7 and negative for CK20 and those positive for CK7, CK20, and CDX-2 favor an ovarian primary tumor, and the immunophenotype that is negative for CK7 and positive for CK20 and CDX-2
Figure 18-48 Metastatic adenocarcinoma from the appendix showing diffuse strong nuclear staining for the transcription factor CDX-2, a useful marker for metastatic intestinal and appendiceal adenocarcinoma. Some primary mucinous carcinomas of the ovary stain for CDX-2, but staining is often weaker or patchier than that seen in metastatic intestinal carcinomas.
Ovary and Fallopian Tubes
strongly favors a metastasis.337,482,615 Another antibody that may be of utility is SMAD4 (formerly DPC4), a nuclear factor that is absent in about 50% of pancreatic adenocarcinomas.338 We will discuss the use of these antibodies in the “Mucinous Tumors” section. Metastatic adenocarcinoma from the stomach tends to be bilateral and of the signet-ring cell (Krukenberg tumor) or poorly differentiated type, although gland formation is present in some tumors, and rare tumors are composed entirely of intestinal-type glands.616 Signet-ring cells are rare in primary ovarian tumors,617,618 and their presence indicates that the tumor is almost certainly metastatic, with stomach being the most likely primary site.619 Metastatic stomach cancer tends to be CK7 positive (56% of cases in one study), and a substantial proportion of cases (33%) are also positive for CK20.615 Morphology can therefore be more important than IHC in establishing the correct diagnosis. Metastases from the pancreas, biliary tract, and even the GI tract can occasionally mimic clear cell carcinoma.620 Such tumors can also be evaluated with antibodies against CK7, CK20, and SMAD4. Pancreatic ductal adenocarcinoma shows a variable staining pattern for CK7 and CK20, and in some cases, its staining pattern overlaps that of primary mucinous carcinoma of the ovary. Primary ovarian carcinomas are not known to show loss of staining for SMAD4, so this finding is very helpful in identifying metastatic pancreatic ductal adenocarcinoma. Pancreatic acinar adenocarcinoma is rare, but it can metastasize to the ovary.621 The tumor cells have granular eosinophilic cytoplasm and are arranged in acini. Positive staining for trypsin and chymotrypsin facilitates recognition of this type of metastatic adenocarcinoma. Metastatic clear cell carcinoma of the kidney can also mimic primary ovarian clear cell carcinoma, but this is a rare diagnostic problem, because renal tumors usually do not metastasize to the ovaries. Metastatic RCC of clear cell type is CK7 negative, but it shows strong membrane staining with antibodies to CD10 and RCC antigen. Primary clear cell carcinoma is CK7 positive, CD10 negative, and RCC negative. Finally, when transitional cell tumors do not contain a benign Brenner component, distinction from a metastatic urothelial tumor is important. Primary ovarian transitional cell carcinoma is CK7 positive. Although metastatic UCa is also CK7 positive, it can stain for CK20, thrombomodulin, or uroplakin, with the latter results differentiating it from ovarian transitional cell carcinoma. Transitional cell carcinoma is WT1 positive and is more likely than UCa to stain for estrogen and progesterone receptors. Other carcinomas that metastasize to the ovary, and for which IHC is helpful in the differential diagnosis, include breast, lung, and thyroid. Breast cancer only rarely poses a differential diagnostic problem, because the patient is usually known to have a history of a breast neoplasm,622,623 and the morphology of metastatic breast cancer differs from that of most ovarian cancers. However, some breast cancers, particularly those with a micropapillary appearance, can be difficult to differentiate from a primary ovarian carcinoma.624 Considerable overlap is seen in the immunophenotypes of breast and ovarian cancer. Both are CK7 positive and CK20
705
negative, and they may show staining for estrogen and progesterone receptors. Some breast cancers show staining for gross cystic disease fluid protein 15 (GCDFP15), making this a potentially useful stain for differentiating between breast and ovarian cancers.330,625 In our practice, and as noted in reports by others,343 GCDFP-15 lacks sensitivity, thus it has not proved to be particularly useful as a marker of metastatic breast cancer. We find mammaglobin to be a more useful marker for metastatic breast cancer.626 WT1 is more commonly expressed by serous ovarian cancer than by metastatic breast carcinoma. CA 125 is also more likely to stain primary ovarian carcinomas, but staining is seen in a significant minority of breast cancers. An IHC panel that includes WT1, CA 125, mammaglobin, and GCDFP-15 generally helps resolve the differential diagnosis between metastatic breast cancer and primary ovarian cancer. Pax-8 is extremely useful in this differential diagnosis, because it is reported to stain all types of ovarian cancer, including mucinous tumor (approximately 50% to 60%) and it is negative in breast cancer.474 TTF-1 is a nuclear transcription factor with staining that is specific for tumors of the thyroid and lung. Thyroid tumors occasionally metastasize to the ovaries, and their nature can be confirmed by positive staining for TTF-1, as well as for thyroglobulin or calcitonin,627 depending upon their histologic type. Adenocarcinoma and small cell carcinoma of the lung also stain for TTF-1 in a high percentage of cases, and staining for this marker can be helpful when a lung primary is considered for a metastatic tumor in the ovary.627-629 TTF-1 positivity is confirmatory of lung origin for metastatic adenocarcinoma. Nonpulmonary small cell carcinomas can also stain for TTF-1,93,630,631 so a positive TTF-1 stain does not prove that a small cell carcinoma in the ovary is metastatic from the lung; it could be primary or metastatic from some other site.632 Rare gynecologic tract tumors can express TTF-1 as well as napsin. Pax-8 is a useful adjunct in these cases. Clinicopathologic correlation is necessary to establish the primary site. Metastases from other genital tract sites can be difficult to differentiate from primary ovarian neoplasms. Morphologic criteria have been developed to decide whether tumors in the endometrium and ovary are separate synchronous primaries or an endometrial primary with ovarian metastases.201 For most tumor types, IHC is not helpful in resolving this diagnostic dilemma, because endometrioid and clear cell endometrial and ovarian neoplasms have similar immunophenotypes. IHC may be helpful in the evaluation of serous carcinomas; although a high proportion of ovarian primary tumors show strong nuclear staining for WT1, most167,471 but not all168 authors have found that staining is detected in a much lower proportion of endometrial serous carcinomas. Thus lack of staining for WT1 favors an endometrial primary with ovarian metastasis. Endocervical adenocarcinomas occasionally metastasize to the ovaries, where they can mimic a primary endometrioid or mucinous adenocarcinoma.504,633 Cervical tumors that metastasize to the ovaries are often deeply invasive, but metastases can arise from surprisingly superficial cervical adenocarcinomas. Cervical adenocarcinomas are often CEA positive and, because of the
706
Immunohistology of the Female Genital Tract
involvement of human papilloma virus (HPV) infection in their pathogenesis, they are often p16 positive (see the section entitled “Cervix”). Positive staining for CEA and diffuse strong staining for p16 in an ovarian tumor with an endometrioid appearance is compatible with a metastasis from the cervix; in this context, a positive result is strong cytoplasmic and/or nuclear staining in more than 75% of tumor cells.505 When metastatic cervical adenocarcinoma mimics an ovarian mucinous carcinoma, staining for CEA is not helpful, because ovarian mucinous adenocarcinoma is also CEA positive, but staining for p16 is compatible with metastatic cervical adenocarcinoma. Some have found p16 to be of value in identifying metastatic cervical adenocarcinoma in the ovary and have observed little staining in primary ovarian neoplasms.505 Others have reported staining of a substantial percentage of primary endometrioid and mucinous carcinomas, all of which were negative for HPV DNA.506 These conflicting results indicate that the use of p16 staining to determine whether an ovarian neoplasm is metastatic from the cervix requires further investigation. Virtually any type of tumor, including soft tissue tumors and hematopoietic neoplasms such as lymphoma and leukemia, can involve the ovaries, either primarily or as metastases from a distant site. Many of these have specific IHC features, as detailed elsewhere in this book, which can be used to assist in their diagnosis. KEY DIAGNOSTIC POINTS Metastatic Tumors • From 5% to 10% of malignant ovarian tumors are metastases. • Colorectal adenocarcinoma is the most frequent metastasis, and it can mimic endometrioid and mucinous adenocarcinoma of the ovary. • The most useful stains for identifying metastases in the ovary are CK7, CK20, Pax-8, and CDX-2. • Primary ovarian carcinomas are usually CK20 negative, CK7 positive, Pax-8 positive, and CDX-2 negative except for mucinous carcinoma, which is CK7 positive and variably positive for CK20 and CDX-2. Also, 60% of primary ovarian mucinous tumors are Pax-8 positive. • Metastatic tumors are usually, but not always, Pax-8 negative. The chief exceptions are renal cell carcinoma, thyroid carcinoma, and thymic carcinoma. • Metastases from colorectal adenocarcinoma are usually, but not always, CK20 positive.
Peritoneal Mesothelioma Serous tumors that involve the ovaries and peritoneal surfaces can clinically and pathologically raise the possibility of mesothelioma. Although uncommon, peritoneal mesothelioma occurs in women and occasionally in children,634 and rarely it can be confined to the surfaces of the ovaries.635-637 Peritoneal mesotheliomas tend to be of the epithelioid type and papillary; sarcomatoid and mixed mesotheliomas are uncommon.638 A deciduoid variant of mesothelioma, in which the tumor cells are large with abundant cytoplasm, can occur in young women.639-641 These tumors are rarely associated with asbestos exposure. The differential diagnosis between peritoneal mesothelioma and serous carcinoma typically requires IHC analysis. Currently, a panel of IHC stains is performed (Table 18-11),339,341 including stains that are usually positive in mesothelioma and negative in serous carcinoma—such as calretinin, D2-40, and h-caldesmon642—and stains that are generally positive in serous carcinoma but negative in mesothelioma such as BerEP4, MOC-31, Pax-8, and ER (Fig. 18-49).643-645 A small percentage of mesotheliomas show positive staining, which is usually focal, for BerEP4 or MOC-31.646 It is worth noting that some of the stains used to evaluate pleural mesotheliomas are less useful in evaluating peritoneal mesotheliomas. CD15 is positive in only a minority of serous carcinomas, CEA is rarely positive in serous carcinoma, and WT1 is typically positive in both mesothelioma and serous carcinoma; therefore none of these antibodies are used in the IHC panel. Because CK5/6 and occasionally D2-40 can also be expressed in low-grade müllerian serous proliferations, these markers also have limited utility in this differential diagnosis in this site.
Fallopian Tube and Broad Ligament Tumors of the fallopian tube and broad ligament are much less common than ovarian neoplasms. Most occur in the fallopian tube, and epithelial tumors are by far the most common.647 Benign and borderline tumors are rare, and most fallopian tube tumors are carcinomas. The same types of carcinoma that occur in the ovary also occur in the fallopian tube. Certain types that are relatively common in the ovary, such as clear cell carcinoma and mucinous carcinoma, rarely arise in the tube. The most common histologic type of tubal carcinoma is serous carcinoma. Endometrioid carcinoma is the second most common, and most of the remaining cases
TABLE 18-11 Peritoneal Mesothelioma vs. Serous Carcinoma Calretinin
BerEP4
MOC-31
ER
Pax-8
Mesothelioma
+
−
−
−
−*
Serous carcinoma
−
+
+
+
+
*Weak and/or strong, patchy staining may be seen. +, Almost always positive; −, negative. ER, Estrogen receptors.
Peritoneal Mesothelioma
A
707
B
Figure 18-49 Mesothelioma. A, The tubular growth pattern and cuboidal appearance of the tumor cells usually differentiate mesothelioma from serous carcinoma, but in some cases the histologic patterns overlap. B, Mesothelioma shows strong cytoplasmic and nuclear staining for calretinin, as shown.
are transitional cell and undifferentiated carcinomas. In one large series, the frequency of the various types of carcinoma was 50% serous, 25% endometrioid, 11% transitional, 8% undifferentiated, 4% mixed cell types, and 2% clear cell.315 Other studies have reported a higher proportion of serous carcinomas.316-318 The IHC features of fallopian tube carcinomas are similar to those of their ovarian counterparts, which are discussed earlier in the chapter. Serous carcinoma of the fallopian tube, the most frequent type, has the same immunophenotype as serous carcinoma of the ovary, and most tumors show positive immunostaining for WT1, p53, CK7, and CA 125. In recent years, studies of risk-reducing salpingooophorectomy specimens from women with BRCA mutations have shown that incidentally discovered intraepithelial and invasive carcinomas, almost invariably of the serous type, are found mainly in the fallopian tubes rather than the ovaries.319-321 Detection of small, grossly invisible carcinomas in these patients requires
A
sectioning of all tissue. Tubal intraepithelial carcinoma (TIC) is characterized by marked nuclear atypia, nuclear stratification and disorder, and mitotic activity (Fig. 18-50, A). Small invasive carcinomas form masses of malignant cells in the tubal mucosa. The neoplasms, which are preferentially located in the fimbriae of the tubes,322 typically show diffuse strong nuclear staining for p53, MIB-1, and WT1 (see Fig. 18-50, B). The p53 and MIB-1 stains help delineate the neoplasms, often highlighting subtle foci of intraepithelial carcinoma that can then be confirmed by careful evaluation of routine H&E-stained sections. A possible precursor of intraepithelial carcinoma, the so-called p53 signature lesion, is p53 positive but lacks the atypia and proliferative activity present in intraepithelial carcinoma.323,324 Staining for MIB-1 differentiates the signature lesion from intraepithelial carcinoma, because staining is low in the former, whereas most nuclei are positive in the latter. Serial sectioning studies of the fallopian tube have shown that intraepithelial and invasive carcinomas,
B
Figure 18-50 Tubal intraepithelial carcinoma (TIC). A, The columnar tubal lining cells have stratified, enlarged, hyperchromatic atypical nuclei. B, Intraepithelial carcinoma, shown here, and invasive serous carcinoma typically show diffuse strong nuclear staining for p53 that helps to confirm the diagnosis and to identify small inconspicuous foci of TIC. Also, TIC is strongly positive for the proliferation marker MIB-1, which shows a similar pattern of staining to that shown here.
708
Immunohistology of the Female Genital Tract
usually of the serous type, are far more frequent than was previously appreciated. Serous neoplasia is frequently present in the fallopian tubes of women with serous carcinoma of the ovaries or peritoneum, and it has been proposed that many carcinomas that involve these sites represent metastases from a carcinoma of the fallopian tube rather than primary neoplasms.325Alternatively, this could be an example of multifocal tumorigenesis.326 Two tumor types occur preferentially in the region of the tube and broad ligament that rarely occur in the ovary. The first is the adenomatoid tumor, and the second is the female adnexal tumor of wolffian origin (FATWO).
KEY DIAGNOSTIC POINTS Fallopian Tube and Broad Ligament • Epithelial tumors of the tube and peritubal region resemble ovarian tumors and have similar immunophenotypic features. • No stains differentiate between primary tumors of the ovary and those of the fallopian tube. • Staining for cytokeratin and calretinin helps with the identification of adenomatoid tumors. • The immunophenotype of the FATWO overlaps with that of epithelial and sex cord–stromal tumors, but positive cytoplasmic staining for CD10 may help differentiate it. Lack of staining for EMA and positive staining for calretinin differentiate the FATWO from an endometrioid carcinoma variant.
FEMALE ADNEXAL TUMOR OF WOLFFIAN ORIGIN Most Useful Antibodies: Cytokeratin 7, Epithelial Membrane Antigen, Inhibin, Calretinin, CD10
The female adnexal tumor of wolffian origin (FATWO) is a distinctive tumor that arises in the broad ligament, attached to the mesosalpinx, or rarely in the ovary.655-657 It may be derived from mesonephric remnants, which are common in the area. Most adnexal tumors of wolffian origin are found in middle-aged women and are benign. Rare malignant variants show increased mitotic activity or cytologic atypia, overgrowth of spindle cells or lymphovascular space invasion, or may unexpectedly metastasize. The FATWO is a solid neoplasm that ranges from 2 to 20 cm in diameter. It is composed of uniform polygonal or spindled epithelial cells that grow in diffuse, trabecular, tubular, retiform, and microcystic patterns. The nuclei are uniform and darkly stained, and mitotic figures and cytologic atypia are typically absent. The main differential diagnostic considerations are a variant of endometrioid carcinoma of the fallopian tube658-660 and a sex cord–stromal tumor, such as a granulosa cell tumor or a Sertoli cell tumor. The immunophenotype is similar to that of the rete ovarii, and it overlaps between that of an epithelial tumor and that of a sex cord–stromal tumor.661 The FATWO is CK positive, but most are EMA negative,661-663 and immunostains for inhibin and calretinin are frequently positive.366,661 The FATWO is CD10 positive and shows cytoplasmic staining, which helps to differentiate it from other diagnostic considerations.518
Genomic Applications ADENOMATOID TUMOR Most Useful Antibodies: Cytokeratin, Calretinin
Adenomatoid tumor is the most common benign tumor of the fallopian tube,648 generally viewed as of mesothelial origin, although origin from submesothelial mesenchymal cells has also been considered.649-651 Adenomatoid tumors are small neoplasms that are usually only 1 to 2 cm in diameter. Microscopically, they grow as cords and tubules, lined by cuboidal cells with eosinophilic cytoplasm, or as glandlike cystic spaces, lined by flattened cells. Some tumor cells have prominent cytoplasmic vacuoles; these are sometimes mistaken for signet-ring cells. The epithelial elements can have a somewhat infiltrative appearance, raising the differential diagnosis of adenocarcinoma. The gross circumscription, bland cytology, and lack of mitotic activity are features that distinguish adenomatoid tumors from malignant neoplasms. IHC studies indicate that adenomatoid tumors are of mesothelial origin. Immunostains for CK5/6, calretinin, and WT1 are positive.652,653 Stains that mark epithelial tumors—such as BerEP4, CEA, and B72.3—are usually negative or at most are weakly and focally positive.654 In one study of uterine adenomatoid tumors, a high frequency of staining was found for BerEP4,145 which is inconsistent with other reports and experience with mesothelioma in general.
Although endometrial adenocarcinoma is the most common gynecologic cancer in Lynch syndrome, approximately 12% of women with Lynch syndrome will develop ovarian cancer.664-667 The same types of testing performed on endometrial cancer specimens can also be performed on ovarian tumors. Given the comparatively lower incidence of this carcinoma in patients with Lynch syndrome, routine screening of women with ovarian cancers with IHC for MMR proteins is probably not warranted; however, all women with a suggestive clinical history should be tested.
Theranostic Applications Clinical and routine pathologic findings are of prognostic significance in tumors of the ovary and fallopian tube. Pathologic stage is the most significant prognostic factor. The tumor grade and tumor type are of some importance, and IHC can provide assistance in the accurate classification of ovarian tumors, as detailed earlier in the chapter. No IHC stain has been identified that is generally accepted to have independent prognostic significance in ovarian or fallopian tube cancer, and we do not routinely perform any immunostains for such a purpose. A variety of markers have been tested in ovarian and tubal neoplasms, including p53, Bcl-2, and BAX.668,669 In general, standard clinical-pathologic
Summary
parameters have provided more significant prognostic information than IHC.670 The authors of a recent study concluded that the various histologic types of ovarian cancer are different diseases, and that they should not be grouped together, as is usually done, in assessing the prognostic value of various biomarkers.671,672 IHC studies have suffered from problems such as variable definitions of a positive result, use of different antibodies, and variations in fixation and testing parameters. A recent centrally tested and centrally reviewed, multi-institutional case study of ER and PR expression in ovarian serous carcinoma has demonstrated impaired prognosis for these tumors expressing PR.672 Similarly designed, largescale controlled studies may provide additional prognoses and therapeutic clinical assays.
Beyond Immunohistochemistry: Diagnostic Applications of Molecular Pathology As discussed earlier in this chapter, Lynch syndrome or hereditary nonpolyposis colon cancer syndrome (HNPCC) is a cancer predisposition syndrome caused by mutation of an MMR gene. Although IHC and molecular testing for abnormalities of the MMR genes and proteins is important, mutations in these genes account for only a small percentage of ovarian cancer cases. In contrast, mutation of one of the BRCA genes, BRCA1 or BRCA2, is responsible for up to 10% of ovarian cancers. Women with germline BRCA mutations have a high risk of developing breast and ovarian cancer.673 Mutation of the BRCA1 gene, located on chromosome 17 (17q12-21), accounts for approximately 70% of hereditary ovarian cancer cases and approximately 7% of ovarian cancer cases overall. Women with a BRCA1 mutation have a 36% to 40% risk of developing ovarian cancer by age 70, with most cancers developing after age 40 years. The BRCA2 gene is located on chromosome 13 (13q12-13), and women with mutations of this gene have a 10% to 27% risk of developing ovarian cancer by age 70 years, with most cancers developing after age 50 years. BRCA2 mutations account for about 2% of ovarian cancers. Women with BRCA mutations also have a high risk of developing breast cancer. No IHC tests can detect abnormalities of the BRCA genes, nor are there any simple screening tests, so identification of a mutation requires molecular testing. Some mutations are common in specific populations. If the patient belongs to a kindred with a specific known mutation, such as women of Ashkenazi Jewish heritage, the pathologist can test for the known mutations.674 Otherwise, full sequencing of both genes is necessary to identify abnormalities. Genetic counseling and testing for BRCA mutations have become increasingly important, because women with such mutations are often offered risk-reducing surgery, usually bilateral laparoscopic salpingo-oophorectomy, as a means of eliminating their risk of developing ovarian cancer. Clinical studies of women with BRCA mutations or strong family histories of ovarian cancer have shown that prophylactic bilateral
709
salpingo-oophorectomy is highly effective in reducing the risk of pelvic cancer, and that the risk of breast cancer is also reduced.675,676 Not all cancers are prevented, because some women have primary peritoneal cancer develop after prophylactic bilateral salpingooophorectomy. Thorough pathologic study of prophylactic salpingo-oophorectomy specimens has revealed occult intraepithelial or invasive carcinoma, usually high-grade serous-type lesions, in 5% to 10% of patients who underwent the procedure. Interestingly, occult cancers occur more often in the fallopian tubes than in the ovaries.319-322,677 Molecular pathologic studies of epithelial tumors of the ovary are an area of extensive research and will doubtless result in additional tests that help in the diagnosis, treatment, and prognosis of ovarian cancer. At the present time, however, the two areas discussed earlier in this section are the only ones with wide clinical applications. Molecular pathologic studies of germ cell and sex cord–stromal tumors are less established, mainly because these tumors are rare. Abnormalities of chromosome 12p, usually an isochromosome 12p, are frequent in some GCTs, particularly in dysgerminoma. These abnormalities can be detected by FISH and by standard genetic analysis. In one study, chromosome 12p abnormalities were detected by FISH in 81% of dysgerminomas.678 This type of testing can be used for diagnostic purposes; however, because the diagnosis is usually readily established by routine light microscopy with or without the use of IHC, FISH is rarely performed. Moreover, a test for chromosome 12p abnormalities is not readily available. Somatic mutations in ARID1A have been implicated in the pathogenesis of endometriosis-associated ovarian endometrioid and clear cell carcinomas. Mutation of the ARID1A gene is associated with loss of the corresponding protein BAF250a in clear cell and endometrioid carcinomas of the ovary. Loss of BAF250a is also seen in some endometrial carcinomas but is infrequent in other types of malignancies. Mutations in ARID1A and loss of BAF250a have been identified in atypical endometriosis adjacent to clear cell and endometrioid carcinomas and, in some cases, in atypical endometriosis preceding the development of endometrioid carcinoma. Testing for loss of BAF250a is not currently utilized for diagnosis, but a possible role in prognosis of endometriotic lesions is under investigation.679
Summary IHC has added a great deal to the understanding of gynecologic tumor pathogenesis and proper diagnoses in gynecologic pathology. Special attention to detail is necessary for IHC interpretation throughout the müllerian tract, because different antibodies have boutique diagnostic uses in various regions of the müllerian tract. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Sano T, Oyama T, Kashiwabara K, et al: Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions. Am J Pathol. 153:1741– 1748, 1998. 2. Agoff SN, Lin P, Morihara J, et al: p16(INK4a) expression correlates with degree of cervical neoplasia: A comparison with Ki-67 expression and detection of high-risk HPV types. Mod Pathol. 16:665–673, 2003. 3. Ishikawa M, Fujii T, Masumoto N, et al: Correlation of p16INK4A overexpression with human papillomavirus infection in cervical adenocarcinomas. Int J Gynecol Pathol. 22:378–385, 2003. 4. Keating JT, Cviko A, Riethdorf S, et al: Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia. Am J Surg Pathol. 25:884–891, 2001. 5. Klaes R, Benner A, Friedrich T, et al: p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia. Am J Surg Pathol. 26:1389– 1399, 2002. 6. Negri G, Egarter-Vigl E, Kasal A, et al: p16INK4a is a useful marker for the diagnosis of adenocarcinoma of the cervix uteri and its precursors: An immunohistochemical study with immunocytochemical correlations. Am J Surg Pathol. 27:187–193, 2003. 7. Riethdorf L, Riethdorf S, Lee KR, et al: Human papillomaviruses, expression of p16, and early endocervical glandular neoplasia. Hum Pathol. 33:899–904, 2002. 8. Tringler B, Gup CJ, Singh M, et al: Evaluation of p16INK4a and pRb expression in cervical squamous and glandular neoplasia. Hum Pathol. 35:689–696, 2004. 9. O’Neill CJ, McCluggage WG: p16 expression in the female genital tract and its value in diagnosis. Adv Anat Pathol. 13:8–15, 2006. 10. Armes JE, Lourie R, de SM, et al: Abnormalities of the RB1 pathway in ovarian serous papillary carcinoma as determined by overexpression of the p16(INK4A) protein. Int J Gynecol Pathol. 24:363–368, 2005. 11. Zeng HA, Cartun R, Ricci A, Jr: Potential diagnostic utility of CDX-2 immunophenotyping in extramammary Paget’s disease. Appl Immunohistochem Mol Morphol. 13:342–346, 2005. 12. Battles OE, Page DL, Johnson JE: Cytokeratins, CEA, and mucin histochemistry in the diagnosis and characterization of extramammary Paget’s disease. Am J Clin Pathol. 108:6–12, 1997. 13. Goldblum JR, Hart WR: Vulvar Paget’s disease: A clinicopathologic and immunohistochemical study of 19 cases. Am J Surg Pathol. 21:1178–1187, 1997. 14. Nowak MA, Guerriere-Kovach P, Pathan A, et al: Perianal Paget’s disease: Distinguishing primary and secondary lesions using immunohistochemical studies including gross cystic disease fluid protein-15 and cytokeratin 20 expression. Arch Pathol Lab Med. 122:1077–1081, 1998. 15. Olson DJ, Fujimura M, Swanson P, et al: Immunohistochemical features of Paget’s disease of the vulva with and without adenocarcinoma. Int J Gynecol Pathol. 10:285–295, 1991. 16. Crawford D, Nimmo M, Clement PB, et al: Prognostic factors in Paget’s disease of the vulva: A study of 21 cases. Int J Gynecol Pathol. 18:351–359, 1999. 17. Brummer O, Stegner HE, Bohmer G, et al: HER-2/neu expression in Paget disease of the vulva and the female breast. Gynecol Oncol. 95:336–340, 2004. 18. Zhang C, Zhang P, Sung CJ, et al: Overexpression of p53 is correlated with stromal invasion in extramammary Paget’s disease of the vulva. Hum Pathol. 34:880–885, 2003. 19. Ohnishi T, Watanabe S: The use of cytokeratins 7 and 20 in the diagnosis of primary and secondary extramammary Paget’s disease. Br J Dermatol. 142:243–247, 2000. 20. Brown HM, Wilkinson EJ: Uroplakin-III to distinguish primary vulvar Paget disease from Paget disease secondary to urothelial carcinoma. Hum Pathol. 33:545–548, 2002. 21. Lopez-Beltran A, Luque RJ, Moreno A, et al: The pagetoid variant of bladder urothelial carcinoma in situ A clinicopathological study of 11 cases. Virchows Arch. 441:148–153, 2002. 22. Diaz de Leon E, Carcangiu ML, Prieto VG, et al: Extramammary Paget disease is characterized by the consistent lack of estrogen
709.e1
and progesterone receptors but frequently expresses androgen receptor. Am J Clin Pathol. 113:572–575, 2000. 23. Glasgow BJ, Wen DR, Al-Jitawi S, et al: Antibody to S-100 protein aids the separation of pagetoid melanoma from mammary and extramammary Paget’s disease. J Cutan Pathol. 14:223–226, 1987. 24. Armes JE, Lourie R, Bowlay G, et al: Pagetoid squamous cell carcinoma in situ of the vulva: Comparison with extramammary Paget disease and nonpagetoid squamous cell neoplasia. Int J Gynecol Pathol. 27:118–124, 2008. 25. Raju RR, Goldblum JR, Hart WR: Pagetoid squamous cell carcinoma in situ (pagetoid Bowen’s disease) of the external genitalia. Int J Gynecol Pathol. 22:127–135, 2003. 26. Williamson JD, Colome MI, Sahin A, et al: Pagetoid bowen disease: A report of 2 cases that express cytokeratin 7. Arch Pathol Lab Med. 124:427–430, 2000. 27. McCluggage WG: Recent developments in vulvovaginal pathology. Histopathology. 2008. 28. Iwasa Y, Fletcher CD: Cellular angiofibroma: Clinicopathologic and immunohistochemical analysis of 51 cases. Am J Surg Pathol. 28:1426–1435, 2004. 29. McCluggage WG, Ganesan R, Hirschowitz L, et al: Cellular angiofibroma and related fibromatous lesions of the vulva: Report of a series of cases with a morphological spectrum wider than previously described. Histopathology. 45:360–368, 2004. 30. Lam MM, Corless CL, Goldblum JR, et al: Extragastrointestinal stromal tumors presenting as vulvovaginal/rectovaginal septal masses: A diagnostic pitfall. Int J Gynecol Pathol. 25:288–292, 2006. 31. Guillou L, Wadden C, Coindre JM, et al: Proximal-type” epithelioid sarcoma, a distinctive aggressive neoplasm showing rhabdoid features. Clinicopathologic, immunohistochemical, and ultrastructural study of a series. Am J Surg Pathol. 21:130–146, 1997. 32. Cessna MH, Zhou H, Perkins SL, et al: Are myogenin and myoD1 expression specific for rhabdomyosarcoma? A study of 150 cases, with emphasis on spindle cell mimics. Am J Surg Pathol. 25:1150–1157, 2001. 33. Morotti RA, Nicol KK, Parham DM, et al: An immunohistochemical algorithm to facilitate diagnosis and subtyping of rhabdomyosarcoma: the Children’s Oncology Group experience. Am J Surg Pathol. 30:962–968, 2006. 34. Medeiros F, Erickson-Johnson MR, Keeney GL, et al: Frequency and characterization of HMGA2 and HMGA1 rearrangements in mesenchymal tumors of the lower genital tract. Genes Chromosomes Cancer. 46:981–990, 2007. 35. Medeiros F, Oliveira AM, Lloyd R: HMGA2 expression as a biomarker for aggressive angiomyxoma. Mod Pathol. 21:214A, 2008. 36. Nucci MR, Weremowicz S, Neskey DM, et al: Chromosomal translocation t(8;12) induces aberrant HMGIC expression in aggressive angiomyxoma of the vulva. Genes Chromosomes Cancer. 32:172–176, 2001. 37. Rabban JT, Dal CP, Oliva E: HMGA2 rearrangement in a case of vulvar aggressive angiomyxoma. Int J Gynecol Pathol. 25:403– 407, 2006. 38. Horowitz IR, Copas P, Majmudar B: Granular cell tumors of the vulva. Am J Obstet Gynecol. 173:1710–1713, 1995. 39. Le BH, Boyer PJ, Lewis JE, et al: Granular cell tumor: Immunohistochemical assessment of inhibin-alpha, protein gene product 9.5, S100 protein, CD68, and Ki-67 proliferative index with clinical correlation. Arch Pathol Lab Med. 128:771–775, 2004. 40. Brustmann H, Naude S: Expression of topoisomerase II alpha, Ki-67, proliferating cell nuclear antigen, p53, and argyrophilic nucleolar organizer regions in vulvar squamous lesions. Gynecol Oncol. 86:192–199, 2002. 41. Pirog EC, Chen YT, Isacson C: MIB-1 immunostaining is a beneficial adjunct test for accurate diagnosis of vulvar condyloma acuminatum. Am J Surg Pathol. 24:1393–1399, 2000. 42. Scurry J, Beshay V, Cohen C, et al: Ki67 expression in lichen sclerosus of vulva in patients with and without associated squamous cell carcinoma. Histopathology. 32:399–404, 1998. 43. van Hoeven KH, Kovatich AJ: Immunohistochemical staining for proliferating cell nuclear antigen, BCL2, and Ki-67 in vulvar tissues. Int J Gynecol Pathol. 15:10–16, 1996.
709.e2
Immunohistology of the Female Genital Tract
44. Bai H, Cviko A, Granter S, et al: Immunophenotypic and viral (human papillomavirus) correlates of vulvar seborrheic keratosis. Hum Pathol. 34:559–564, 2003. 45. Riethdorf S, Neffen EF, Cviko A, et al: p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias. Hum Pathol. 35:1477–1483, 2004. 46. Santos M, Montagut C, Mellado B, et al: Immunohistochemical staining for p16 and p53 in premalignant and malignant epithelial lesions of the vulva. Int J Gynecol Pathol. 23:206–214, 2004. 47. Ruhul QM, Xu C, Steinhoff MM, et al: Simplex (differentiated) type VIN: Absence of p16INK4 supports its weak association with HPV and its probable precursor role in non-HPV related vulvar squamous cancers. Histopathology. 46:718–720, 2005. 48. Mulvany NJ, Allen DG: Differentiated intraepithelial neoplasia of the vulva. Int J Gynecol Pathol. 27:125–135, 2008. 49. Yang B, Hart WR: Vulvar intraepithelial neoplasia of the simplex (differentiated) type: A clinicopathologic study including analysis of HPV and p53 expression. Am J Surg Pathol. 24:429–241, 2000. 50. Liegl B, Regauer S: p53 immunostaining in lichen sclerosus is related to ischaemic stress and is not a marker of differentiated vulvar intraepithelial neoplasia (d-VIN). Histopathology. 48:268– 274, 2006. 51. McCluggage WG, Oliva E, Herrington CS, et al: CD10 and calretinin staining of endocervical glandular lesions, endocervical stroma and endometrioid adenocarcinomas of the uterine corpus: CD10 positivity is characteristic of, but not specific for, mesonephric lesions and is not specific for endometrial stroma. Histopathology. 43:144–150, 2003. 52. Ordi J, Romagosa C, Tavassoli FA, et al: CD10 expression in epithelial tissues and tumors of the gynecologic tract: A useful marker in the diagnosis of mesonephric, trophoblastic, and clear cell tumors. Am J Surg Pathol. 27:178–186, 2003. 53. Silver SA, Devouassoux-Shisheboran M, Mezzetti TP, et al: Mesonephric adenocarcinomas of the uterine cervix: A study of 11 cases with immunohistochemical findings. Am J Surg Pathol. 25:379–387, 2001. 54. Mittal K, Mesia A, Demopoulos RI: MIB-1 expression is useful in distinguishing dysplasia from atrophy in elderly women. Int J Gynecol Pathol. 18:122–124, 1999. 55. Kruse AJ, Baak JP, Helliesen T, et al: Evaluation of MIB-1positive cell clusters as a diagnostic marker for cervical intraepithelial neoplasia. Am J Surg Pathol. 26:1501–1507, 2002. 56. McCluggage WG, Buhidma M, Tang L, et al: Monoclonal antibody MIB1 in the assessment of cervical squamous intraepithelial lesions. Int J Gynecol Pathol. 15:131–136, 1996. 57. Pirog EC, Baergen RN, Soslow RA, et al: Diagnostic Accuracy of Cervical Low-Grade Squamous Intraepithelial Lesions Is Improved With MIB-1 Immunostaining. Am J Surg Pathol. 26:70–75, 2002. 58. Klaes R, Friedrich T, Spitkovsky D, et al: Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer. 92:276–284, 2001. 59. Benevolo M, Mottolese M, Marandino F, et al: Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol. 19:384–391, 2006. 60. Kong CS, Balzer BL, Troxell ML, et al: p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia. Am J Surg Pathol. 31:33–43, 2007. 61. Shi J, Liu H, Wilkerson M, et al: Evaluation of p16INK4a, minichromosome maintenance protein 2, DNA topoisomerase II alpha, ProEX C, and p16INK4a/ProEX C in cervical squamous intraepithelial lesions. Hum Pathol. 38:1335–1344, 2007. 62. Pinto AP, Schlecht NF, Woo TY, et al: Biomarker (ProEx C, p16(INK4A), and MiB-1) distinction of high-grade squamous intraepithelial lesion from its mimics. Mod Pathol. 21:1067– 1074, 2008. 63. Cina SJ, Richardson MS, Austin RM, et al: Immunohistochemical staining for Ki-67 antigen, carcinoembryonic antigen, and p53 in the differential diagnosis of glandular lesions of the cervix. Mod Pathol. 10:176–180, 1997.
64. Marques T, Andrade LA, Vassallo J: Endocervical tubal metaplasia and adenocarcinoma in situ: Role of immunohistochemistry for carcinoembryonic antigen and vimentin in differential diagnosis. Histopathology. 28:549–550, 1996. 65. Cameron RI, Maxwell P, Jenkins D, et al: Immunohistochemical staining with MIB1, bcl2 and p16 assists in the distinction of cervical glandular intraepithelial neoplasia from tuboendometrial metaplasia, endometriosis and microglandular hyperplasia. Histopathology. 41:313–321, 2002. 66. McCluggage WG, Maxwell P, McBride HA, et al: Monoclonal antibodies Ki-67 and MIB1 in the distinction of tuboendometrial metaplasia from endocervical adenocarcinoma and adenocarcinoma in situ in formalin-fixed material. Int J Gynecol Pathol. 14:209–216, 1995. 67. Ansari-Lari MA, Staebler A, Zaino RJ, et al: Distinction of endocervical and endometrial adenocarcinomas: Immunohistochemical p16 expression correlated with human papillomavirus (HPV) DNA detection. Am J Surg Pathol. 28:160–167, 2004. 68. Castrillon DH, Lee KR, Nucci MR: Distinction between endometrial and endocervical adenocarcinoma: An immunohistochemical study. Int J Gynecol Pathol. 21:4–10, 2002. 69. Kamoi S, Al-Juboury MI, Akin MR, et al: Immunohistochemical staining in the distinction between primary endometrial and endocervical adenocarcinomas: Another viewpoint. Int J Gynecol Pathol. 21:217–223, 2002. 70. McCluggage WG, Sumathi VP, McBride HA, et al: A panel of immunohistochemical stains, including carcinoembryonic antigen, vimentin, and estrogen receptor, aids the distinction between primary endometrial and endocervical adenocarcinomas. Int J Gynecol Pathol. 21:11–15, 2002. 71. McCluggage WG, Jenkins D: p16 immunoreactivity may assist in the distinction between endometrial and endocervical adenocarcinoma. Int J Gynecol Pathol. 22:231–235, 2003. 72. O’Neill CJ, McBride HA, Connolly LE, et al: High-grade ovarian serous carcinoma exhibits significantly higher p16 expression than low-grade serous carcinoma and serous borderline tumour. Histopathology. 50:773–779, 2007. 73. Qiu W, Mittal K: Comparison of morphologic and immunohistochemical features of cervical microglandular hyperplasia with low-grade mucinous adenocarcinoma of the endometrium. Int J Gynecol Pathol. 22:261–265, 2003. 74. McCluggage WG, Shah R, Connolly LE, et al: Intestinal-type cervical adenocarcinoma in situ and adenocarcinoma exhibit a partial enteric immunophenotype with consistent expression of CDX2. Int J Gynecol Pathol. 27:92–100, 2008. 75. Sullivan LM, Smolkin ME, Frierson HF, Jr, et al: Comprehensive Evaluation of CDX2 in Invasive Cervical Adenocarcinomas: Immunopositivity in the Absence of Overt Colorectal Morphology. Am J Surg Pathol. 32(11):1608–1612, 2008. 76. Gilks CB, Young RH, Aguirre P, et al: Adenoma malignum (minimal deviation adenocarcinoma) of the uterine cervix. A clinicopathological and immunohistochemical analysis of 26 cases. Am J Surg Pathol. 13:717–729, 1989. 77. Mikami Y, Kiyokawa T, Hata S, et al: Gastrointestinal immunophenotype in adenocarcinomas of the uterine cervix and related glandular lesions: A possible link between lobular endocervical glandular hyperplasia/pyloric gland metaplasia and “adenoma malignum”. Mod Pathol. 17:962–972, 2004. 78. Xu JY, Hashi A, Kondo T, et al: Absence of human papillomavirus infection in minimal deviation adenocarcinoma and lobular endocervical glandular hyperplasia. Int J Gynecol Pathol. 24:296– 302, 2005. 79. Mino M, Pilch BZ, Faquin WC: Expression of KIT (CD117) in neoplasms of the head and neck: An ancillary marker for adenoid cystic carcinoma. Mod Pathol. 16:1224–1231, 2003. 80. Grayson W, Taylor LF, Cooper K: Adenoid cystic and adenoid basal carcinoma of the uterine cervix: Comparative morphologic, mucin, and immunohistochemical profile of two rare neoplasms of putative “reserve cell” origin. Am J Surg Pathol. 23:448–458, 1999. 81. Grayson W, Cooper K: A reappraisal of “basaloid carcinoma” of the cervix, and the differential diagnosis of basaloid cervical neoplasms. Adv Anat Pathol. 9:290–300, 2002. 82. Emanuel P, Wang B, Wu M, et al: p63 Immunohistochemistry in the distinction of adenoid cystic carcinoma from basaloid squamous cell carcinoma. Mod Pathol. 18:645–650, 2005.
References 83. Ferry JA, Scully RE: “Adenoid cystic” carcinoma and adenoid basal carcinoma of the uterine cervix. A study of 28 cases. Am J Surg Pathol. 12:134–144, 1988. 84. Russell MJ, Fadare O: Adenoid basal lesions of the uterine cervix: Evolving terminology and clinicopathological concepts. Diagn Pathol. 1:18, 2006. 85. Cviko A, Briem B, Granter SR, et al: Adenoid basal carcinomas of the cervix: A unique morphological evolution with cell cycle correlates. Hum Pathol. 31:740–744, 2000. 86. Parwani AV, Smith Sehdev AE, Kurman RJ, et al: Cervical adenoid basal tumors comprised of adenoid basal epithelioma associated with various types of invasive carcinoma: Clinicopathologic features, human papillomavirus DNA detection, and P16 expression. Hum Pathol. 36:82–90, 2005. 87. Albores-Saavedra J, Latif S, Carrick KS, et al: CD56 reactivity in small cell carcinoma of the uterine cervix. Int J Gynecol Pathol. 24:113–117, 2005. 88. Gilks CB, Young RH, Gersell DJ, et al: Large cell neuroendocrine carcinoma of the uterine cervix: A clinicopathologic study of 12 cases. Am J Surg Pathol. 21:905–914, 1997. 89. Horn LC, Lindner K, Szepankiewicz G, et al: p16, p14, p53, and cyclin D1 expression and HPV analysis in small cell carcinomas of the uterine cervix. Int J Gynecol Pathol. 25:182–186, 2006. 90. Masumoto N, Fujii T, Ishikawa M, et al: P16 overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix. Hum Pathol. 34:778–783, 2003. 91. Wang HL, Lu DW: Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix. Am J Surg Pathol. 28:901–908, 2004. 92. Wang TY, Chen BF, Yang YC, et al: Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: A study of 250 cases. Hum Pathol. 32:479– 486, 2001. 93. Agoff SN, Lamps LW, Philip AT, et al: Thyroid transcription factor-1 is expressed in extrapulmonary small cell carcinomas but not in other extrapulmonary neuroendocrine tumors. Mod Pathol. 13:238–242, 2000. 94. McCluggage WG, Sargent A, Bailey A, et al: Large cell neuroendocrine carcinoma of the uterine cervix exhibiting TTF1 immunoreactivity. Histopathology. 51:405–407, 2007. 95. Guimaraes MC, Goncalves MA, Soares CP, et al: Immunohistochemical expression of p16INK4a and bcl-2 according to HPV type and to the progression of cervical squamous intraepithelial lesions. J Histochem Cytochem. 53:509–516, 2005. 96. Hariri J, Oster A: The negative predictive value of p16INK4a to assess the outcome of cervical intraepithelial neoplasia 1 in the uterine cervix. Int J Gynecol Pathol. 26:223–228, 2007. 97. Negri G, Vittadello F, Romano F, et al: p16INK4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri. Virchows Arch. 445:616–620, 2004. 98. Negri G, Bellisano G, Zannoni GF, et al: p16 ink4a and HPV L1 immunohistochemistry is helpful for estimating the behavior of low-grade dysplastic lesions of the cervix uteri. Am J Surg Pathol. 32:1715–1720, 2008. 99. Farhood AI, Abrams J: Immunohistochemistry of endometrial stromal sarcoma. Hum Pathol. 22:224–230, 1991. 100. Franquemont DW, Frierson HF, Jr, Mills SE: An immunohistochemical study of normal endometrial stroma and endometrial stromal neoplasms. Evidence for smooth muscle differentiation. Am J Surg Pathol. 15:861–870, 1991. 101. Oliva E, Young RH, Clement PB, et al: Cellular benign mesenchymal tumors of the uterus: A comparative morphologic and immunohistochemical analysis of 33 highly cellular leiomyomas and six endometrial stromal nodules, two frequently confused tumors. Am J Surg Pathol. 19:757–768, 1995. 102. Bitterman P, Chun B, Kurman RJ: The significance of epithelial differentiation in mixed mesodermal tumors of the uterus. A clinicopathological and immunohistochemical study. Am J Surg Pathol. 14:317–328, 1990. 103. George E, Manivel JC, Dehner LP, et al: Malignant mixed mullerian tumors: An immunohistochemical study of 47 cases, with histogenetic considerations and clinical correlation. Hum Pathol. 22:215–223, 1991.
709.e3
104. Ordonez NG: Value of PAX 8 immunostaining in tumor diagnosis: a review and update. Adv Anat Pathol. 19(3):140–151, 2012. 105. Dabbs DJ, Sturtz K, Zaino RJ: The immunohistochemical discrimination of endometrioid adenocarcinomas. Hum Pathol. 27:172–177, 1996. 106. Dabbs DJ, Geisinger KR, Norris HT: Intermediate filaments in endometrial and endocervical carcinomas. The diagnostic utility of vimentin patterns. Am J Surg Pathol. 10:568–576, 1986. 107. Sherman ME, Bur ME, Kurman RJ: p53 in endometrial cancer and its putative precursors: Evidence for diverse pathways of tumorigenesis. Hum Pathol. 26:1268–1274, 1995. 108. Tashiro H, Isacson C, Levine R, et al: p53 gene mutations are common in uterine serous carcinoma and occur early in their pathogenesis. Am J Pathol. 150:177–185, 1997. 109. Zheng WX, Khurana R, Farahmand S, et al: p53 immunostaining as a significant adjunct diagnostic method for uterine surface carcinoma—Precursor of uterine papillary serous carcinoma. Am J Surg Pathol. 22:1463–1473, 1998. 110. Kohler MF, Kerns B-JM, Humphrey PA, et al: Mutation and overexpression of p53 in early-stage epithelial ovarian cancer. Obstet Gynecol. 81:643–650, 1993. 111. Yu CC, Wilkinson N, Brito MJ, et al: Patterns of immunohistochemical staining for proliferating cell nuclear antigen and p53 in benign and neoplastic human endometrium. Histopathology. 23:367–371, 1993. 112. Lax SF, Pizer ES, Ronnett BM, et al: Comparison of estrogen and progesterone receptor, Ki-67, and p53 immunoreactivity in uterine endometrioid carcinoma and endometrioid carcinoma with squamous, mucinous, secretory, and ciliated cell differentiation. Hum Pathol. 29:924–931, 1998. 113. Lax SF, Kendall B, Tashiro H, et al: The frequency of p53, K-ras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma—Evidence of distinct molecular genetic pathways. Cancer. 88:814–824, 2000. 114. Soslow RA, Shen PU, Chung MH, et al: Distinctive p53 and mdm2 immunohistochemical expression profiles suggest different pathogenetic pathways in poorly differentiated endometrial carcinoma. Int J Gynecol Pathol. 17:129–134, 1998. 115. Carcangiu ML, Chambers JT, Voynick IM, et al: Immunohistochemical evaluation of estrogen and progesterone receptor content in 183 patients with endometrial carcinoma. Part I: Clinical and histologic correlations. Am J Clin Pathol. 94:247– 254, 1990. 116. Chambers JT, Carcangiu ML, Voynick IM, et al: Immunohistochemical evaluation of estrogen and progesterone receptor content in 183 patients with endometrial carcinoma. Part II: Correlation between biochemical and immunohistochemical methods and survival. Am J Clin Pathol. 94:255–260, 1990. 117. Soslow RA, Shen PU, Chung MH, et al: Cyclin D1 expression in high-grade endometrial carcinomas—Association with histologic subtype. Int J Gynecol Pathol. 19:329–334, 2000. 118. Nei H, Saito T, Yamasaki H, et al: Nuclear localization of betacatenin in normal and carcinogenic endometrium. Mol Carcinog. 25:207–218, 1999. 119. Schlosshauer PW, Ellenson LH, Soslow RA: Beta-catenin and E-cadherin expression patterns in high-grade endometrial carcinoma are associated with histological subtype. Mod Pathol. 15:1032–1037, 2002. 120. Risinger JI, Hayes AK, Berchuck A, et al: PTEN/MMAC1 mutations in endometrial cancers. Cancer Res. 57:4736–4738, 1997. 121. Tashiro H, Blazes MS, Wu R, et al: Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res. 57:3935–3940, 1997. 122. Obata K, Morland SJ, Watson RH, et al: Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors. Cancer Res. 58:2095–2097, 1998. 123. Simpkins SB, Peiffer-Schneider S, Mutch DG, et al: PTEN mutations in endometrial cancers with 10q LOH: Additional evidence for the involvement of multiple tumor suppressors. Gynecol Oncol. 71:391–395, 1998. 124. Yokoyama Y, Wan X, Shinohara A, et al: Expression of PTEN and PTEN pseudogene in endometrial carcinoma. Int J Mol Med. 6:47–50, 2000. 125. Darvishian F, Hummer AJ, Thaler HT, et al: Serous endometrial cancers that mimic endometrioid adenocarcinomas:
709.e4
Immunohistology of the Female Genital Tract
A clinicopathologic and immunohistochemical study of a group of problematic cases. Am J Surg Pathol. 28:1568–1578, 2004. 126. Lax SF, Pizer ES, Ronnett BM, et al: Clear cell carcinoma of the endometrium is characterized by a distinctive profile of p53, Ki-67, estrogen, and progesterone receptor expression. Hum Pathol. 29:551–558, 1998. 127. Vang R, Whitaker BP, Farhood AI, et al: Immunohistochemical analysis of clear cell carcinoma of the gynecologic tract. Int J Gynecol Pathol. 20:252–259, 2001. 128. Reid-Nicholson M, Iyengar P, Hummer AJ, et al: Immunophenotypic diversity of endometrial adenocarcinomas: Implications for differential diagnosis. Mod Pathol. 19:1091–1100, 2006. 129. Fukuchi T, Sakamoto M, Tsuda H, et al: Beta-catenin mutation in carcinoma of the uterine endometrium. Cancer Res. 58:3526– 3528, 1998. 130. Schlosshauer PW, Pirog EC, Levine RL, et al: Mutational analysis of the CTNNB1 and APC genes in uterine endometrioid carcinoma. Mod Pathol. 13:1066–1071, 2000. 131. Saegusa M, Hamano M, Kuwata T, et al: Up-regulation and nuclear localization of beta-catenin in endometrial carcinoma in response to progesterone therapy. Cancer Sci. 94:103–111, 2003. 132. Bussaglia E, Del Rio E, Matias-Guiu X, et al: PTEN mutations in endometrial carcinomas: A molecular and clinicopathologic analysis of 38 cases. Hum Pathol. 31:312–317, 2000. 133. Oliva E, Young RH, Clement PB, et al: Myxoid and fibrous endometrial stromal tumors of the uterus: A report of 10 cases. Int J Gynecol Pathol. 18:310–319, 1999. 134. Oliva E, Clement PB, Young RH, et al: Mixed endometrial stromal and smooth muscle tumors of the uterus—A clinicopathologic study of 15 cases. Am J Surg Pathol. 22:997–1005, 1998. 135. Nucci MR, O’Connell JT, Huettner PC, et al: h-Caldesmon expression effectively distinguishes endometrial stromal tumors from uterine smooth muscle tumors. Am J Surg Pathol. 25:455– 463, 2001. 136. Rush DS, Tan JY, Baergen RN, et al: h-caldesmon, a novel smooth muscle-specific antibody, distinguishes between cellular leiomyoma and endometrial stromal sarcoma. Am J Surg Pathol. 25:253–258, 2001. 137. Baker RJ, Hildebrandt RH, Rouse RV, et al: Inhibin and CD99 (MIC2) expression in uterine stromal neoplasms with sex-cordlike elements. Hum Pathol. 30:671–679, 1999. 138. Krishnamurthy S, Jungbluth AA, Busam KJ, et al: Uterine tumors resembling ovarian sex-cord tumors have an immunophenotype consistent with true sex-cord differentiation. Am J Surg Pathol. 22:1078–1082, 1998. 139. Oliva E, Young RH, Amin MB, et al: An immunohistochemical analysis of endometrial stromal and smooth muscle tumors of the uterus—A study of 54 cases emphasizing the importance of using a panel because of overlap in immunoreactivity for individual antibodies. Am J Surg Pathol. 26:403–412, 2002. 140. Czernobilsky B: Uterine tumors resembling ovarian sex cord tumors: An update. Int J Gynecol Pathol. 27:229–235, 2008. 141. Meis JM, Lawrence WD: The immunohistochemical profile of malignant mixed müllerian tumor. Overlap with endometrial adenocarcinoma. Am J Clin Pathol. 94:1–7, 1990. 142. Chu PG, Arber DA, Weiss LM, et al: Utility of CD10 in distinguishing between endometrial stromal sarcoma and uterine smooth muscle tumors: An immunohistochemical comparison of 34 cases. Mod Pathol. 14:465–471, 2001. 143. McCluggage WG, Sumathi VP, Maxwell P: CD10 is a sensitive and diagnostically useful immunohistochemical marker of normal endometrial stroma and of endometrial stromal neoplasms. Histopathology. 39:273–278, 2001. 144. Sumathi VP, Al-Hussaini M, Connolly LE, et al: Endometrial stromal neoplasms are immunoreactive with WT-1 antibody. Int J Gynecol Pathol. 23:241–247, 2004. 145. Otis CN: Uterine adenomatoid tumors: Immunohistochemical characteristics with emphasis on Ber-EP4 immunoreactivity and distinction from adenocarcinoma. Int J Gynecol Pathol. 15:146– 151, 1996. 146. Thor A, Viglione MJ, Muraro R, et al: Monoclonal antibody B72.3 reactivity with human endometrium: A study of normal and malignant tissues. Int J Gynecol Pathol. 6:235–247, 1987.
147. Hareyama H, Sakuragi N, Makinoda S, et al: Serum and tissue measurements of CA72-4 in patients with endometrial carcinoma. J Clin Pathol. 49:967–970, 1996. 148. Dallenbach-Hellweg G, Lang-Averous G, Hahn U: The value of immunohistochemistry in the differential diagnosis of endometrial carcinomas. APMIS. 23(Suppl):91–99, 1991. 149. Yaziji H, Gown AM: Immunohistochemical analysis of gynecologic tumors. Int J Gynecol Pathol. 20:64–78, 2001. 150. Wang NP, Zee S, Zarbo RJ, et al: Coordinate expression of cytokeratins 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem. 3:99–107, 1995. 151. Wani Y, Notohara K, Saegusa M, et al: Aberrant Cdx2 expression in endometrial lesions with squamous differentiation: important role of Cdx2 in squamous morula formation. Hum Pathol. 39:1072–1079, 2008. 152. Park KJ, Bramlage MP, Ellenson LH, et al: Immunoprofile of adenocarcinomas of the endometrium, endocervix, and ovary with mucinous differentiation. Appl Immunohistochem Mol Morphol. 17:8–11, 2009. 153. Bonazzi del Poggetto C, Virtanen I, Lehto VP, et al: Expression of intermediate filaments in ovarian and uterine tumors. Int J Gynecol Pathol. 1:359–366, 1983. 154. Azumi N, Battifora H: The distribution of vimentin and keratin in epithelial and nonepithelial neoplasms. A comprehensive immunohistochemical study on form. Am J Clin Pathol. 88:286– 296, 1987. 155. Koshiyama M, Konishi I, Wang DP, et al: Immunohistochemical analysis of p53 protein over-expression in endometrial carcinomas: Inverse correlation with sex steroid receptor status. Virchows Arch A Pathol Anat Histopathol. 423:265–271, 1993. 156. Chiesa-Vottero AG, Malpica A, Deavers MT, et al: Immunohistochemical overexpression of p16 and p53 in uterine serous carcinoma and ovarian high-grade serous carcinoma. Int J Gynecol Pathol. 26:328–333, 2007. 157. Pallares J, Bussaglia E, Martinez-Guitarte JL, et al: Immunohistochemical analysis of PTEN in endometrial carcinoma: A tissue microarray study with a comparison of four commercial antibodies in correlation with molecular abnormalities. Mod Pathol. 18:719–727, 2005. 158. De Leeuw WJF, Dierssen J, Vasen HFA, et al: Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients. J Pathol. 192:328–335, 2000. 159. Peiro G, Diebold J, Lohse P, et al: Microsatellite instability, loss of heterozygosity, and loss of hMLH1 and hMSH2 protein expression in endometrial carcinoma. Hum Pathol. 33:347–354, 2002. 160. Vasen HF, Hendriks Y, de Jong AE, et al: Identification of HNPCC by molecular analysis of colorectal and endometrial tumors. Dis Markers. 20:207–213, 2004. 161. Modica I, Soslow RA, Black D, et al: Utility of immunohistochemistry in predicting microsatellite instability in endometrial carcinoma. Am J Surg Pathol. 31:744–751, 2007. 162. Sherman ME, Bitterman P, Rosenshein NB, et al: Uterine serous carcinoma: A morphologically diverse neoplasm with unifying clinicopathologic features. Am J Surg Pathol. 16:600–610, 1992. 163. Ambros RA, Sherman ME, Zahn CM, et al: Endometrial intra epithelial carcinoma: A distinctive lesion specifically associated with tumors displaying serous differentiation. Hum Pathol. 26:1260–1267, 1995. 164. Liang SX, Chambers SK, Cheng L, et al: Endometrial glandular dysplasia: A putative precursor lesion of uterine papillary serous carcinoma. Part II: molecular features. Int J Surg Pathol. 12:319– 331, 2004. 165. Zheng W, Liang SX, Yu H, et al: Endometrial glandular dysplasia: A newly defined precursor lesion of uterine papillary serous carcinoma. Part I: morphologic features. Int J Surg Pathol. 12:207–223, 2004. 166. Alkushi A, Clarke BA, Akbari M, et al: Identification of prognostically relevant and reproducible subsets of endometrial adenocarcinoma based on clustering analysis of immunostaining data. Mod Pathol. 20:1156–1165, 2007. 167. Goldstein NS, Uzieblo A: WT1 immunoreactivity in uterine papillary serous carcinomas is different from ovarian serous carcinomas. Am J Clin Pathol. 117:541–545, 2002.
References 168. Acs G, Pasha T, Zhang PJ: WT1 is differentially expressed in serous, endometrioid, clear cell, and mucinous carcinomas of the peritoneum, fallopian tube, ovary, and endometrium. Int J Gynecol Pathol. 23:110–118, 2004. 169. Egan JA, Ionescu MC, Eapen E, et al: Differential expression of WT1 and p53 in serous and endometrioid carcinomas of the endometrium. Int J Gynecol Pathol. 23:119–122, 2004. 170. Silva EG, Young RH: Endometrioid neoplasms with clear cells: A report of 21 cases in which the alteration is not of typical secretory type. Am J Surg Pathol. 31:1203–1208, 2007. 171. An HJ, Logani S, Isacson C, et al: Molecular characterization of uterine clear cell carcinoma. Mod Pathol. 17:530–537, 2004. 172. Catasus L, Bussaglia E, Rodrguez I, et al: Molecular genetic alterations in endometrioid carcinomas of the ovary: Similar frequency of beta-catenin abnormalities but lower rate of microsatellite instability and PTEN alterations than in uterine endometrioid carcinomas. Hum Pathol. 35:1360–1368, 2004. 173. Carcangiu ML, Dorji T, Radice P, et al: HNPCC-related endometrial carcinomas show a high frequency of non-endometrioid types and of high FIGO grade endometrioid carcinomas. Mod Pathol. 19:173A, 2006. 174. Yamamoto S, Tsuda H, Aida S, et al: Immunohistochemical detection of hepatocyte nuclear factor 1beta in ovarian and endometrial clear-cell adenocarcinomas and nonneoplastic endometrium. Hum Pathol. 38:1074–1080, 2007. 175. Kato N, Tamura G, Motoyama T: Hypomethylation of hepatocyte nuclear factor-1beta (HNF-1beta) CpG island in clear cell carcinoma of the ovary. Virchows Archiv: an International Journal of Pathology. 452(2):175–180, 2008. 176. Kato N, Sasou S, Motoyama T: Expression of hepatocyte nuclear factor-1beta (HNF-1beta) in clear cell tumors and endometriosis of the ovary. Mod Pathol. 19(1):83–89, 2006. 177. DeLair D, Han G, Irving JA, et al: HNF-1beta in ovarian carcinomas with serous and clear cell change. Int J Gynecol Pathol. In Press. 178. Mills AM, Ly A, Balzer BL, et al: Cell cycle regulatory markers in uterine atypical leiomyoma and leiomyosarcoma: immunohistochemical study of 60 cases with clinical follow-up. Am J Surg Pathol. In Press. 179. Huntsman DG, Clement PB, Gilks CB, et al: Small-cell carcinoma of the endometrium: A clinicopathological study of sixteen cases. Am J Surg Pathol. 18:364–375, 1994. 180. Katahira A, Akahira J, Niikura H, et al: Small cell carcinoma of the endometrium: Report of three cases and literature review. Int J Gynecol Cancer. 14:1018–1023, 2004. 181. Mulvany NJ, Allen DG: Combined large cell neuroendocrine and endometrioid carcinoma of the endometrium. Int J Gynecol Pathol. 27:49–57, 2008. 182. Carlson JW, Nucci MR, Brodsky J, et al: Biomarker-assisted diagnosis of ovarian, cervical and pulmonary small cell carcinomas: The role of TTF-1, WT-1 and HPV analysis. Histopathology. 51:305–312, 2007. 183. Siami K, McCluggage WG, Ordonez NG, et al: Thyroid transcription factor-1 expression in endometrial and endocervical adenocarcinomas. Am J Surg Pathol. 31:1759–1763, 2007. 184. Altrabulsi B, Malpica A, Deavers MT, et al: Undifferentiated carcinoma of the endometrium. Am J Surg Pathol. 29:1316– 1321, 2005. 185. Silva EG, Deavers MT, Bodurka DC, et al: Association of lowgrade endometrioid carcinoma of the uterus and ovary with undifferentiated carcinoma: A new type of dedifferentiated carcinoma? Int J Gynecol Pathol. 25:52–58, 2006. 186. Broaddus RR, Lynch HT, Chen LM, et al: Pathologic features of endometrial carcinoma associated with HNPCC: A comparison with sporadic endometrial carcinoma. Cancer. 106:87–94, 2006. 187. Garg K, Kauff N, Hansen J, et al: IHC for DNA-MMR in EEC in a selected group of patients. Mod Pathol. 21:205A, 2008. 188. Albannai RA, Alkushi A, Gilks CB: Serous and endometrioid components of mixed endometrial adenocarcinoma show similar immunostaining profiles. Mod Pathol. 21:194A, 2009. 189. Nascimento AF, Hirsch MS, Cviko A, et al: The role of CD10 staining in distinguishing invasive endometrial adenocarcinoma from adenocarcinoma involving adenomyosis. Mod Pathol. 16:22–27, 2003.
709.e5
190. Srodon M, Klein WM, Kurman RJ: CD10 immunostaining does not distinguish endometrial carcinoma invading myometrium from carcinoma involving adenomyosis. Am J Surg Pathol. 27:786–789, 2003. 191. Tsuruchi N, Kaku T, Kamura T, et al: The prognostic significance of lymphovascular space invasion in endometrial cancer when conventional hemotoxylin and eosin staining is compared to immunohistochemical staining. Gynecol Oncol. 57:307–312, 1995. 192. Alexander-Sefre F, Salvesen HB, Ryan A, et al: Molecular assessment of depth of myometrial invasion in stage I endometrial cancer: A model based on K-ras mutation analysis. Gynecol Oncol. 91:218–225, 2003. 193. Lim CS, Alexander-Sefre F, Allam M, et al: Clinical value of immunohistochemically detected lymphovascular space invasion in early stage cervical carcinoma. Ann Surg Oncol. 15:2581– 2588, 2008. 194. Ali A, Black D, Soslow RA: Difficulties in assessing the depth of myometrial invasion in endometrial carcinoma. Int J Gynecol Pathol. 26:115–123, 2007. 195. Murray SK, Young RH, Scully RE: Unusual epithelial and stromal changes in myoinvasive endometrioid adenocarcinoma: A study of their frequency, associated diagnostic problems, and prognostic significance. Int J Gynecol Pathol. 22:324–333, 2003. 196. McKenney JK, Kong CS, Longacre TA: Endometrial adenocarcinoma associated with subtle lymph-vascular space invasion and lymph node metastasis: A histologic pattern mimicking intravascular and sinusoidal histiocytes. Int J Gynecol Pathol. 24:73–78, 2005. 197. Frumovitz M, Bodurka DC, Broaddus RR, et al: Lymphatic mapping and sentinel node biopsy in women with high-risk endometrial cancer. Gynecol Oncol. 104:100–103, 2007. 198. Baergen RN, Warren CD, Isacson C, et al: Early uterine serous carcinoma: Clonal origin of extrauterine disease. Int J Gynecol Pathol. 20:214–219, 2001. 199. Euscher ED, Malpica A, Deavers MT, et al: Differential expression of WT-1 in serous carcinomas in the peritoneum with or without associated serous carcinoma in endometrial polyps. Am J Surg Pathol. 29:1074–1078, 2005. 200. Dupont J, Wang X, Marshall DS, et al: Wilms Tumor Gene (WT1) and p53 expression in endometrial carcinomas: A study of 130 cases using a tissue microarray. Gynecol Oncol. 94:449– 455, 2004. 201. Irving JA, Catasus L, Gallardo A, et al: Synchronous endometrioid carcinomas of the uterine corpus and ovary: Alterations in the beta-catenin (CTNNB1) pathway are associated with independent primary tumors and favorable prognosis. Hum Pathol. 36:605–619, 2005. 202. Coosemans A, Nik SA, Caluwaerts S, et al: Upregulation of Wilms’ tumour gene 1 (WT1) in uterine sarcomas. Eur J Cancer. 43:1630–1637, 2007. 203. Zhai YL, Nikaido T, Toki T, et al: Prognostic significance of bcl-2 expression in leiomyosarcoma of the uterus. Br J Cancer. 80:1658–1664, 1999. 204. Leiser AL, Anderson SE, Nonaka D, et al: Apoptotic and cell cycle regulatory markers in uterine leiomyosarcoma. Gynecol Oncol. 101:86–91, 2006. 205. Iwasa Y, Haga H, Konishi I, et al: Prognostic factors in uterine carcinosarcoma—A clinicopathologic study of 25 patients. Cancer. 82:512–519, 1998. 206. Sutton GP, Stehman FB, Michael H, et al: Estrogen and progesterone receptors in uterine sarcomas. Obstet Gynecol. 68:709– 714, 1986. 207. Zhai YL, Kobayashi Y, Mori A, et al: Expression of steroid receptors, Ki-67, and p53 in uterine leiomyosarcomas. Int J Gynecol Pathol. 18:20–28, 1999. 208. Mittal K, Demopoulos RI: MIB-1 (Ki-67), p53, estrogen receptor, and progesterone receptor expression in uterine smooth muscle tumors. Hum Pathol. 32:984–987, 2001. 209. Leitao MM, Soslow RA, Nonaka D, et al: Tissue microarray immunohistochemical expression of estrogen, progesterone, and androgen receptors in uterine leiomyomata and leiomyosarcoma. Cancer. 101:1455–1462, 2004. 210. de Leval L, Waltregny D, Boniver J, et al: Use of histone deacetylase 8 (HDAC8), a new marker of smooth muscle
709.e6
Immunohistology of the Female Genital Tract
differentiation, in the classification of mesenchymal tumors of the uterus. Am J Surg Pathol. 30:319–327, 2006. 211. Loddenkemper C, Mechsner S, Foss HD, et al: Use of oxytocin receptor expression in distinguishing between uterine smooth muscle tumors and endometrial stromal sarcoma. Am J Surg Pathol. 27:1458–1462, 2003. 212. Rizeq MN, Van de Rijn M, Hendrickson MR, et al: A comparative immunohistochemical study of uterine smooth muscle neoplasms with emphasis on the epithelioid variant. Hum Pathol. 25:671–677, 1994. 213. Blom R, Guerrieri C, Stal O, et al: Leiomyosarcoma of the uterus: A clinicopathologic, DNA flow cytometric, p53, and mdm-2 analysis of 49 cases. Gynecol Oncol. 68:54–61, 1998. 214. Nordal RR, Kristensen GB, Stenwig AE, et al: Immunohistochemical analysis of p53 protein in uterine sarcomas. Gynecol Oncol. 70:45–48, 1998. 215. Leitao MM, Soslow RA, Baergen RN, et al: Mutation and expression of the TP53 gene in early stage epithelial ovarian carcinoma. Gynecol Oncol. 93:301–306, 2004. 216. Anderson SE, Nonaka D, Chuai S, et al: p53, epidermal growth factor, and platelet-derived growth factor in uterine leiomyosarcoma and leiomyomas. Int J Gynecol Cancer. 16:849–853, 2006. 217. Novelli M, Rossi S, Rodriguez-Justo M, et al: DOG1 and CD117 are the antibodies of choice in the diagnosis of gastrointestinal stromal tumours. Histopathology. 57:259–270, 2010. 218. Toledo G, Growdon WB, Palmeri ML, et al: Utility of the mitosis marker anti-phosphohistone H3 (PHH3) in precise and rapid evaluation of mitotic index in leiomyomas with bizarre nuclei. Mod Pathol. 21:226A–227A, 2008. 219. Agoff SN, Grieco VS, Garcia R, et al: Immunohistochemical distinction of endometrial stromal sarcoma and cellular leiomyoma. Appl Immunohistochem Mol Morphol. 9:164–169, 2001. 220. Bhargava R, Shia J, Hummer AJ, et al: Distinction of endometrial stromal sarcomas from “hemangiopericytomatous” tumors using a panel of immunohistochemical stains. Mod Pathol. 18:40–47, 2005. 221. Ng TL, Gown AM, Barry TS, et al: Nuclear beta-catenin in mesenchymal tumors. Mod Pathol. 18:68–74, 2005. 222. Jung CK, Jung JH, Lee A, et al: Diagnostic use of nuclear betacatenin expression for the assessment of endometrial stromal tumors. Mod Pathol. 21:756–763, 2008. 223. Kurihara S, Oda Y, Ohishi Y, et al: Endometrial stromal sarcomas and related high-grade sarcomas: Immunohistochemical and molecular genetic study of 31 cases. Am J Surg Pathol. 32:1228– 1238, 2008. 224. Moinfar F, Regitnig P, Tabrizi AD, et al: Expression of androgen receptors in benign and malignant endometrial stromal neoplasms. Virchows Arch. 444:410–414, 2004. 225. Devaney K, Tavassoli FA: Immunohistochemistry as a diagnostic aid in the interpretation of unusual mesenchymal tumors of the uterus. Mod Pathol. 4:225–231, 1991. 226. Yilmaz A, Rush DS, Soslow RA: Endometrial stromal sarcomas with unusual histologic features: A report of 24 primary and metastatic tumors emphasizing fibroblastic and smooth muscle differentiation. Am J Surg Pathol. 26:1142–1150, 2002. 227. Clement PB, Scully RE: Endometrial stromal sarcomas of the uterus with extensive glandular differentiation: A report of three cases that caused problems in differential diagnosis. Int J Gynecol Pathol. 11:163–173, 1992. 228. Oliva E, Clement PB, Young RH: Epithelioid endometrial and endometrioid stromal tumors: A report of four cases emphasizing their distinction from epithelioid smooth muscle tumors and other oxyphilic uterine and extrauterine tumors. Int J Gynecol Pathol. 21:48–55, 2002. 229. Ferguson SE, Tornos C, Hummer A, et al: Prognostic features of surgical stage I uterine carcinosarcoma. Am J Surg Pathol. 31:1653–1661, 2007. 230. Nogales FF, Bergeron C, Carvia RE, et al: Ovarian endometrioid tumors with yolk sac tumor component, an unusual form of ovarian neoplasm—Analysis of six cases. Am J Surg Pathol. 20:1056–1066, 1996. 231. Ehrmann RL, Weidner N, Welch WR, et al: Malignant mixed müllerian tumor of the ovary with prominent neuroectodermal differentiation (teratoid carcinosarcoma). Int J Gynecol Pathol. 9:272–282, 1990.
232. Fukunaga M, Nomura K, Endo Y, et al: Carcinosarcoma of the uterus with extensive neuroectodermal differentiation. Histopathology. 29:565–570, 1996. 233. Mikami Y, Hata S, Kiyokawa T, et al: Expression of CD10 in malignant müllerian mixed tumors and adenosarcomas: An immunohistochemical study. Mod Pathol. 15:923–930, 2002. 234. Amant F, Steenkiste E, Schurmans K, et al: Immunohistochemical expression of CD10 antigen in uterine adenosarcoma. Int J Gynecol Cancer. 14:1118–1121, 2004. 235. Soslow RA, Ali A, Oliva E: Mullerian adenosarcomas: An immunophenotypic analysis of 35 cases. Am J Surg Pathol. 32:1013– 1021, 2008. 236. Tedeschi A, Di Mezza G, D’Amico O, et al: A case of pelvic actinomycosis presenting as cutaneous fistula. Eur J Obstet Gynecol Reprod Biol. 108:103–105, 2003. 237. Vang R, Kempson RL: Perivascular epithelioid cell tumor (“PEComa”) of the uterus: A subset of HMB-45-positive epithelioid mesenchymal neoplasms with an uncertain relationship to pure smooth muscle tumors. Am J Surg Pathol. 26:1–13, 2002. 238. Folpe AL, Mentzel T, Lehr HA, et al: Perivascular epithelioid cell neoplasms of soft tissue and gynecologic origin: a clinicopathologic study of 26 cases and review of the literature. Am J Surg Pathol. 29:1558–1575, 2005. 239. Fadare O: Perivascular epithelioid cell tumors (PEComas) and smooth muscle tumors of the uterus. Am J Surg Pathol. 31:1454– 1455, 2007. 240. Silva EG, Deavers MT, Bodurka DC, et al: Uterine epithelioid leiomyosarcomas with clear cells: Reactivity with HMB-45 and the concept of PEComa. Am J Surg Pathol. 28:244–249, 2004. 241. Oliva E, Wang WL, Branton P, et al: Expression of melanocytic (“PEComa”) markers in smooth muscle tumors of the uterus: An immunohistochemical analysis of 86 cases. Mod Pathol. 19:191A, 2006. 242. Clement PB, Scully RE: Uterine tumors resembling ovarian sexcord tumors. A clinicopathologic analysis of 14 cases. Am J Clin Pathol. 66:512–525, 1976. 243. Wingen CB, Pauwels PA, Debiec-Rychter M, et al: Uterine gastrointestinal stromal tumour (GIST). Gynecol Oncol. 97:970– 972, 2005. 244. Klein WM, Kurman RJ: Lack of expression of c-kit protein (CD117) in mesenchymal tumors of the uterus and ovary. Int J Gynecol Pathol. 22:181–184, 2003. 245. Winter WE, III, Seidman JD, Krivak TC, et al: Clinicopathological analysis of c-kit expression in carcinosarcomas and leiomyosarcomas of the uterine corpus. Gynecol Oncol. 91:3–8, 2003. 246. Rushing RS, Shajahan S, Chendil D, et al: Uterine sarcomas express KIT protein but lack mutation(s) in exon 11 or 17 of c-KIT. Gynecol Oncol. 91:9–14, 2003. 247. Wang L, Felix JC, Lee JL, et al: The proto-oncogene c-kit is expressed in leiomyosarcomas of the uterus. Gynecol Oncol. 90:402–406, 2003. 248. Rabban JT, Zaloudek CJ, Shekitka KM, et al: Inflammatory myofibroblastic tumor of the uterus: a clinicopathologic study of 6 cases emphasizing distinction from aggressive mesenchymal tumors. Am J Surg Pathol. 29:1348–1355, 2005. 249. Daya D, Sabet L: The use of cytokeratin as a sensitive and reliable marker for trophoblastic tissue. Am J Clin Pathol. 95:137– 141, 1991. 250. Shih IM, Kurman RJ: Immunohistochemical localization of inhibin-alpha in the placenta and gestational trophoblastic lesions. Int J Gynecol Pathol. 18:144–150, 1999. 251. Kommoss F, Schmidt D, Coerdt W, et al: Immunohistochemical expression analysis of inhibin-alpha and -beta subunits in partial and complete moles, trophoblastic tumors, and endometrial decidua. Int J Gynecol Pathol. 20:380–385, 2001. 252. Ino K, Suzuki T, Uehara C, et al: The expression and localization of neutral endopeptidase 24.11/CD10 in human gestational trophoblastic diseases. Lab Invest. 80:1729–1738, 2000. 253. Mao TL, Kurman RJ, Jeng YM, et al: HSD3B1 as a novel trophoblast-associated marker that assists in the differential diagnosis of trophoblastic tumors and tumorlike lesions. Am J Surg Pathol. 32:236–242, 2008. 254. Shih IM, Kurman RJ: p63 Expression is useful in the distinction of epithelioid trophoblastic and placental site trophoblastic
References tumors by profiling trophoblastic subpopulations. Am J Surg Pathol. 28:1177–1183, 2004. 255. Shih I: Trophogram, an immunohistochemistry-based algorithmic approach, in the differential diagnosis of trophoblastic tumors and tumorlike lesions. Ann Diagn Pathol. 11:228–234, 2007. 256. Shih IM, Kurman RJ: Expression of melanoma cell adhesion molecule in intermediate trophoblast. Lab Invest. 75:377–388, 1996. 257. Singer G, Kurman RJ, McMaster MT, et al: HLA-G immunoreactivity is specific for intermediate trophoblast in gestational trophoblastic disease and can serve as a useful marker in differential diagnosis. Am J Surg Pathol. 26:914–920, 2002. 258. Kurman RJ, Young RH, Norris HJ, et al: Immunocytochemical localization of placental lactogen and chorionic gonadotropin in the normal placenta and trophoblastic tumors, with emphasis on intermediate trophoblast and the placental site trophoblastic tumor. Int J Gynecol Pathol. 3:101–121, 1984. 259. Huettner PC, Gersell DJ: Placental site nodule: A clinicopathologic study of 38 cases. Int J Gynecol Pathol. 13:191–198, 1994. 260. Yeh IT, O’Connor DM, Kurman RJ: Vacuolated cytotrophoblast: A subpopulation of trophoblast in the chorion laeve. Placenta. 10:429–438, 1989. 261. Li HW, Cheung AN, Tsao SW, et al: Expression of e-cadherin and beta-catenin in trophoblastic tissue in normal and pathological pregnancies. Int J Gynecol Pathol. 22:63–70, 2003. 262. Wong SC, Chan AT, Chan JK, et al: Nuclear beta-catenin and Ki-67 expression in choriocarcinoma and its pre-malignant form. J Clin Pathol. 59:387–392, 2006. 263. Mao TL, Kurman RJ, Huang CC, et al: Immunohistochemistry of choriocarcinoma: An aid in differential diagnosis and in elucidating pathogenesis. Am J Surg Pathol. 31:1726–1732, 2007. 264. Castrillon DH, Sun DQ, Weremowicz S, et al: Discrimination of complete hydatidiform mole from its mimics by immunohistochemistry of the paternally imprinted gene product p57KIP2. Am J Surg Pathol. 25:1225–1230, 2001. 265. Fisher RA, Hodges MD, Rees HC, et al: The maternally transcribed gene p57(KIP2) (CDNK1C) is abnormally expressed in both androgenetic and biparental complete hydatidiform moles. Hum Mol Genet. 11:3267–3272, 2002. 266. Fukunaga M: Immunohistochemical characterization of p57(KIP2) expression in early hydatidiform moles. Hum Pathol. 33:1188–1192, 2002. 267. Genest DR, Dorfman DM, Castrillon DH: Ploidy and imprinting in hydatidiform moles. Complementary use of flow cytometry and immunohistochemistry of the imprinted gene product p57KIP2 to assist molar classification. J Reprod Med. 47:342– 346, 2002. 268. Hoffner L, Dunn J, Esposito N, et al: P57KIP2 immunostaining and molecular cytogenetics: Combined approach aids in diagnosis of morphologically challenging cases with molar phenotype and in detecting androgenetic cell lines in mosaic/chimeric conceptions. Hum Pathol. 39:63–72, 2008. 269. Keep D, Zaragoza MV, Hassold T, et al: Very early complete hydatidiform mole. Hum Pathol. 27:708–713, 1996. 270. Kim MJ, Kim KR, Ro JY, et al: Diagnostic and pathogenetic significance of increased stromal apoptosis and incomplete vasculogenesis in complete hydatidiform moles in very early pregnancy periods. Am J Surg Pathol. 30:362–369, 2006. 271. Shih IM, Seidman JD, Kurman RJ: Placental site nodule and characterization of distinctive types of intermediate trophoblast. Hum Pathol. 30:687–694, 1999. 272. Shih IM, Kurman RJ: Ki-67 labeling index in the differential diagnosis of exaggerated placental site, placental site trophoblastic tumor, and choriocarcinoma: A double immunohistochemical staining technique using Ki-67 and Mel-CAM antibodies. Hum Pathol. 29:27–33, 1998. 273. Shih IM, Kurman RJ: Epithelioid trophoblastic tumor—A neoplasm distinct from choriocarcinoma and placental site trophoblastic tumor simulating carcinoma. Am J Surg Pathol. 22:1393–1403, 1998. 274. Mao TL, Seidman JD, Kurman RJ, et al: Cyclin E and p16 immunoreactivity in epithelioid trophoblastic tumor—An aid in differential diagnosis. Am J Surg Pathol. 30:1105–1110, 2006.
709.e7
275. Mazur MT: Metastatic gestational choriocarcinoma. Unusual pathologic variant following therapy. Cancer. 63:1370–1377, 1989. 276. Bacchi CE, Coelho KI, Goldberg J: Expression of beta-human chorionic gonadotropin (beta-hCG) in non-trophoblastic elements of transitional cell carcinoma of the bladder: Possible relationship with the prognosis. Rev Paul Med. 111:412–416, 1993. 277. Hameed A, Miller DS, Muller CY, et al: Frequent expression of beta-human chorionic gonadotropin (beta-hCG) in squamous cell carcinoma of the cervix. Int J Gynecol Pathol. 18:381–386, 1999. 278. Lynch HT, Smyrk TC, Watson P, et al: Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: An updated review. Gastroenterology. 104:1535–1549, 1993. 279. Lynch HT, Lanspa S, Smyrk T, et al: Hereditary nonpolyposis colorectal cancer (Lynch syndromes I & II). Genetics, pathology, natural history, and cancer control. Part I. Cancer Genet Cytogenet. 53:143–160, 1991. 280. Lynch HT, Smyrk T: An update on Lynch syndrome. Curr Opin Oncol. 10:349–356, 1998. 281. Shia J, Black D, Hummer AJ, et al: Routinely assessed morphological features correlate with microsatellite instability status in endometrial cancer. Hum Pathol. 39:116–125, 2008. 282. Kwon JS, Scott JL, Gilks CB, et al: Testing women with endometrial cancer to detect Lynch syndrome. J Clin Oncol. 29:2247– 2252, 2011. 283. Mvundura M, Grosse SD, Hampel H, et al: The cost-effectiveness of genetic testing strategies for Lynch syndrome among newly diagnosed patients with colorectal cancer. Genet Med. 12:93– 104, 2010. 284. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med. 11:35–41, 2009. 285. Lu KH, Schorge JO, Rodabaugh KJ, et al: Prospective determination of prevalence of Lynch syndrome in young women with endometrial cancer. J Clin Oncol. 25:5158–5164, 2007. 286. Simpkins SB, Bocker T, Swisher EM, et al: MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers. Hum Mol Genet. 8:661–666, 1999. 287. Buttin BM, Powell MA, Mutch DG, et al: Increased risk for hereditary nonpolyposis colorectal cancer-associated synchronous and metachronous malignancies in patients with microsatellite instability-positive endometrial carcinoma lacking MLH1 promoter methylation. Clin Cancer Res. 10:481–490, 2004. 288. Shia J: Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn. 10:293–300, 2008. 289. Ito K, Watanabe K, Nasim S, et al: Prognostic significance of p53 overexpression in endometrial cancer. Cancer Res. 54:4667– 4670, 1994. 290. Sung CJ, Zheng Y, Quddus MR, et al: p53 as a significant prognostic marker in endometrial carcinoma. Int J Gynecol Cancer. 10:119–127, 2000. 291. Alkushi A, Lim P, Coldman A, et al: Interpretation of p53 immunoreactivity in endometrial carcinoma: Establishing a clinically relevant cut-off level. Int J Gynecol Pathol. 23:129–137, 2004. 292. Santin AD, Bellone S, Gokden M, et al: Overexpression of HER-2/neu in uterine serous papillary cancer. Clin Cancer Res. 8:1271–1279, 2002. 293. Lax SF: Molecular genetic pathways in various types of endometrial carcinoma: From a phenotypical to a molecular-based classification. Virchows Arch. 444:213–223, 2004. 294. Slomovitz BM, Broaddus RR, Burke TW, et al: Her-2/neu overexpression and amplification in uterine papillary serous carcinoma. J Clin Oncol. 22:3126–3132, 2004. 295. Santin AD, Bellone S, Van SS, et al: Determination of HER2/neu status in uterine serous papillary carcinoma: Comparative analysis of immunohistochemistry and fluorescence in situ hybridization. Gynecol Oncol. 98:24–30, 2005.
709.e8
Immunohistology of the Female Genital Tract
296. Odicino FE, Bignotti E, Rossi E, et al: HER-2/neu overexpression and amplification in uterine serous papillary carcinoma: Comparative analysis of immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and fluorescence in situ hybridization. Int J Gynecol Cancer. 18:14–21, 2008. 297. Bodner-Adler B, Bodner K, Czerwenka K, et al: Expression of p16 protein in patients with uterine smooth muscle tumors: An immunohistochemical analysis. Gynecol Oncol. 96:62–66, 2005. 298. O’Neill CJ, McBride HA, Connolly LE, et al: Uterine leiomyosarcomas are characterized by high p16, p53 and MIB1 expression in comparison with usual leiomyomas, leiomyoma variants and smooth muscle tumours of uncertain malignant potential. Histopathology. 50:851–858, 2007. 299. Atkins KA, Arronte N, Darus CJ, et al: The Use of p16 in enhancing the histologic classification of uterine smooth muscle tumors. Am J Surg Pathol. 32:98–102, 2008. 300. Gannon BR, Manduch M, Childs TJ: Differential Immunoreactivity of p16 in Leiomyosarcomas and Leiomyoma Variants. Int J Gynecol Pathol. 27:68–73, 2008. 301. Koontz JI, Soreng AL, Nucci M, et al: Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors. Proc Natl Acad Sci U S A. 98:6348–6353, 2001. 302. Goodfellow PJ, Buttin BM, Herzog TJ, et al: Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers. Proc Natl Acad Sci U S A. 100:5908–5913, 2003. 303. Ollikainen M, Abdel-Rahman WM, Moisio AL, et al: Molecular analysis of familial endometrial carcinoma: A manifestation of hereditary nonpolyposis colorectal cancer or a separate syndrome? J Clin Oncol. 23:4609–4616, 2005. 304. Hampel H, Frankel W, Panescu J, et al: Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients. Cancer Res. 66:7810–7817, 2006. 305. Micci F, Walter CU, Teixeira MR, et al: Cytogenetic and molecular genetic analyses of endometrial stromal sarcoma: Nonrandom involvement of chromosome arms 6p and 7p and confirmation of JAZF1/JJAZ1 gene fusion in t(7;17). Cancer Genet Cytogenet. 144:119–124, 2003. 306. Huang HY, Ladanyi M, Soslow RA: Molecular detection of JAZF1-JJAZ1 gene fusion in endometrial stromal neoplasms with classic and variant histology: Evidence for genetic heterogeneity. Am J Surg Pathol. 28:224–232, 2004. 307. Hrzenjak A, Moinfar F, Tavassoli FA, et al: JAZF1/JJAZ1 gene fusion in endometrial stromal sarcomas: Molecular analysis by reverse transcriptase-polymerase chain reaction optimized for paraffin-embedded tissue. J Mol Diagn. 7:388–395, 2005. 308. Nucci MR, Harburger D, Koontz J, et al: Molecular analysis of the JAZF1-JJAZ1 gene fusion by RT-PCR and fluorescence in situ hybridization in endometrial stromal neoplasms. Am J Surg Pathol. 31:65–70, 2007. 309. Oliva E, de Leval L, Soslow RA, et al: High frequency of JAZF1JJAZ1 gene fusion in endometrial stromal tumors with smooth muscle differentiation by interphase FISH detection. Am J Surg Pathol. 31:1277–1284, 2007. 310. Fukunaga M: Immunohistochemical characterization of p57Kip2 expression in tetraploid hydropic placentas. Arch Pathol Lab Med. 128:897–900, 2004. 311. Lage JM, Mark SD, Roberts DJ, et al: A flow cytometric study of 137 fresh hydropic placentas: Correlation between types of hydatidiform moles and nuclear DNA ploidy. Obstet Gynecol. 79:403–410, 1992. 312. Zaragoza MV, Surti U, Redline RW, et al: Parental origin and phenotype of triploidy in spontaneous abortions: Predominance of diandry and association with the partial hydatidiform mole. Am J Hum Genet. 66:1807–1820, 2000. 313. Vang R, Gupta M, Wu LS, et al: Diagnostic reproducibility of hydatidiform moles: ancillary techniques (p57 immunohistochemistry and molecular genotyping) improve morphologic diagnosis. Am J Surg Pathol. 36:443–453, 2012. 314. Tavassoli FA, Devilee P, International Agency for Research on Cancer, et al: Pathology and genetics of tumours of the breast and female genital organs, Lyon, 2003, International Agency for Research on Cancer. 315. Alvarado-Cabrero I, Young RH, Vamvakas EC, et al: Carcinoma of the fallopian tube: A clinicopathological study of 105 cases
with observations on staging and prognostic factors. Gynecol Oncol. 72:367–379, 1999. 316. Baekelandt M, Nesbakken AJ, Kristensen GB, et al: Carcinoma of the fallopian tube—Clinicopathologic study of 151 patients treated at the Norwegian Radium Hospital. Cancer. 89:2076– 2084, 2000. 317. Piura B, Rabinovich A: Primary carcinoma of the fallopian tube: Study of 11 cases. Eur J Obstet Gynecol Reprod Biol. 91:169–175, 2000. 318. di Re E, Grosso G, Raspagliesi F, et al: Fallopian tube cancer: Incidence and role of lymphatic spread. Gynecol Oncol. 62:199– 202, 1996. 319. Callahan MJ, Crum CP, Medeiros F, et al: Primary fallopian tube malignancies in BRCA-positive women undergoing surgery for ovarian cancer risk reduction. J Clin Oncol. 25:3985–3990, 2007. 320. Carcangiu ML, Peissel B, Pasini B, et al: Incidental carcinomas in prophylactic specimens in BRCA1 and BRCA2 germ-line mutation carriers, with emphasis on fallopian tube lesions: Report of 6 cases and review of the literature. Am J Surg Pathol. 30:1222– 1230, 2006. 321. Finch A, Shaw P, Rosen B, et al: Clinical and pathologic findings of prophylactic salpingo-oophorectomies in 159 BRCA1 and BRCA2 carriers. Gynecol Oncol. 100:58–64, 2006. 322. Medeiros F, Muto MG, Lee Y, et al: The tubal fimbria is a preferred site for early adenocarcinoma in women with familial ovarian cancer syndrome. Am J Surg Pathol. 30:230–236, 2006. 323. Folkins AK, Jarboe EA, Saleemuddin A, et al: A candidate precursor to pelvic serous cancer (p53 signature) and its prevalence in ovaries and fallopian tubes from women with BRCA mutations. Gynecol Oncol. 109:168–173, 2008. 324. Jarboe E, Folkins A, Nucci MR, et al: Serous carcinogenesis in the fallopian tube: A descriptive classification. Int J Gynecol Pathol. 27:1–9, 2008. 325. Kindelberger DW, Lee Y, Miron A, et al: Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma: Evidence for a causal relationship. Am J Surg Pathol. 31:161–169, 2007. 326. Bannatyne P, Russell P: Early adenocarcinoma of the fallopian tubes. A case for multifocal tumorigenesis. Diagn Gynecol Obstet. 3:49–60, 1981. 327. Moll R, Franke WW, Schiller DL, et al: The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell. 31:11–24, 1982. 328. Moll R, Levy R, Czernobilsky B, et al: Cytokeratins of normal epithelia and some neoplasms of the female genital tract. Lab Invest. 49:599–610, 1983. 329. Berezowski K, Stastny JF, Kornstein MJ: Cytokeratins 7 and 20 and carcinoembryonic antigen in ovarian and colonic carcinoma. Mod Pathol. 9:426–429, 1996. 330. Lagendijk JH, Mullink H, Van Diest PJ, et al: Immunohistochemical differentiation between primary adenocarcinomas of the ovary and ovarian metastases of colonic and breast origin. Comparison between a statistical and an intuitive approach. J Clin Pathol. 52:283–290, 1999. 331. Cathro HP, Stoler MH: Expression of cytokeratins 7 and 20 in ovarian neoplasia. Am J Clin Pathol. 117:944–951, 2002. 332. Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases. Mod Pathol. 13:962–972, 2000. 333. Raspollini MR, Amunni G, Villanucci A, et al: Utility of CDX-2 in distinguishing between primary and secondary (intestinal) mucinous ovarian carcinoma: An immunohistochemical comparison of 43 cases. Appl Immunohistochem Mol Morphol. 12:127–131, 2004. 334. Moll R, Lowe A, Laufer J, et al: Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol. 140:427–447, 1992. 335. Miettinen M: Keratin 20: Immunohistochemical marker for gastrointestinal, urothelial, and Merkel cell carcinomas. Mod Pathol. 8:384–388, 1995. 336. Wauters CC, Smedts F, Gerrits LG, et al: Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary. Hum Pathol. 26:852–855, 1995. 337. Loy TS, Calaluce RD, Keeney GL: Cytokeratin immunostaining in differentiating primary ovarian carcinoma from metastatic colonic adenocarcinoma. Mod Pathol. 9:1040–1044, 1996.
References 338. Ji H, Isacson C, Seidman JD, et al: Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 21:391–400, 2002. 339. Ordonez NG: Role of immunohistochemistry in distinguishing epithelial peritoneal mesotheliomas from peritoneal and ovarian serous carcinomas. Am J Surg Pathol. 22:1203–1214, 1998. 340. Ramalingam P, Malpica A, Silva EG, et al: The use of cytokeratin 7 and EMA in differentiating ovarian yolk sac tumors from endometrioid and clear cell carcinomas. Am J Surg Pathol. 28:1499–1505, 2004. 341. Attanoos RL, Webb R, Dojcinov SD, et al: Value of mesothelial and epithelial antibodies in distinguishing diffuse peritoneal mesothelioma in females from serous papillary carcinoma of the ovary and peritoneum. Histopathology. 40:237–244, 2002. 342. Charpin C, Bhan AK, Zurawski VRJ, et al: Carcinoembryonic antigen (CEA) and carbohydrate determinant 19-9 (CA 19-9) localization in 121 primary and metastatic ovarian tumors: An immunohistochemical study with the use of monoclonal antibodies. Int J Gynecol Pathol. 1:231–245, 1982. 343. Brown RW, Campagna LB, Dunn JK, et al: Immunohistochemical identification of tumor markers in metastatic adenocarcinoma. A diagnostic adjunct in the determination of primary site. Am J Clin Pathol. 107:12–19, 1997. 344. Lagendijk JH, Mullink H, Van Diest PJ, et al: Tracing the origin of adenocarcinomas with unknown primary using immunohistochemistry: Differential diagnosis between colonic and ovarian carcinomas as primary sites. Hum Pathol. 29:491–497, 1998. 345. Ulfig N: Calcium-binding proteins in the human developing brain. Adv Anat Embryol Cell Biol. 165:1–92:III–IX, 2002. 346. Chou YY, Jeng YM, Kao HL, et al: Differentiation of ovarian mucinous carcinoma and metastatic colorectal adenocarcinoma by immunostaining with beta-catenin. Histopathology. 43:151– 156, 2003. 347. Goldstein NS, Bassi D, Uzieblo A: WT1 is an integral component of an antibody panel to distinguish pancreaticobiliary and some ovarian epithelial neoplasms. Am J Clin Pathol. 116:246–252, 2001. 348. Multhaupt HAB, Arenas-Elliott CP, Warhol MJ: Comparison of glycoprotein expression between ovarian and colon adenocarcinomas. Arch Pathol Lab Med. 123:909–916, 1999. 349. Thor A, Gorstein F, Ohuchi N, et al: Tumor-associated glycoprotein (TAG-72) in ovarian carcinomas defined by monoclonal antibody B72.3. J Natl Cancer Inst. 76:995–1006, 1986. 350. Bollinger DJ, Wick MR, Dehner LP, et al: Peritoneal malignant mesothelioma versus serous papillary adenocarcinoma. A histochemical and immunohistochemical comparison. Am J Surg Pathol. 13:659–670, 1989. 351. O’Brien TJ, Beard JB, Underwood LJ, et al: The CA 125 gene: An extracellular superstructure dominated by repeat sequences. Tumor Biol. 22:348–366, 2001. 352. O’Brien TJ, Beard JB, Underwood LJ, et al: The CA 125 gene: A newly discovered extension of the glycosylated N-terminal domain doubles the size of this extracellular superstructure. Tumor Biol. 23:154–169, 2002. 353. Bast RC, Jr, Xu FJ, Yu YH, et al: CA 125: The past and the future. Int J Biol Markers. 13:179–187, 1998. 354. Loy TS, Quesenberry JT, Sharp SC: Distribution of CA 125 in adenocarcinomas: An immunohistochemical study of 481 cases. Am J Clin Pathol. 98:175–179, 1992. 355. Koelma IA, Nap M, Rodenburg CJ, et al: The value of tumour marker CA 125 in surgical pathology. Histopathology. 11:287– 294, 1987. 356. Keen CE, Szakacs S, Okon E, et al: CA125 and thyroglobulin staining in papillary carcinomas of thyroid and ovarian origin is not completely specific for site of origin. Histopathology. 34:113– 117, 1999. 357. Nap M, Vitali A, Nustad K, et al: Immunohistochemical characterization of 22 monoclonal antibodies against the CA125 antigen: 2nd report from the ISOBM TD-1 workshop. Tumor Biol. 17:325–331, 1996. 358. Leake J, Woolas RP, Daniel J, et al: Immunocytochemical and serological expression of CA 125: A clinicopathological study of 40 malignant ovarian epithelial tumours. Histopathology. 24:57– 64, 1994.
709.e9
359. Zheng W, Senturk BZ, Parkash V: Inhibin immunohistochemical staining: A practical approach for the surgical pathologist in the diagnoses of ovarian sex cord-stromal tumors. Adv Anat Pathol. 10:27–38, 2003. 360. Rivier C, Meunier H, Roberts V, et al: Inhibin: Role and secretion in the rat. Recent Prog Horm Res. 46:231–257, 1990. 361. Vale W, Rivier C, Hsueh A, et al: Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res. 44:1–34, 1988. 362. Welt CK: The physiology and pathophysiology of inhibin, activin and follistatin in female reproduction. Curr Opin Obstet Gynecol. 14:317–323, 2002. 363. Rishi M, Howard LN, Bratthauer GL, et al: Use of monoclonal antibody against human inhibin as a marker for sex cord stromal tumors of the ovary. Am J Surg Pathol. 21:583–589, 1997. 364. Costa MJ, Ames PF, Walls J, et al: Inhibin immunohistochemistry applied to ovarian neoplasms: A novel, effective, diagnostic tool. Hum Pathol. 28:1247–1254, 1997. 365. Hildebrandt RH, Rouse RV, Longacre TA: Value of inhibin in the identification of granulosa cell tumors of the ovary. Hum Pathol. 28:1387–1395, 1997. 366. Kommoss F, Oliva E, Bhan AK, et al: Inhibin expression in ovarian tumors and tumor-like lesions: An immunohistochemical study. Mod Pathol. 11:656–664, 1998. 367. McCluggage WG: Value of inhibin staining in gynecological pathology. Int J Gynecol Pathol. 20:79–85, 2001. 368. Deavers MT, Malpica A, Liu J, et al: Ovarian sex cord-stromal tumors: An immunohistochemical study including a comparison of calretinin and inhibin. Mod Pathol. 16:584–590, 2003. 369. Pelkey TJ, Frierson HFJ, Mills SE, et al: The diagnostic utility of inhibin staining in ovarian neoplasms. Int J Gynecol Pathol. 17:97–105, 1998. 370. Matias-Guiu X, Pons C, Prat J: Mullerian inhibiting substance, alpha-inhibin, and CD99 expression in sex cord-stromal tumors and endometrioid ovarian carcinomas resembling sex cordstromal tumors. Hum Pathol. 29:840–845, 1998. 371. Yao DX, Soslow RA, Hedvat CV, et al: Melan-A (A103) and inhibin expression in ovarian neoplasms. Appl Immunohistochem Mol Morphol. 11:244–249, 2003. 372. McCluggage WG, Burton J, Maxwell P, et al: Immunohistochemical staining of normal, hyperplastic, and neoplastic adrenal cortex with a monoclonal antibody against alpha inhibin. J Clin Pathol. 51:114–116, 1998. 373. Pelkey TJ, Frierson HFJ, Mills SE, et al: The alpha subunit of inhibin in adrenal cortical neoplasia. Mod Pathol. 11:516–524, 1998. 374. Chivite A, MatiasGuiu X, Pons C, et al: Inhibin A expression in adrenal neoplasms—A new immunohistochemical marker for adrenocortical tumors. Appl Immunohistochem. 6:42–49, 1998. 375. Cho EY, Ahn GH: Immunoexpression of inhibin alpha-subunit in adrenal neoplasms. Appl Immunohistochem Mol Morphol. 9:222–228, 2001. 376. Jorda M, De MB, Nadji M: Calretinin and inhibin are useful in separating adrenocortical neoplasms from pheochromocytomas. Appl Immunohistochem Mol Morphol. 10:67–70, 2002. 377. Zhang PJ, Genega EM, Tomaszewski JE, et al: The role of calretinin, inhibin, melan-A, BCL-2, and C-kit in differentiating adrenal cortical and medullary tumors: An immunohistochemical study. Mod Pathol. 16:591–597, 2003. 378. Rogers JH: Calretinin: A gene for a novel calcium-binding protein expressed principally in neurons. J Cell Biol. 105:1343– 1353, 1987. 379. Tos AP, Doglioni C: Calretinin: A novel tool for diagnostic immunohistochemistry. Adv Anat Pathol. 5:61–66, 1998. 380. Rogers J, Khan M, Ellis J: Calretinin and other CaBPs in the nervous system. Adv Exp Med Biol. 269:195–203, 1990. 381. Cao QJ, Jones JG, Li M: Expression of calretinin in human ovary, testis, and ovarian sex cord-stromal tumors. Int J Gynecol Pathol. 20:346–352, 2001. 382. McCluggage WG, Maxwell P: Immunohistochemical staining for calretinin is useful in the diagnosis of ovarian sex cord-stromal tumours. Histopathology. 38:403–408, 2001. 383. Movahedi-Lankarani S, Kurman RJ: Calretinin, a more sensitive but less specific marker than alpha-inhibin for ovarian sex cordstromal neoplasms—An immunohistochemical study of 215 cases. Am J Surg Pathol. 26:1477–1483, 2002.
709.e10
Immunohistology of the Female Genital Tract
384. Al-Agha OM, Huwait HF, Chow C, et al: FOXL2 is a sensitive and specific marker for sex cord-stromal tumors of the ovary. Am J Surg Pathol. 35:484–494, 2011. 385. Zhao C, Vinh TN, McManus K, et al: Identification of the most sensitive and robust immunohistochemical markers in different categories of ovarian sex cord-stromal tumors. Am J Surg Pathol. 33:354–366, 2009. 386. Zhao C, Barner R, Vinh TN, et al: SF-1 is a diagnostically useful immunohistochemical marker and comparable to other sex cord-stromal tumor markers for the differential diagnosis of ovarian sertoli cell tumor. Int J Gynecol Pathol. 27:507–514, 2008. 387. Lee SB, Haber DA: Wilms tumor and the WT1 gene. Exp Cell Res. 264:74–99, 2001. 388. Charles AK, Mall S, Watson J, et al: Expression of the Wilms’ tumour gene WT1 in the developing human and in paediatric renal tumours: an immunohistochemical study. Mol Pathol. 50:138–144, 1997. 389. Ordonez NG: Desmoplastic small round cell tumor II: An ultrastructural and immunohistochemical study with emphasis on new immunohistochemical markers. Am J Surg Pathol. 22:1314– 1327, 1998. 390. Hill DA, Pfeifer JD, Marley EF, et al: WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing sarcoma/primitive neuroectodermal tumor—An immunohistochemical and molecular diagnostic study. Am J Clin Pathol. 114:345–353, 2000. 391. Lae ME, Roche PC, Jin L, et al: Desmoplastic small round cell tumor—A clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol. 26:823–835, 2002. 392. Hwang H, Quenneville L, Yaziji H, et al: Wilms tumor gene product: Sensitive and contextually specific marker of serous carcinomas of ovarian surface epithelial origin. Appl Immunohistochem Mol Morphol. 12:122–126, 2004. 393. Ordonez NG: Value of thyroid transcription factor-1, E-cadherin, BG8, WT1, and CD44S immunostaining in distinguishing epithelial pleural mesothelioma from pulmonary and nonpulmonary adenocarcinoma. Am J Surg Pathol. 24:598–606, 2000. 394. Shimizu M, Toki T, Takagi Y, et al: Immunohistochemical detection of the Wilms’ tumor gene (WT1) in epithelial ovarian tumors. Int J Gynecol Pathol. 19:158–163, 2000. 395. Logani S, Oliva E, Amin MB, et al: Immunoprofile of ovarian tumors with putative transitional cell (urothelial) differentiation using novel urothelial markers: Histogenetic and diagnostic implications. Am J Surg Pathol. 27:1434–1441, 2003. 396. McCluggage WG, Oliva E, Connolly LE, et al: An immunohistochemical analysis of ovarian small cell carcinoma of hypercalcemic type. Int J Gynecol Pathol. 23:330–336, 2004. 397. Nakopoulou L, Stefanaki K, Janinis J, et al: Immunohistochemical expression of placental alkaline phosphatase and vimentin in epithelial ovarian neoplasms. Acta Oncol. 34:511–515, 1995. 398. Nouwen EJ, Hendrix PG, Dauwe S, et al: Tumor markers in the human ovary and its neoplasms. A comparative immunohistochemical study. Am J Pathol. 126:230–242, 1987. 399. Lifschitz-Mercer B, Walt H, Kushnir I, et al: Differentiation potential of ovarian dysgerminoma: An immunohistochemical study of 15 cases. Hum Pathol. 26:62–66, 1995. 400. Hustin J, Gillerot Y, Collette J, et al: Placental alkaline phosphatase in developing normal and abnormal gonads and in germ-cell tumours. Virchows Arch A Pathol Anat Histopathol. 417:67–72, 1990. 401. Arber DA, Tamayo R, Weiss LM: Paraffin section detection of the c-kit gene product (CD117) in human tissues: Value in the diagnosis of mast cell disorders. Hum Pathol. 29:498–504, 1998. 402. Gibson PC, Cooper K: CD117 (KIT): A diverse protein with selective applications in surgical pathology. Adv Anat Pathol. 9:65–69, 2002. 403. Sarlomo-Rikala M, Kovatich AJ, Barusevicius A, et al: CD117: A sensitive marker for gastrointestinal stromal tumors that is more specific than CD34. Mod Pathol. 11:728–734, 1998. 404. Miettinen M, Sarlomo-Rikala M, Lasota J: Gastrointestinal stromal tumors: Recent advances in understanding of their biology. Hum Pathol. 30:1213–1220, 1999. 405. Lee JR, Joshi V, Griffin JW, Jr, et al: Gastrointestinal autonomic nerve tumor—Immunohistochemical and molecular identity
with gastrointestinal stromal tumor. Am J Surg Pathol. 25:979– 987, 2001. 406. Leroy X, Augusto D, Leteurtre E, et al: CD30 and CD117 (c-kit) used in combination are useful for distinguishing embryonal carcinoma from seminoma. J Histochem Cytochem. 50:283– 285, 2002. 407. Sever M, Jones TD, Roth LM, et al: Expression of CD117 (c-kit) receptor in dysgerminoma of the ovary: Diagnostic and therapeutic implications. Mod Pathol. 18:1411–1416, 2005. 408. Cheng L, Sung MT, Cossu-Rocca P, et al: OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. J Pathol. 211:1–9, 2007. 409. Cheng L, Thomas A, Roth LM, et al: OCT4: A novel biomarker for dysgerminoma of the ovary. Am J Surg Pathol. 28:1341–1346, 2004. 410. De Jong J, Stoop H, Dohle GR, et al: Diagnostic value of OCT3/4 for pre-invasive and invasive testicular germ cell tumours. J Pathol. 206:242–249, 2005. 411. Rajpert-DeMeyts E, Hanstein R, Jorgensen N, et al: Developmental expression of POU5F1 (OCT-3/4) in normal and dysgenetic human gonads. Hum Reprod. 19:1338–1344, 2004. 412. Hoei-Hansen CE, Kraggerud SM, Abeler VM, et al: Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers. Mol Cancer. 6:12, 2007. 413. Harms D, Janig U: Germ cell tumours of childhood. Report of 170 cases including 59 pure and partial yolk-sac tumours. Virchows Arch A. 409:223–239, 1986. 414. Clement PB, Young RH, Scully RE: Endometrioid-like variant of ovarian yolk sac tumor. A clinicopathological analysis of eight cases. Am J Surg Pathol. 11:767–778, 1987. 415. Kurman RJ, Norris HJ: Endodermal sinus tumor of the ovary. A clinical and pathologic analysis of 71 cases. Cancer. 38:2404– 2419, 1976. 416. Prat J, Bhan AK, Dickersin GR, et al: Hepatoid yolk sac tumor of the ovary (endodermal sinus tumor with hepatoid differentiation): A light microscopic, ultrastructural and immunohistochemical study of seven cases. Cancer. 50:2355–2368, 1982. 417. Zirker TA, Silva EG, Morris M, et al: Immunohistochemical differentiation of clear-cell carcinoma of the female genital tract and endodermal sinus tumor with the use of alpha-fetoprotein and Leu-M1. Am J Clin Pathol. 91:511–514, 1989. 418. Devouassoux-Shisheboran M, Schammel DP, Tavassoli FA: Ovarian hepatoid yolk sac tumours: Morphological, immunohistochemical and ultrastructural features. Histopathology. 34:462– 469, 1999. 419. Esheba GE, Pate LL, Longacre TA: Oncofetal protein glypican-3 distinguishes yolk sac tumor from clear cell carcinoma of the ovary. Am J Surg Pathol. 32:600–607, 2008. 420. Ishikura H, Scully RE: Hepatoid carcinoma of the ovary. A newly described tumor. Cancer. 60:2775–2784, 1987. 421. Pitman MB, Triratanachat S, Young RH, et al: Hepatocyte paraffin 1 antibody does not distinguish primary ovarian tumors with hepatoid differentiation from metastatic hepatocellular carcinoma. Int J Gynecol Pathol. 23:58–64, 2004. 422. Tochigi N, Kishimoto T, Supriatna Y, et al: Hepatoid carcinoma of the ovary: A report of three cases admixed with a common surface epithelial carcinoma. Int J Gynecol Pathol. 22:266–271, 2003. 423. Young RH, Gersell DJ, Clement PB, et al: Hepatocellular carcinoma metastatic to the ovary: A report of three cases discovered during life with discussion of the differential diagnosis of hepatoid tumors of the ovary. Hum Pathol. 23:574–580, 1992. 424. Gagnon S, Tëtu B, Silva EG, et al: Frequency of alpha-fetoprotein production by Sertoli-Leydig cell tumors of the ovary: An immunohistochemical study of eight cases. Mod Pathol. 2:63–67, 1989. 425. Hammad A, Jasnosz KM, Olson PR: Expression of alphafetoprotein by ovarian Sertoli-Leydig cell tumors—Case report and review of the literature. Arch Pathol Lab Med. 119:1075– 1079, 1995. 426. Mooney EE, Nogales FF, Tavassoli FA: Hepatocytic differentiation in retiform Sertoli-Leydig cell tumors: distinguishing a heterologous element from Leydig cells. Hum Pathol. 30:611–617, 1999.
References 427. Niehans GA, Manivel JC, Copland GT, et al: Immunohistochemistry of germ cell and trophoblastic neoplasms. Cancer. 62:1113–1123, 1988. 428. Perrone T, Steeper TA, Dehner LP: Alpha-fetoprotein localization in pure ovarian teratoma. An immunohistochemical study of 12 cases. Am J Clin Pathol. 88:713–717, 1987. 429. Kurman RJ, Norris HJ: Embryonal carcinoma of the ovary: A clinicopathologic entity distinct from endodermal sinus tumor resembling embryonal carcinoma of the adult testis. Cancer. 38:2420–2433, 1976. 430. Ueda G, Abe Y, Yoshida M, et al: Embryonal carcinoma of the ovary: A six-year survival. Int J Gynaecol Obstet. 31:287–292, 1990. 431. Nakashima N, Fukatsu T, Nagasaka T, et al: The frequency and histology of hepatic tissue in germ cell tumors. Am J Surg Pathol. 11:682–692, 1987. 432. Furumoto M: Cellular localization of AFP, hCG and its free subunits, and SP1 in embryonal carcinoma of the testis and ovary. Pathol Res Pract. 173:12–21, 1981. 433. Lopez JM, Malpica A, Deavers MT, et al: Ovarian yolk sac tumor associated with endometrioid carcinoma and mucinous cystadenoma of the ovary. Ann Diagn Pathol. 7:300–305, 2003. 434. Zynger DL, McCallum JC, Luan C, et al: Glypican 3 has a higher sensitivity than alpha-fetoprotein for testicular and ovarian yolk sac tumour: immunohistochemical investigation with analysis of histological growth patterns. Histopathology. 56:750–757, 2010. 435. Maeda D, Ota S, Takazawa Y, et al: Glypican-3 expression in clear cell adenocarcinoma of the ovary. Mod Pathol. 22:824–832, 2009. 436. Cao D, Guo S, Allan RW, et al: SALL4 is a novel sensitive and specific marker of ovarian primitive germ cell tumors and is particularly useful in distinguishing yolk sac tumor from clear cell carcinoma. Am J Surg Pathol. 33:894–890, 2009. 437. Kurman RJ, Scardino PT, McIntire KR, et al: Cellular localization of alpha-fetoprotein and human chorionic gonadotropin in germ cell tumors of the testis using an indirect immunoperoxidase technique. Cancer. 40:2136–2151, 1977. 438. Hustin J, Reuter AM, Franchimont P: Immunohistochemical localization of HCG and its subunits in testicular germ cell tumours. Virchows Arch A Pathol Anat Histopathol. 406:333– 338, 1985. 439. Zaloudek CJ, Tavassoli FA, Norris HJ: Dysgerminoma with syncytiotrophoblastic giant cells: A histologically and clinically distinctive subtype of dysgerminoma. Am J Surg Pathol. 5:361–367, 1981. 440. Oliva E, Andrada E, Pezzica E, et al: Ovarian carcinomas with choriocarcinomatous differentiation. Cancer. 72:2441–2446, 1993. 441. Matias-Guiu X, Prat J: Ovarian tumors with functioning stroma. An immunohistochemical study of 100 cases with human chorionic gonadotropin monoclonal and polyclonal antibodies. Cancer. 65:2001–2005, 1990. 442. Mohabeer J, Buckley CH, Fox H: An immunohistochemical study of the incidence and significance of human chorionic gonadotrophin synthesis by epithelial ovarian neoplasms. Gynecol Oncol. 16:78–84, 1983. 443. Donato R: S100: A multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. Int J Biochem Cell Biol. 33:637–668, 2001. 444. Donato R: Intracellular and extracellular roles of S100 proteins. Microsc Res Tech. 60:540–551, 2003. 445. Heizmann CW, Fritz G, Schafer BW: S100 proteins: Structure, functions and pathology. Front Biosci. 7:d1356–d1368, 2002. 446. Gupta D, Deavers MT, Silva EG, et al: Malignant melanoma involving the ovary: A clinicopathologic and immunohistochemical study of 23 cases. Am J Surg Pathol. 28:771–780, 2004. 447. Drier JK, Swanson PE, Cherwitz DL, et al: S100 protein immunoreactivity in poorly differentiated carcinomas. Immunohistochemical comparison with malignant melanoma. Arch Pathol Lab Med. 111:447–452, 1987. 448. Herrera GA, Turbat-Herrera EA, Lott RL: S-100 protein expression by primary and metastatic adenocarcinomas. Am J Clin Pathol. 89:168–176, 1988. 449. Costa MJ, DeRose PB, Roth LM, et al: Immunohistochemical phenotype of ovarian granulosa cell tumors: Absence of
709.e11
epithelial membrane antigen has diagnostic value. Hum Pathol. 25:60–66, 1994. 450. Oliva E, Alvarez T, Young RH: Sertoli cell tumors of the ovary: A clinicopathologic and immunohistochemical study of 54 cases. Am J Surg Pathol. 29:143–156, 2005. 451. Jungbluth AA, Busam KJ, Gerald WL, et al: A103—An antiMelan-A monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues. Am J Surg Pathol. 22:595–602, 1998. 452. Bacchi CE, Bonetti F, Pea M, et al: HMB-45: A review. Appl Immunohistochem. 4:73–85, 1996. 453. Gown AM, Vogel AM, Hoak D, et al: Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Pathol. 123:195–203, 1986. 454. Wick MR, Swanson PE, Rocamora A: Recognition of malignant melanoma by monoclonal antibody HMB-45. An immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol. 15:201–207, 1988. 455. Hoon V, Thung SN, Kaneko M, et al: HMB-45 reactivity in renal angiomyolipoma and lymphangioleiomyomatosis. Arch Pathol Lab Med. 118:732–734, 1994. 456. Gyure KA, Hart WR, Kennedy AW: Lymphangiomyomatosis of the uterus associated with tuberous sclerosis and malignant neoplasia of the female genital tract: A report of two cases. Int J Gynecol Pathol. 14:344–351, 1995. 457. Longacre TA, Hendrickson MR, Kapp DS, et al: Lymphangioleiomyomatosis of the uterus simulating high-stage endometrial stromal sarcoma. Gynecol Oncol. 63:404–410, 1996. 458. Anderson AE, Yang X, Young RH: Epithelioid angiomyolipoma of the ovary: A case report and literature review. Int J Gynecol Pathol. 21:69–73, 2002. 459. Makhlouf HR, Ishak KG, Shekar R, et al: Melanoma markers in angiomyolipoma of the liver and kidney—A comparative study. Arch Pathol Lab Med. 126:49–55, 2002. 460. Stone CH, Lee MW, Amin MB, et al: Renal angiomyolipoma— Further immunophenotypic characterization of an expanding morphologic spectrum. Arch Pathol Lab Med. 125:751–758, 2001. 461. Matsui K, Tatsuguchi A, Valencia J, et al: Extrapulmonary lymphangioleiomyomatosis (LAM): Clinicopathologic features in 22 cases. Hum Pathol. 31:1242–1248, 2000. 462. Busam KJ, Iversen K, Coplan KA, et al: Immunoreactivity for A103, an antibody to Melan-A (MART-1), in adrenocortical and other steroid tumors. Am J Surg Pathol. 22:57–63, 1998. 463. Deavers MT, Malpica A, Ordonez NG, et al: Ovarian steroid cell tumors: An immunohistochemical study including a comparison of calretinin with inhibin. Int J Gynecol Pathol. 22:162–167, 2003. 464. Weiss LM, Chang KL: CD 45: A review. Appl Immunohistochem. 1:166–181, 1993. 465. Hanselaar AG, Vooijs GP, Mayall B, et al: Epithelial markers to detect occult microinvasion in serous ovarian tumors. Int J Gynecol Pathol. 12:20–27, 1993. 466. Halperin R, Zehavi S, Hadas E, et al: Immunohistochemical comparison of primary peritoneal and primary ovarian serous papillary carcinoma. Int J Gynecol Pathol. 20:341–345, 2001. 467. Geisler JP, Geisler HE, Wiemann MC, et al: Quantification of p53 in epithelial ovarian cancer. Gynecol Oncol. 66:435–438, 1997. 468. Köbel M, Kalloger SE, Carrick J, et al: A limited panel of immunomarkers can reliably distinguish between clear cell and highgrade serous carcinoma of the ovary. Am J Surg Pathol. 33:14–21, 2009. 469. O’Neill CJ, Deavers MT, Malpica A, et al: An immunohistochemical comparison between low-grade and high-grade ovarian serous carcinomas: Significantly higher expression of p53, MIB1, BCL2, HER-2/neu, and C-KIT in high-grade neoplasms. Am J Surg Pathol. 29:1034–1041, 2005. 470. Hashi A, Yuminamochi T, Murata S, et al: Wilms tumor gene immunoreactivity in primary serous carcinomas of the fallopian tube, ovary, endometrium, and peritoneum. Int J Gynecol Pathol. 22:374–377, 2003. 471. Al Hussaini M, Stockman A, Foster H, et al: WT-1 assists in distinguishing ovarian from uterine serous carcinoma and in
709.e12
Immunohistology of the Female Genital Tract
distinguishing between serous and endometrioid ovarian carcinoma. Histopathology. 44:109–115, 2004. 472. Waldstrom M, Grove A: Immunohistochemical expression of wilms tumor gene protein in different histologic subtypes of ovarian carcinomas. Arch Pathol Lab Med. 129:85–88, 2005. 473. Tong GX, Chiriboga L, Hamele-Bena D, et al: Expression of PAX2 in papillary serous carcinoma of the ovary: Immunohistochemical evidence of fallopian tube or secondary Müllerian system origin? Mod Pathol. 20:856–863, 2007. 474. Nonaka D, Chiriboga L, Soslow RA: Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol. 32:1566–1571, 2008. 475. Lin X, Lindner JL, Silverman JF, et al: Intestinal type and endocervical-like ovarian mucinous neoplasms are immunophenotypically distinct entities. Appl Immunohistochem Mol Morphol. 16:453–458, 2008. 476. Vang R, Gown AM, Barry TS, et al: Cytokeratins 7 and 20 in primary and secondary mucinous tumors of the ovary: Analysis of coordinate immunohistochemical expression profiles and staining distribution in 179 cases. Am J Surg Pathol. 30:1130– 1139, 2006. 477. Werling RW, Yaziji H, Bacchi CE, et al: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: An immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 27:303–310, 2003. 478. Moskaluk CA, Zhang H, Powell SM, et al: Cdx2 protein expression in normal and malignant human tissues: An immunohistochemical survey using tissue microarrays. Mod Pathol. 16:913–919, 2003. 479. Groisman GM, Meir A, Sabo E: The value of Cdx2 immunostaining in differentiating primary ovarian carcinomas from colonic carcinomas metastatic to the ovaries. Int J Gynecol Pathol. 23:52–57, 2004. 480. Tornillo L, Moch H, Diener PA, et al: CDX-2 immunostaining in primary and secondary ovarian carcinomas. J Clin Pathol. 57:641–643, 2004. 481. Logani S, Oliva E, Arnell PM, et al: Use of novel immunohistochemical markers expressed in colonic adenocarcinoma to distinguish primary ovarian tumors from metastatic colorectal carcinoma. Mod Pathol. 18:19–25, 2005. 482. Vang R, Gown AM, Wu LS, et al: Immunohistochemical expression of CDX2 in primary ovarian mucinous tumors and metastatic mucinous carcinomas involving the ovary: Comparison with CK20 and correlation with coordinate expression of CK7. Mod Pathol. 19:1421–1428, 2006. 483. Vang R, Gown AM, Zhao C, et al: Ovarian mucinous tumors associated with mature cystic teratomas: Morphologic and immunohistochemical analysis identifies a subset of potential teratomatous origin that shares features of lower gastrointestinal tract mucinous tumors more commonly encountered as secondary tumors in the ovary. Am J Surg Pathol. 31:854–869, 2007. 484. Zhang PJ, Shah M, Spiegel GW, et al: Cytokeratin 7 immunoreactivity in rectal adenocarcinomas. Appl Immunohistochem Mol Morphol. 11:306–310, 2003. 485. Rutgers JL, Bell DA: Immunohistochemical characterization of ovarian borderline tumors of intestinal and mullerian types. Mod Pathol. 5:367–371, 1992. 486. Vang R, Gown AM, Barry TS, et al: Immunohistochemistry for estrogen and progesterone receptors in the distinction of primary and metastatic mucinous tumors in the ovary: An analysis of 124 cases. Mod Pathol. 19:97–105, 2006. 487. Ronnett BM, Shmookler BM, Diener-West M, et al: Immunohistochemical evidence supporting the appendiceal origin of pseudomyxoma peritonei in women. Int J Gynecol Pathol. 16:1– 9, 1997. 488. O’Connell JT, Tomlinson JS, Roberts AA, et al: Pseudomyxoma peritonei is a disease of MUC2-expressing goblet cells. Am J Pathol. 161:551–564, 2002. 489. Prat J, Young RH, Scully RE: Ovarian mucinous tumors with foci of anaplastic carcinoma. Cancer. 50:300–304, 1982. 490. Provenza C, Young RH, Prat J: Anaplastic carcinoma in mucinous ovarian tumors: A clinicopathologic study of 34 cases emphasizing the crucial impact of stage on prognosis, their histologic spectrum, and overlap with sarcomalike mural nodules. Am J Surg Pathol. 32:383–389, 2008.
491. Prat J, Scully RE: Sarcomas in ovarian mucinous tumors. A report of two cases. Cancer. 44:1327–1331, 1979. 492. Prat J, Scully RE: Ovarian mucinous tumors with sarcoma-like mural nodules. A report of seven cases. Cancer. 44:1332–1344, 1979. 493. Bague S, Rodriguez IM, Prat J: Sarcoma-like mural nodules in mucinous cystic tumors of the ovary revisited—A clinicopathologic analysis of 10 additional cases. Am J Surg Pathol. 26:1467– 1476, 2002. 494. Nichols GE, Mills SE, Ulbright TM, et al: Spindle cell mural nodules in cystic ovarian mucinous tumors: A clinicopathologic and immunohistochemical study of five cases. Am J Surg Pathol. 15:1055–1062, 1991. 495. Chan YF, Ho HC, Yau SM, et al: Ovarian mucinous tumor with mural nodules of anaplastic carcinoma. Gynecol Oncol. 35:112– 119, 1989. 496. Baergen RN, Rutgers JL: Mural nodules in common epithelial tumors of the ovary. Int J Gynecol Pathol. 13:62–71, 1994. 497. Matias-Guiu X, Aranda I, Prat J: Immunohistochemical study of sarcoma-like mural nodules in a mucinous cystadenocarcinoma of the ovary. Virchows Arch A Pathol Anat Histopathol. 419:89– 92, 1991. 498. Lee KR, Nucci MR: Ovarian mucinous and mixed epithelial carcinomas of mullerian (endocervical-like) type: A clinicopathologic analysis of four cases of an uncommon variant associated with endometriosis. Int J Gynecol Pathol. 22:42–51, 2003. 499. Sarrio D, Moreno-Bueno G, Sanchez-Estevez C, et al: Expression of cadherins and catenins correlates with distinct histologic types of ovarian carcinomas. Hum Pathol. 37:1042–1049, 2006. 500. Oliva E, Sarrio D, Brachtel EF, et al: High frequency of betacatenin mutations in borderline endometrioid tumours of the ovary. J Pathol. 208:708–713, 2006. 501. Houghton O, Connolly LE, McCluggage WG: Morules in endometrioid proliferations of the uterus and ovary consistently express the intestinal transcription factor CDX2. Histopathology. 53:156–165, 2008. 502. Ordi J, Schammel DP, Rasekh L, et al: Sertoliform endometrioid carcinomas of the ovary: A clinicopathologic and immunohistochemical study of 13 cases. Mod Pathol. 12:933–940, 1999. 503. Misir A, Sur M: Sertoliform endometrioid carcinoma of the ovary: A potential diagnostic pitfall. Arch Pathol Lab Med. 131:979–981, 2007. 504. Elishaev E, Gilks CB, Miller D, et al: Synchronous and metachronous endocervical and ovarian neoplasms: Evidence supporting interpretation of the ovarian neoplasms as metastatic endocervical adenocarcinomas simulating primary ovarian surface epithelial neoplasms. Am J Surg Pathol. 29:281–294, 2005. 505. Vang R, Gown AM, Farinola M, et al: p16 expression in primary ovarian mucinous and endometrioid tumors and metastatic adenocarcinomas in the ovary: Utility for identification of metastatic HPV-related endocervical adenocarcinomas. Am J Surg Pathol. 31:653–663, 2007. 506. Wentzensen N, du Bois AD, Kommoss S, et al: No metastatic cervical adenocarcinomas in a series of p16INK4a-positive mucinous or endometrioid advanced ovarian carcinomas: An analysis of the AGO Ovarian Cancer Study Group. Int J Gynecol Pathol. 27:18–23, 2008. 507. Dellers EA, Valente PT, Edmonds PR, et al: Extrauterine mixed mesodermal tumors: An immunohistochemical study. Arch Pathol Lab Med. 115:918–920, 1991. 508. De Brito PA, Silverberg SG, Orenstein JM: Carcinosarcoma (malignant mixed Müllerian (mesodermal) tumor) of the female genital tract: Immunohistochemical and ultrastructural analysis of 28 cases. Hum Pathol. 24:132–142, 1993. 509. Chang KL, Crabtree GS, Lim-Tan SK, et al: Primary extrauterine endometrial stromal neoplasms: A clinicopathologic study of 20 cases and a review of the literature. Int J Gynecol Pathol. 12:282– 296, 1993. 510. Cameron RI, Ashe P, O’Rourke DM, et al: A panel of immunohistochemical stains assists in the distinction between ovarian and renal clear cell carcinoma. Int J Gynecol Pathol. 22:272–276, 2003.
References 511. Kato N, Motoyama T: Overexpression of osteopontin in clear cell carcinoma of the ovary: Close association with HNF-1beta expression. Histopathology. 52:682–688, 2008. 512. Ho ES, Lai CR, Hsieh YT, et al: p53 mutation is infrequent in clear cell carcinoma of the ovary. Gynecol Oncol. 80:189–193, 2001. 513. Shimizu M, Nikaido T, Toki T, et al: Clear cell carcinoma has an expression pattern of cell cycle regulatory molecules that is unique among ovarian adenocarcinomas. Cancer. 85:669–677, 1999. 514. Kwon TJ, Ro JY, Tornos C, et al: Reduplicated basal lamina in clear-cell carcinoma of the ovary: An immunohistochemical and electron microscopic study. Ultrastruct Pathol. 20:529–536, 1996. 515. Mikami Y, Hata S, Melamed J, et al: Basement membrane material in ovarian clear cell carcinoma: Correlation with growth pattern and nuclear grade. Int J Gynecol Pathol. 18:52–57, 1999. 516. Offman S, Longacre TA: Clear cell carcinoma of the female genital tract. Adv Anat Pathol. 19:296–312, 2012. 517. Nolan LP, Heatley MK: The value of immunocytochemistry in distinguishing between clear cell carcinoma of the kidney and ovary. Int J Gynecol Pathol. 20:155–159, 2001. 518. Ordonez NG, Mackay B: Brenner tumor of the ovary: A comparative immunohistochemical and ultrastructural study with transitional cell carcinoma of the bladder. Ultrastruct Pathol. 24:157–167, 2000. 519. Riedel I, Czernobilsky B, Lifschitz-Mercer B, et al: Brenner tumors but not transitional cell carcinomas of the ovary show urothelial differentiation: Immunohistochemical staining of urothelial markers, including cytokeratins and uroplakins. Virchows Arch Int J Pathol. 438:181–191, 2001. 520. Soslow RA, Rouse RV, Hendrickson MR, et al: Transitional cell neoplasms of the ovary and urinary bladder: A comparative immunohistochemical analysis. Int J Gynecol Pathol. 15:257– 265, 1996. 521. Kaufmann O, Volmerig J, Dietel M: Uroplakin III is a highly specific and moderately sensitive immunohistochemical marker for primary and metastatic urothelial carcinomas. Am J Clin Pathol. 113:683–687, 2000. 522. Ogawa K, Johansson SL, Cohen SM: Immunohistochemical analysis of uroplakins, urothelial specific proteins, in ovarian Brenner tumors, normal tissues, and benign and neoplastic lesions of the female genital tract. Am J Pathol. 155:1047–1050, 1999. 523. Liao XY, Xue WC, Shen DH, et al: p63 expression in ovarian tumours: A marker for Brenner tumours but not transitional cell carcinomas. Histopathology. 51:477–483, 2007. 524. Ordonez NG: Transitional cell carcinomas of the ovary and bladder are immunophenotypically different. Histopathology. 36:433–438, 2000. 525. Ordonez NG: Application of mesothelin immunostaining in tumor diagnosis. Am J Surg Pathol. 27:1418–1428, 2003. 526. Ohishi Y, Kaku T, Oya M, et al: CD56 expression in ovarian granulosa cell tumors, and its diagnostic utility and pitfalls. Gynecol Oncol. 107:30–38, 2007. 527. McCluggage WG, McKenna M, McBride HA: CD56 is a sensitive and diagnostically useful immunohistochemical marker of ovarian sex cord-stromal tumors. Int J Gynecol Pathol. 26:322– 327, 2007. 528. Riopel MA, Perlman EJ, Seidman JD, et al: Inhibin and epithelial membrane antigen immunohistochemistry assist in the diagnosis of sex cord-stromal tumors and provide clues to the histogenesis of hypercalcemic small cell carcinomas. Int J Gynecol Pathol. 17:46–53, 1998. 529. Young RH, Oliva E, Scully RE: Small cell carcinoma of the ovary, hypercalcemic type: A clinicopathological analysis of 150 cases. Am J Surg Pathol. 18:1102–1116, 1994. 530. Aguirre P, Thor AD, Scully RE: Ovarian small cell carcinoma. Histogenetic considerations based on immunohistochemical and other findings. Am J Clin Pathol. 92:140–149, 1989. 531. Young RH, Scully RE: Malignant melanoma metastatic to the ovary: A clinicopathologic analysis of 20 cases. Am J Surg Pathol. 15:849–860, 1991. 532. McCluggage WG, Bissonnette JP, Young RH: Primary malignant melanoma of the ovary: A report of 9 definite or probable cases
709.e13
with emphasis on their morphologic diversity and mimicry of other primary and secondary ovarian neoplasms. Int J Gynecol Pathol. 25:321–329, 2006. 533. Young RH, Eichhorn JH, Dickersin GR, et al: Ovarian involvement by the intra-abdominal desmoplastic small round cell tumor with divergent differentiation: A report of three cases. Hum Pathol. 23:454–464, 1992. 534. Zaloudek C, Miller TR, Stern JL: Desmoplastic small cell tumor of the ovary: A unique polyphenotypic tumor with an unfavorable prognosis. Int J Gynecol Pathol. 14:260–265, 1995. 535. Fang X, Rodabaugh K, Penetrante R, et al: Desmoplastic small round cell tumor (DSRCT) with ovarian involvement in 2 young women. Appl Immunohistochem Mol Morphol. 16:94–99, 2008. 536. Kawauchi S, Fukuda T, Miyamoto S, et al: Peripheral primitive neuroectodermal tumor of the ovary confirmed by CD99 immunostaining, karyotypic analysis, and RT-PCR for EWS/FLI-1 chimeric mRNA. Am J Surg Pathol. 22:1417–1422, 1998. 537. Young RH, Scully RE: Alveolar rhabdomyosarcoma metastatic to the ovary. A report of two cases and a discussion of the differential diagnosis of small cell malignant tumors of the ovary. Cancer. 64:899–904, 1989. 538. Nielsen GP, Oliva E, Young RH, et al: Primary ovarian rhabdomyosarcoma: A report of 13 cases. Int J Gynecol Pathol. 17:113– 119, 1998. 539. Paler RJ, Felix JC: Desmin, myoglobin, and muscle-specific actin immunohistochemical staining in a case of embryonal rhabdomyosarcoma of the ovary. Appl Immunohistochem Mol Morphol. 7:237–241, 1999. 540. Katsube Y, Berg JW, Silverberg SG: Epidemiologic pathology of ovarian tumors: A histopathologic review of primary ovarian neoplasms diagnosed in the Denver Standard Metropolitan Statistical Area, 1 July–31 December 1969 and 1 July–31 December 1979. Int J Gynecol Pathol. 1:3–16, 1982. 541. Koonings PP, Campbell K, Mishell DR, Jr, et al: Relative frequency of primary ovarian neoplasms: A 10-year review. Obstet Gynecol. 74:921–926, 1989. 542. Shah SP, Köbel M, Senz J, et al: Mutation of FOXL2 in granulosacell tumors of the ovary. N Engl J Med. 360:2719–2729, 2009. 543. Stewart CJR, Jeffers MD, Kennedy A: Diagnostic value of inhibin immunoreactivity in ovarian gonadal stromal tumours and their histological mimics. Histopathology. 31:67–74, 1997. 544. Oliva E, Garcia-Miralles N, Vu Q, et al: CD10 expression in pure stromal and sex cord-stromal tumors of the ovary: An immunohistochemical analysis of 101 cases. Int J Gynecol Pathol. 26:359– 367, 2007. 545. He H, Luthringer DJ, Hui P, et al: Expression of CD56 and WT1 in ovarian stroma and ovarian stromal tumors. Am J Surg Pathol. 32:884–890, 2008. 546. Tiltman AJ, Haffajee Z: Sclerosing stromal tumors, thecomas, and fibromas of the ovary: An immunohistochemical profile. Int J Gynecol Pathol. 18:254–258, 1999. 547. Chalvardjian A, Scully RE: Sclerosing stromal tumors of the ovary. Cancer. 31:664–670, 1973. 548. Kawauchi S, Tsuji T, Kaku T, et al: Sclerosing stromal tumor of the ovary. A clinicopathologic, immunohistochemical, ultrastructural, and cytogenetic analysis with special reference to its vasculature. Am J Surg Pathol. 22:83–92, 1998. 549. Sabah M, Leader M, Kay E: The problem with KIT: Clinical implications and practical difficulties with CD117 immunostaining. Appl Immunohistochem Mol Morphol. 11:56–61, 2003. 550. Saitoh A, Tsutsumi Y, Osamura RY, et al: Sclerosing stromal tumor of the ovary. Immunohistochemical and electron microscopic demonstration of smooth-muscle differentiation. Arch Pathol Lab Med. 113:372–376, 1989. 551. Shaw JA, Dabbs DJ, Geisinger KR: Sclerosing stromal tumor of the ovary: An ultrastructural and immunohistochemical analysis with histogenetic considerations. Ultrastruct Pathol. 16:363–377, 1992. 552. Ishioka S, Sagae S, Saito T, et al: A case of a sclerosing stromal ovarian tumor that expresses VEGF. J Obstet Gynaecol Res. 26:35–38, 2000. 553. Miettinen M, Wang ZF, Paetau A, et al: ERG transcription factor as an immunohistochemical marker for vascular endothelial
709.e14
Immunohistology of the Female Genital Tract
tumors and prostatic carcinoma. Am J Surg Pathol. 35:432–441, 2011. 554. Fox H: Pathologic prognostic factors in early stage adult-type granulosa cell tumors of the ovary. Int J Gynecol Cancer. 13:1–4, 2003. 555. Schumer ST, Cannistra SA: Granulosa cell tumor of the ovary. J Clin Oncol. 21:1180–1189, 2003. 556. Hines JF, Khalifa MA, Moore JL, et al: Recurrent granulosa cell tumor of the ovary 37 years after initial diagnosis: A case report and review of the literature. Gynecol Oncol. 60:484–488, 1996. 557. Shah VI, Freites NO, Maxwell P, et al: Inhibin is more specific than calretinin as an immunohistochemical marker for differentiating sarcomatoid granulosa cell tumour of the ovary from other spindle cell neoplasms. J Clin Pathol. 56:221–224, 2003. 558. Gitsch G, Kohlberger P, Steiner A, et al: Expression of cytokeratins in granulosa cell tumors and ovarian carcinomas. Arch Gynecol Obstet. 251:193–197, 1992. 559. Otis CN, Powell JL, Barbuto D, et al: Intermediate filamentous proteins in adult granulosa cell tumors: An immunohistochemical study of 25 cases. Am J Surg Pathol. 16:962–968, 1992. 560. Choi YL, Kim HS, Ahn G: Immunoexpression of inhibin alpha subunit, inhibin/activin betaA subunit and CD99 in ovarian tumors. Arch Pathol Lab Med. 124:563–569, 2000. 561. Gordon MD, Corless C, Renshaw AA, et al: CD99, keratin, and vimentin staining of sex cord-stromal tumors, normal ovary, and testis. Mod Pathol. 11:769–773, 1998. 562. Cathro HP, Stoler MH: The utility of calretinin, inhibin, and WT1 immunohistochemical staining in the differential diagnosis of ovarian tumors. Hum Pathol. 36:195–201, 2005. 563. Farinola MA, Gown AM, Judson K, et al: Estrogen receptor alpha and progesterone receptor expression in ovarian adult granulosa cell tumors and Sertoli-Leydig cell tumors. Int J Gynecol Pathol. 26:375–382, 2007. 564. Young RH, Dickersin GR, Scully RE: Juvenile granulosa cell tumor of the ovary. A clinicopathological analysis of 125 cases. Am J Surg Pathol. 8:575–596, 1984. 565. Zaloudek CJ, Norris HJ: Granulosa tumors of the ovary in children: A clinical and pathologic study of 32 cases. Am J Surg Pathol. 6:503–512, 1982. 566. Rakheja D, Sharma S: Pathologic quiz case—Cystic and solid ovarian tumor in a 43-year-old woman—Pathologic diagnosis: Cystic juvenile-type granulosa cell tumor of the ovary in an adult. Arch Pathol Lab Med. 126:1123–1124, 2002. 567. McCluggage WG: Immunoreactivity of ovarian juvenile granulosa cell tumours with epithelial membrane antigen. Histopathology. 46:235–236, 2005. 568. Young RH: Sertoli-Leydig cell tumors of the ovary: Review with emphasis on historical aspects and unusual forms. Int J Gynecol Pathol. 12:141–147, 1993. 569. Zaloudek C, Norris HJ: Sertoli-Leydig tumors of the ovary. A clinicopatholgic study of 64 intermediate and poorly differentiated neoplasms. Am J Surg Pathol. 8:405–418, 1984. 570. Young RH, Scully RE: Well-differentiated ovarian Sertoli-Leydig cell tumors: A clinicopathological analysis of 23 tumors. Int J Gynecol Pathol. 3:277–290, 1984. 571. Young RH, Scully RE: Ovarian Sertoli-Leydig cell tumors. A clinicopathological analysis of 207 cases. Am J Surg Pathol. 9:543–569, 1985. 572. Young RH, Scully RE: Ovarian Sertoli-Leydig cell tumors with a retiform pattern—A problem in diagnosis: A report of 25 cases. Am J Surg Pathol. 7:755–771, 1983. 573. Mooney EE, Nogales FF, Bergeron C, et al: Retiform SertoliLeydig cell tumours: Clinical, morphological and immunohistochemical findings. Histopathology. 41:110–117, 2002. 574. Guerrieri C, Franlund B, Malmstrom H, et al: Ovarian endometrioid carcinomas simulating sex cord-stromal tumors: A study using inhibin and cytokeratin 7. Int J Gynecol Pathol. 17:266– 271, 1998. 575. McCluggage WG, Young RH: Ovarian sertoli-leydig cell tumors with pseudoendometrioid tubules (pseudoendometrioid sertolileydig cell tumors). Am J Surg Pathol. 31:592–597, 2007. 576. Young RH, Prat J, Scully RE: Ovarian Sertoli-Leydig cell tumors with heterologous elements. I. Gastrointestinal epithelium and carcinoid: A clinicopathologic analysis of 36 cases. Cancer. 50:2448–2456, 1982.
577. Aguirre P, Scully RE, DeLellis RA: Ovarian heterologous SertoliLeydig cell tumors with gastrointestinal-type epithelium. An immunohistochemical analysis. Arch Pathol Lab Med. 110:528– 533, 1986. 578. Prat J, Young RH, Scully RE: Ovarian Sertoli-Leydig cell tumors with heterologous elements. II. Cartilage and skeletal muscle. A clinicopathologic analysis of twelve cases. Cancer. 50:2465– 2475, 1982. 579. Roth LM, Liban E, Czernobilsky B: Ovarian endometrioid tumors mimicking Sertoli and Sertoli-Leydig cell tumors: Sertoliform variant of endometrioid carcinoma. Cancer. 50:1322– 1331, 1982. 580. McCluggage WG, Young RH: Ovarian sertoli-leydig cell tumors with pseudoendometrioid tubules (pseudoendometrioid sertolileydig cell tumors). Am J Surg Pathol. 31:592–597, 2007. 581. Young RH, Scully RE: Ovarian Sertoli cell tumors. A report of 10 cases. Int J Gynecol Pathol. 2:349–363, 1984. 582. Ferry JA, Young RH, Engel G, et al: Oxyphilic Sertoli cell tumor of the ovary: A report of three cases, two in patients with the Peutz-Jeghers syndrome. Int J Gynecol Pathol. 13:259–266, 1994. 583. Aguirre P, Thor AD, Scully RE: Ovarian endometrioid carcinomas resembling sex cord-stromal tumors. An immunohistochemical study. Int J Gynecol Pathol. 8:364–373, 1989. 584. Young RH, Prat J, Scully RE: Ovarian endometrioid carcinomas resembling sex cord-stromal tumors. A clinicopathologic analysis of 13 cases. Am J Surg Pathol. 6:513–522, 1982. 585. Benjamin E, Law S, Bobrow LG: Intermediate filaments cytokeratin and vimentin in ovarian sex cord-stromal tumours with correlative studies in adult and fetal ovaries. J Pathol. 152:253– 263, 1987. 586. Stewart GJR, Nandini CL, Richmond JA: Value of A103 (melanA) immunostaining in the differential diagnosis of ovarian sex cord stromal tumours. J Clin Pathol. 53:206–211, 2000. 587. Seidman JD, Abbondanzo SL, Bratthauer GL: Lipid cell (steroid cell) tumor of the ovary: Immunophenotype with analysis of potential pitfall due to endogenous biotin-like activity. Int J Gynecol Pathol. 14:331–338, 1995. 588. Lau SK, Weiss LM, Chu PG: D2-40 immunohistochemistry in the differential diagnosis of seminoma and embryonal carcinoma: A comparative immunohistochemical study with KIT (CD117) and CD30. Mod Pathol. 20:320–325, 2007. 589. Cossu-Rocca P, Jones TD, Roth LM, et al: Cytokeratin and CD30 expression in dysgerminoma. Hum Pathol. 37:1015–1021, 2006. 590. McCluggage WG, Ashe P, McBride H, et al: Localization of the cellular expression of inhibin in trophoblastic tissue. Histopathology. 32:252–256, 1998. 591. Pelkey TJ, Frierson HFJ, Mills SE, et al: Detection of the alphasubunit of inhibin in trophoblastic neoplasia. Hum Pathol. 30:26–31, 1999. 592. Oliva E, Musulen E, Prat J, et al: Transitional cell carcinoma of the renal pelvis with symptomatic ovarian metastases. Int J Surg Pathol. 2:231–236, 1995. 593. Hirabayashi K, Yasuda M, Osamura RY, et al: Ovarian nongestational choriocarcinoma mixed with various epithelial malignancies in association with endometriosis. Gynecol Oncol. 102:111–117, 2006. 594. Bing Z, Pasha TL, Lal P, et al: Expression of glial cell line-derived neurotropic factor receptor alpha-1 in immature teratomas. Am J Clin Pathol. 130:892–896, 2008. 595. Nogales FF, Avila IR, Concha A, et al: Immature endodermal teratoma of the ovary: Embryologic correlations and immunohistochemistry. Hum Pathol. 24:364–370, 1993. 596. Szyfelbein WM, Young RH, Scully RE: Cystic struma ovarii: A frequently unrecognized tumor: A report of 20 cases. Am J Surg Pathol. 18:785–788, 1994. 597. Szyfelbein WM, Young RH, Scully RE: Struma ovarii simulating ovarian tumors of other types. A report of 30 cases. Am J Surg Pathol. 19:21–29, 1995. 598. Devaney K, Snyder R, Norris HJ, et al: Proliferative and histologically malignant struma ovarii: A clinicopathologic study of 54 cases. Int J Gynecol Pathol. 12:333–343, 1993. 599. Sporrong B, Falkmer S, Robboy SJ, et al: Neurohormonal peptides in ovarian carcinoids: An immunohistochemical study of 81 primary carcinoids and of intraovarian metastases from six mid-gut carcinoids. Cancer. 49:68–74, 1982.
References 600. Sidhu J, Sánchez RL: Prostatic acid phosphatase in strumal carcinoids of the ovary: An immunohistochemical study. Cancer. 72:1673–1678, 1993. 601. Stagno PA, Petras RE, Hart WR: Strumal carcinoids of the ovary. An immunohistologic and ultrastructural study. Arch Pathol Lab Med. 111:440–446, 1987. 602. Snyder RR, Tavassoli FA: Ovarian strumal carcinoid: Immunohistochemical, ultrastructural, and clinicopathologic analysis. Int J Gynecol Pathol. 5:187–201, 1986. 603. Matsuda K, Maehama T, Kanazawa K: Strumal carcinoid tumor of the ovary: A case exhibiting severe constipation associated with PYY. Gynecol Oncol. 87:143–145, 2002. 604. Baker PM, Oliva E, Young RH, et al: Ovarian mucinous carcinoids including some with a carcinomatous component—A report of 17 cases. Am J Surg Pathol. 25:557–568, 2001. 605. Rabban JT, Lerwill MF, McCluggage WG, et al: Primary ovarian carcinoid tumors may express CDX-2: A potential pitfall in distinction from metastatic intestinal carcinoid tumors involving the ovary. Int J Gynecol Pathol. 28:41–48, 2009. 606. Hussong J, Crussi FG, Chou PM: Gonadoblastoma: Immunohistochemical localization of Müllerian-inhibiting substance, inhibin, WT-1, and p53. Mod Pathol. 10:1101–1105, 1997. 607. Roth LM, Eglen DE: Gonadoblastoma. Immunohistochemical and ultrastructural observations. Int J Gynecol Pathol. 8:72–81, 1989. 608. Moore RG, Chung M, Granai CO, et al: Incidence of metastasis to the ovaries from nongenital tract primary tumors. Gynecol Oncol. 93:87–91, 2004. 609. Seidman JD, Kurman RJ, Ronnett BM: Primary and metastatic mucinous adenocarcinomas in the ovaries: Incidence in routine practice with a new approach to improve intraoperative diagnosis. Am J Surg Pathol. 27:985–993, 2003. 610. Yemelyanova AV, Vang R, Judson K, et al: Distinction of primary and metastatic mucinous tumors involving the ovary: Analysis of size and laterality data by primary site with reevaluation of an algorithm for tumor classification. Am J Surg Pathol. 32:128– 138, 2008. 611. Lewis MR, Deavers MT, Silva EG, et al: Ovarian involvement by metastatic colorectal adenocarcinoma: Still a diagnostic challenge. Am J Surg Pathol. 30:177–184, 2006. 612. Ronnett BM, Kurman RJ, Shmookler BM, et al: The morphologic spectrum of ovarian metastases of appendiceal adenocarcinomas— A clinicopathologic and immunohistochemical analysis of tumors often misinterpreted as primary ovarian tumors or metastatic tumors from other gastrointestinal sites. Am J Surg Pathol. 21:1144–1155, 1997. 613. Khunamornpong S, Siriaunkgul S, Suprasert P, et al: Intrahepatic cholangiocarcinoma metastatic to the ovary: A report of 16 cases of an underemphasized form of secondary tumor in the ovary that may mimic primary neoplasia. Am J Surg Pathol. 31:1788– 1799, 2007. 614. Khunamornpong S, Lerwill MF, Siriaunkgul S, et al: Carcinoma of extrahepatic bile ducts and gallbladder metastatic to the ovary: A report of 16 cases. Int J Gynecol Pathol. 27:366–379, 2008. 615. Park SY, Kim HS, Hong EK, et al: Expression of cytokeratins 7 and 20 in primary carcinomas of the stomach and colorectum and their value in the differential diagnosis of metastatic carcinomas to the ovary. Hum Pathol. 33:1078–1085, 2002. 616. Lerwill MF, Young RH: Ovarian metastases of intestinal-type gastric carcinoma: A clinicopathologic study of 4 cases with contrasting features to those of the Krukenberg tumor. Am J Surg Pathol. 30:1382–1388, 2006. 617. McCluggage WG, Young RH: Primary ovarian mucinous tumors with signet ring cells: Report of 3 cases with discussion of so-called primary Krukenberg tumor. Am J Surg Pathol. 32:1373– 1379, 2008. 618. Reichert RA: Primary ovarian adenofibromatous neoplasms with mucin-containing signet-ring cells: A report of 2 cases. Int J Gynecol Pathol. 26:165–172, 2007. 619. Kiyokawa T, Young RH, Scully RE: Krukenberg tumors of the ovary: A clinicopathologic analysis of 120 cases with emphasis on their variable pathologic manifestations. Am J Surg Pathol. 30:277–299, 2006.
709.e15
620. Young RH, Hart WR: Metastatic intestinal carcinomas simulating primary ovarian clear cell carcinoma and secretory endometrioid carcinoma—A clinicopathologic and immunohistochemical study of five cases. Am J Surg Pathol. 22:805–815, 1998. 621. Vakiani E, Young RH, Carcangiu ML, et al: Acinar cell carcinoma of the pancreas metastatic to the ovary: A report of 4 cases. Am J Surg Pathol. 32:1540–1545, 2008. 622. Gagnon Y, Tëtu B: Ovarian metastases of breast carcinoma. A clinicopathologic study of 59 cases. Cancer. 64:892–898, 1989. 623. Young RH, Carey RW, Robboy SJ: Breast carcinoma masquerading as primary ovarian neoplasm. Cancer. 48:210–212, 1981. 624. Moritani S, Ichihara S, Hasegawa M, et al: Serous papillary adenocarcinoma of the female genital organs and invasive micropapillary carcinoma of the breast. Are WT1, CA125, and GCDFP-15 useful in differential diagnosis? Hum Pathol. 39:666– 671, 2008. 625. Monteagudo C, Merino MJ, LaPorte N, et al: Value of gross cystic disease fluid protein-15 in distinguishing metastatic breast carcinomas among poorly differentiated neoplasms involving the ovary. Hum Pathol. 22:368–372, 1991. 626. Kanner WA, Galgano MT, Stoler MH, et al: Distinguishing breast carcinoma from Müllerian serous carcinoma with mammaglobin and mesothelin. Int J Gynecol Pathol. 27:491–495, 2008. 627. Lau SK, Luthringer DJ, Eisen RN: Thyroid transcription factor-1: A review. Appl Immunohistochem Mol Morphol. 10:97–102, 2002. 628. Howell NR, Zheng W, Cheng L, et al: Carcinomas of ovary and lung with clear cell features: can immunohistochemistry help in differential diagnosis? Int J Gynecol Pathol. 26:134–140, 2007. 629. Irving JA, Young RH: Lung Carcinoma Metastatic to the Ovary: A clinicopathologic study of 32 cases emphasizing their morphologic spectrum and problems in differential diagnosis. Am J Surg Pathol. 29:997–1006, 2005. 630. Kaufmann O, Dietel M: Expression of thyroid transcription factor-1 in pulmonary and extrapulmonary small cell carcinomas and other neuroendocrine carcinomas of various primary sites. Histopathology. 36:415–420, 2000. 631. Ordonez NG: Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol. 24:1217–1223, 2000. 632. Cheuk W, Kwan MY, Suster S, et al: Immunostaining for thyroid transcription factor 1 and cytokeratin 20 aids the distinction of small cell carcinoma from Merkel cell carcinoma, but not pulmonary from extrapulmonary small cell carcinomas. Arch Pathol Lab Med. 125:228–231, 2001. 633. Ronnett BM, Yemelyanova AV, Vang R, et al: Endocervical adenocarcinomas with ovarian metastases: Analysis of 29 cases with emphasis on minimally invasive cervical tumors and the ability of the metastases to simulate primary ovarian neoplasms. Am J Surg Pathol. 32:1835–1853, 2008. 634. Moran CA, Albores-Saavedra J, Suster S: Primary peritoneal mesotheliomas in children: A clinicopathological and immunohistochemical study of eight cases. Histopathology. 52:824–830, 2008. 635. Goldblum J, Hart WR: Localized and diffuse mesotheliomas of the genital tract and peritoneum in women—A clinicopathologic study of nineteen true mesothelial neoplasms, other than adenomatoid tumors, multicystic mesotheliomas, and localized fibrous tumors. Am J Surg Pathol. 19:1124–1137, 1995. 636. Clement PB, Young RH, Scully RE: Malignant mesotheliomas presenting as ovarian masses—A report of nine cases, including two primary ovarian mesotheliomas. Am J Surg Pathol. 20:1067– 1080, 1996. 637. Kerrigan SAJ, Turnnir RT, Clement PB, et al: Diffuse malignant epithelial mesotheliomas of the peritoneum in women—A clinicopathologic study of 25 patients. Cancer. 94:378–385, 2002. 638. Baker PM, Clement PB, Young RH: Malignant peritoneal mesothelioma in women: A study of 75 cases with emphasis on their morphologic spectrum and differential diagnosis. Am J Clin Pathol. 123:724–737, 2005. 639. Nascimento AG, Keeney GL, Fletcher CDM: Deciduoid peritoneal mesothelioma: An unusual phenotype affecting young females. Am J Surg Pathol. 18:439–445, 1994. 640. Orosz Z, Nagy P, Szentirmay Z, et al: Epithelial mesothelioma with deciduoid features. Virchows Arch. 434:263–266, 1999.
709.e16
Immunohistology of the Female Genital Tract
641. Shanks JH, Harris M, Banerjee SS, et al: Mesotheliomas with deciduoid morphology—A morphologic spectrum and a variant not confined to young females. Am J Surg Pathol. 24:285–294, 2000. 642. Comin CE, Saieva C, Messerini L: h-caldesmon, calretinin, estrogen receptor, and Ber-EP4: A useful combination of immunohistochemical markers for differentiating epithelioid peritoneal mesothelioma from serous papillary carcinoma of the ovary. Am J Surg Pathol. 31:1139–1148, 2007. 643. Barnetson RJ, Burnett RA, Downie I, et al: Immunohistochemical analysis of peritoneal mesothelioma and primary and secondary serous carcinoma of the peritoneum: Antibodies to estrogen and progesterone receptors are useful. Am J Clin Pathol. 125:67– 76, 2006. 644. Ordonez NG: Value of estrogen and progesterone receptor immunostaining in distinguishing between peritoneal mesotheliomas and serous carcinomas. Hum Pathol. 36:1163–1167, 2005. 645. Ordonez NG: Value of immunohistochemistry in distinguishing peritoneal mesothelioma from serous carcinoma of the ovary and peritoneum: A review and update. Adv Anat Pathol. 13:16– 25, 2006. 646. Ordonez NG: The diagnostic utility of immunohistochemistry and electron microscopy in distinguishing between peritoneal mesotheliomas and serous carcinomas: A comparative study. Mod Pathol. 19:34–48, 2006. 647. Young RH: Neoplasms of the fallopian tube and broad ligament: A selective survey including historical perspective and emphasising recent developments. Pathology. 39:112–124, 2007. 648. Youngs LA, Taylor HB: Adenomatoid tumors of the uterus and fallopian tube. Am J Clin Pathol. 48:537–545, 1967. 649. Stephenson TJ, Mills PM: Adenomatoid tumours: An immunohistochemical and ultrastructural appraisal of their histogenesis. J Pathol. 148:327–335, 1986. 650. Salazar H, Kanbour A, Burgess F: Ultrastructure and observations on the histogenesis of mesotheliomas, “adenomatoid tumors,” of the female genital tract. Cancer. 29:141–152, 1972. 651. Mai KT, Yazdi HM, Perkins DG, et al: Adenomatoid tumor of the genital tract: Evidence of mesenchymal cell origin. Pathol Res Pract. 195:605–610, 1999. 652. Nogales FF, Isaac MA, Hardisson D, et al: Adenomatoid tumors of the uterus: An analysis of 60 cases. Int J Gynecol Pathol. 21:34–40, 2002. 653. Schwartz EJ, Longacre TA: Adenomatoid tumors of the female and male genital tracts express WT1. Int J Gynecol Pathol. 23:123–128, 2004. 654. Delahunt B, Eble JN, King D, et al: Immunohistochemical evidence for mesothelial origin of paratesticular adenomatoid tumour. Histopathology. 36:109–115, 2000. 655. Kariminejad MH, Scully RE: Female adnexal tumor of probable Wolffian origin: A distinctive pathologic entity. Cancer. 31:671– 677, 1973. 656. Young RH, Scully RE: Ovarian tumors of probable Wolffian origin: A report of 11 cases. Am J Surg Pathol. 7:125–136, 1983. 657. Tavassoli FA, Andrade R, Merino M: Retiform wolffian adenoma. In Fenoglio-Preiser CM, Wolffe M, Rilke F, editors: Progress in surgical pathology, vol. XI, New York, 1990, Field and Wood Medical Publishers, pp 121–136. 658. Daya D, Young RH, Scully RE: Endometrioid carcinoma of the fallopian tube resembling an adnexal tumor of probable Wolffian origin: A report of six cases. Int J Gynecol Pathol. 11:122–130, 1992. 659. Karpuz V, Berger SD, Burkhardt K, et al: A case of endometrioid carcinoma of the fallopian tube mimicking an adnexal tumor of probable Wolffian origin. APMIS. 107:550–554, 1999.
660. Fukunaga M, Bisceglia M, Dimitri L: Endometrioid carcinoma of the fallopian tube resembling a female adnexal tumor of probable wolffian origin. Adv Anat Pathol. 11:269–272, 2004. 661. Devouassoux-Shisheboran M, Silver SA, Tavassoli FA: Wolffian adnexal tumor, so-called female adnexal tumor of probable Wolffian origin (FATWO): Immunohistochemical evidence in support of a Wolffian origin. Hum Pathol. 30:856–863, 1999. 662. Rahilly MA, Williams ARW, Krausz T, et al: Female adnexal tumour of probable Wolffian origin: A clinicopathological and immunohistochemical study of three cases. Histopathology. 26:69–74, 1995. 663. Tiltman AJ, Allard U: Female adnexal tumours of probable Wolffian origin: An immunohistochemical study comparing tumours, mesonephric remnants and paramesonephric derivatives. Histopathology. 38:237–242, 2001. 664. van den Bos M, van den Hoven M, Jongejan E, et al: More differences between HNPCC-related and sporadic carcinomas from the endometrium as compared to the colon. Am J Surg Pathol. 28:706–711, 2004. 665. Lu KH, Dinh M, Kohlmann W, et al: Gynecologic cancer as a “sentinel cancer” for women with hereditary nonpolyposis colorectal cancer syndrome. Obstet Gynecol. 105:569–574, 2005. 666. Shannon C, Kirk J, Barnetson R, et al: Incidence of microsatellite instability in synchronous tumors of the ovary and endometrium. Clin Cancer Res. 9:1387–1392, 2003. 667. Soliman PT, Broaddus RR, Schmeler KM, et al: Women with synchronous primary cancers of the endometrium and ovary: Do they have Lynch syndrome? J Clin Oncol. 23:9344–9350, 2005. 668. Ziolkowska-Seta I, Madry R, Kraszewska E, et al: TP53, BCL-2 and BAX analysis in 199 ovarian cancer patients treated with taxane-platinum regimens. Gynecol Oncol. 112:179–184, 2009. 669. Darcy KM, Brady WE, McBroom JW, et al: Associations between p53 overexpression and multiple measures of clinical outcome in high-risk, early stage or suboptimally-resected, advanced stage epithelial ovarian cancers A Gynecologic Oncology Group study. Gynecol Oncol. 111:487–495, 2008. 670. Palmer JE, Sant Cassia LJ, Irwin CJ, et al: P53 and bcl-2 assessment in serous ovarian carcinoma. Int J Gynecol Cancer. 18:241– 248, 2008. 671. Kobel M, Kalloger SE, Boyd N, et al: Ovarian carcinoma subtypes are different diseases: implications for biomarker studies. PLoS Med. 5:e232, 2008. 672. Bacher JW, Flanagan LA, Smalley RL, et al: Development of a fluorescent multiplex assay for detection of MSI-High tumors. Dis Markers. 20:237–250, 2004. 673. Narod SA, Foulkes WD: BRCA1 and BRCA2: 1994 and beyond. Nat Rev Cancer. 4:665–676, 2004. 674. Levine DA, Argenta PA, Yee CJ, et al: Fallopian tube and primary peritoneal carcinomas associated with BRCA mutations. J Clin Oncol. 21:4222–4227, 2003. 675. Kauff ND, Satagopan JM, Robson ME, et al: Risk-reducing salpingo-oophorectomy in women with a BRCA1 or BRCA2 mutation. N Engl J Med. 346:1609–1615, 2002. 676. Rebbeck TR, Lynch HT, Neuhausen SL, et al: Prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations. N Engl J Med. 346:1616–1622, 2002. 677. Agoff SN, Mendelin JE, Grieco VS, et al: Unexpected gynecologic neoplasms in patients with proven or suspected BRCA-1 or -2 mutations: Implications for gross examination, cytology, and clinical follow-up. Am J Surg Pathol. 26:171–178, 2002. 678. Cossu-Rocca P, Zhang S, Roth LM, et al: Chromosome 12p abnormalities in dysgerminoma of the ovary: A FISH analysis. Mod Pathol. 19:611–615, 2006. 679. Wiegand KC, Lee AF, Al-Agha OM, et al: Loss of BAF250a (ARID1A) is frequent in high-grade endometrial carcinomas. J Pathol. 224:328–333, 2011.
C H A P T E R 1 9
IMMUNOHISTOLOGY OF THE BREAST ROHIT BHARGAVA, DAVID J. DABBS
Overview 710 Myoepithelial Cells and Assessment of Stromal Invasion 710 Immunohistochemistry of Papillary Lesions 717 Proliferative Ductal Epithelial Lesions and In Situ Carcinoma 719 Tumor Type Identification by Immunohistochemistry 721 Paget Disease of the Breast 732 Detection of Lymphatic Space Invasion 734 Sentinel Lymph Node Examination 734 Systemic Metastasis of Breast Carcinoma 738 Fibroepithelial Tumors 740 Theranostic Applications 744 Genomic Applications of Immunohistochemistry: Breast Cancer Molecular Classification and Immunogenomics 752 Other Tumor Markers 758 Summary 761
Overview By virtue of the volume and sheer difficulty of cases, perhaps the most frequent use of diagnostic immunohistochemistry (IHC) in the surgical pathology laboratory pertains to breast biopsies. In addition to utilizing IHC for diagnostic problems with breast biopsies, breast biopsies lend themselves to the frequent use of IHC for prognostic and predictive tests. In addition, the diagnosis of breast carcinoma in the metastatic setting remains a challenge even today. This chapter addresses diagnostic issues that involve stromal invasion, papillary lesions, atypical proliferative lesions, discrimination of ductal and lobular neoplasia, and identification of breast tumor types, including Paget disease of the breast, fibroepithelial lesions, and metastatic breast carcinoma. The diagnostic section is followed by a discussion of theranostic applications in breast cancer and a section on immunogenomics. 710
Myoepithelial Cells and Assessment of Stromal Invasion Epithelial lesions of the breast are not only the most frequent lesions encountered by the surgical pathologist but also are the greatest source of concern in the differential diagnosis of benign versus malignant lesions. The lesion categories that typically need to be differentiated include nonneoplastic proliferative lesions versus malignant lesions, in situ carcinoma versus invasive malignancy, and pseudoinvasive lesions (adenosis, radial scar, sclerosing papillary tumors, etc.) versus invasive malignancies.1,2 In addition, epithelial atypical ductal hyperplasia (ADH), papillary lesions, and microinvasive carcinoma (invasive focus ≤1 mm) lend themselves to IHC clarification in many instances. In all of these diagnostic situations, the presence of the myoepithelial cell (MEC) in intimate relationship with the epithelial cells of the lesion is what differentiates in situ from invasive disease and benign pseudoinvasive lesions from invasive carcinoma; microglandular adenosis, a distinct nonorganoid benign form of adenosis, is the only known exception. The presence of MECs that envelop ductal-lobular epithelium, situated on the epithelial basal lamina, has always been considered the important criterion that separates invasive from noninvasive neoplasms.3-8 MECs can be visualized rather easily in normal breast ductules and acini, but when these structures dilate and fill with proliferating cells or are compressed, it is virtually impossible to visualize them on hematoxylin and eosin (H&E) stain. Replacing the yesteryear antibodies to S-100 protein, highmolecular-weight cytokeratin (HMWCK), smooth muscle actin (SMA), calponin, and smooth muscle myosin heavy chain (SMMHC) are the more sensitive and specific antibodies to cytoplasmic components of MECs, along with the nuclear marker, p63 (Table 19-1). Antibodies to S-100 protein are not sensitive or specific for MEC and stain MEC in an erratic manner.9-12 In addition, the recent use of antibodies to mammary serine protease inhibitor (maspin) and CD10 have been tempered by the fact that they stain a variety of cell types, including luminal cells of the terminal duct lobular unit and tumor cells.13-16 Cytokeratin (CK) cocktail antibodies, in addition to CK14 and CK17, are used to identify MEC,17 but they also immunostain acinar cells, which makes it difficult
Myoepithelial Cells and Assessment of Stromal Invasion
711
TABLE 19-1 Antibodies for Myoepithelial Cells in the Breast Antibody
Localization
MEC
Myofibroblast
Microvasculature
Carcinoma
S100
Cytoplasm
Weak
Variable
Negative
Variable
SMA
Cytoplasm
Strong
Moderate
Strong
Rare
Calponin
Cytoplasm
Strong
Weak/moderate
Strong
Rare
SMMHC
Cytoplasm
Strong
Rare
Strong
Negative
p63
Nucleus
Strong
Negative
Negative
Rare nuclei
MEC, Myoepithelial cell; SMA, smooth muscle antigen; SMMHC, smooth muscle myosin heavy chain.
A
B Figure 19-1 Smooth muscle myosin heavy chain (A) and p63 (B) stain myoepithelial cells of a breast lobule.
to differentiate MECs because of their proximity to the acinar cells. Anti-SMAs react with stromal myofibroblasts in addition to MECs18-21 and thus are not specific for MECs. The cross-reaction with myofibroblasts makes it difficult to identify MECs specifically, especially in ductal carcinoma in situ (DCIS), in which there may be periductal stromal desmoplasia. Although anti-SMA and muscle-specific actin (MSA) HHF-35 stain MECs in the majority of benign breast lesions, cross-reaction with stromal myofibroblasts is substantial, especially with SMA. Calponin and SMMHC are two antibodies that are more specific for MECs.22-24 SMMHC is a 200-kD structural component unique to smooth muscle cells that functions within the hexagonal array of the thick-thin filament contractile apparatus.25 Calponin, a 34-kD polypeptide, modulates actomyosin adenosine triphosphatase (ATPase) activity in the smooth muscle contractile apparatus and is unique to smooth muscle.22,24,26 In their analysis of 85 breast lesions, Werling and colleagues23 found that calponin and SMMHC always detected MEC in benign lesions and that SMMHC stained myofibroblasts in 8% of cases compared with calponin, which stained 76% of cases. It is also our experience that SMMHC and calponin are excellent antibodies, but calponin does stain stromal myofibroblasts to a greater extent than SMMHC does. The protein p63, a homolog of the tumor suppressor protein p53, is increasingly used in multiple organs as a multitasker for the detection of MEC, basal cells (prostate), and myoepithelial differentiation (breast
metaplastic carcinoma and salivary gland tumors) and also as a marker for squamous differentiation.23,27,28 The advantage of p63 in the diagnosis of stromal invasion is that it is present only in the nucleus, which renders it most specific for MEC in the breast, and it does not stain myofibroblasts. Some have used a cocktail of dual staining for SMMHC and p63 together. In our experience, using SMMHC and p63 is optimal for discerning MECs on difficult breast biopsies, especially diagnostic core biopsies (Figs. 19-1 through 19-5). Distinguishing DCIS from invasive carcinoma on core biopsy can be
Figure 19-2 Smooth muscle myosin heavy chain stains myoepithelial cells in a fibroadenoma.
712
Immunohistology of the Breast
A
B
C
D
Figure 19-3 A breast core biopsy demonstrates a subtle invasive carcinoma with abundant intratumoral and peritumoral lymphocytic infiltrate (A shows low power, B shows high power). An AE1/AE3 immunostain confirms the epithelial nature of these infiltrating cells (C), and p63 stains confirms absence of myoepithelial cells around these infiltrating cells (D) to establish the diagnosis of invasive carcinoma.
A
B
Figure 19-4 A, This ductal carcinoma in situ is heavily obscured with lymphocytes, but smooth muscle myosin heavy chain clearly reflects the presence of myoepithelial cells. B, Another case in which myoepithelial cells are easy to see with p63 in spite of heavy lymphoid infiltrate on core biopsy, confirming lack of invasion.
Myoepithelial Cells and Assessment of Stromal Invasion
Figure 19-5 This infiltrative-appearing breast core biopsy is clearly invasive carcinoma, as seen with complete lack of smooth muscle myosin heavy chain. A blood vessel serves as a positive internal control.
713
crucial, because almost all patients with invasive carcinoma undergo a sentinel lymph node (SLN) biopsy. An important pitfall to note is that approximately 5% of DCIS cases, especially DCIS in the background of a papillary lesion, completely lack MEC staining using any antibody (Fig. 19-6). In these situations, critical appraisal of the histologic section is crucial to arrive at the correct diagnosis. It is also important to remember that p63 nuclear immunostaining results in apparent staining “gaps” because staining of cytoplasm of the MEC does not occur (Fig. 19-7). Any nuclear staining around nests of tumor cells can be construed as evidence of the presence of MECs. Special care must be taken to exclude nuclear staining of tumor cells around the periphery of neoplastic ducts, because p63 stains tumor cells in approximately 10% of cases (see Fig. 19-7). Lesions that are especially difficult to identify on core biopsies include carcinoma in situ from invasive carcinoma in the presence of prominent periductal stromal desmoplasia (“regressive changes”) or heavy lymphoid infiltrates; lobular growth of rounded sheets of tumor cells (to the pathologist’s eye, invasion is “all or none”); infiltrating cribriform carcinoma; sclerosing adenosis with or without DCIS involvement; cancerization of
A
B
C
D
Figure 19-6 Approximately 5% of morphologically identifiable ductal carcinoma in situ (DCIS) may not show myoepithelial cells. This case of cribriform and papillary DCIS (A) shows lack of staining with p63 (B) and smooth muscle myosin heavy chain (C). Collagen type IV demonstrates strong continuous staining around tumor nests, confirming the in situ nature of the lesion (D).
714
Immunohistology of the Breast
A
B
C
D
Figure 19-7 Pitfalls of p63 and smooth muscle myosin heavy chain. A p63 stain demonstrates apparent gaps in staining around the luminal epithelium of a duct within a radial scar (A). The same duct shows intense continuous staining with smooth muscle myosin heavy chain, but myofibroblastic cells in the background are also positive (B). A p63 stain on core biopsy shows staining of a few tumor cells (C). A rare example of diffuse p63 staining of tumor cells (D).
lobules; radial scars with stromal elastosis-desmoplasia; tubular carcinoma; and sclerosing papillary lesion. The optimal MEC antibodies needed to attack these difficult cases include both SMMHC and p63 (Figs. 19-8 through 19-12).29 A significant pitfall for misinterpretation of MEC antibodies, such as calponin and even SMMHC, is that these antibodies may immunostain the microvasculature around tumor nests. Initial examination of such a case with SMMHC will reveal immunostaining that hugs the tumor nests, which suggests the presence of MECs (Fig. 19-13). Examination at higher magnification will reveal the microvasculature around the tumor nests. When the pathologist then examines the p63, it is negative (see Fig. 19-13). IHC for MEC is useful to help discriminate the three dominant benign lesions of the breast—sclerosing adenosis, microglandular adenosis (MGA), and tubular carcinoma (Table 19-2)—but a detailed morphologic study of the lesion is essential.30,31 The MECs are seen by IHC in all forms of adenosis except the microglandular form, the only benign lesion known not to contain MECs. In addition to the distinct nonorganoid morphology of MGA, tubular adenosis, described by Lee and
colleagues,31 may mimic both MGA and carcinoma but differs from MGA in that it contains MECs. Microglandular adenosis is positive with S-100 protein, whereas sclerosing adenosis and tubular carcinomas are S-100 negative. KEY DIAGNOSTIC POINTS Myoepithelial Cell Antibodies for Stromal Invasion • The presence of MECs that envelop proliferating and sclerosing breast lesions is indicative of a benign or noninvasive process. • A combination of cytoplasmic SMMHC and nuclear p63 antibodies are the best discriminators for the presence of MECs, especially in desmoplastic-sclerotic proliferations. • MEC antibodies may be confirmatory for diagnosing microinvasive carcinoma (i.e., invasive carcinoma measuring no greater than 1 mm in largest dimension). • Microglandular adenosis: S-100 positive, negative for estrogen receptors, SMMHC, and p63. • Pitfall: Immunostaining of vascular walls with SMMHC may occur, and p63 occasionally stains neoplastic cells.
A
B
Figure 19-8 Sclerosing adenosis may simulate carcinoma (A) but demonstrates envelopment of cell nests by myoepithelial cells with smooth muscle myosin heavy chain immunostaining (B).
A
B
C
D
E
F
Figure 19-9 Simulating cancer, this case of radial scar shows prominent elastosis (A and C), but strong smooth muscle myosin heavy chain staining of myoepithelial cells is seen, indicative of a benign process (B and D). Another case of radial scar (E) with strong smooth muscle myosin heavy stain staining in the periphery of the ducts (F) indicative of a benign process.
716
A
Immunohistology of the Breast
B
Figure 19-10 A, Carcinoma in situ involving sclerosing adenosis is always frightful to look at. B, Diagnosis is confirmed with smooth muscle myosin heavy chain to document the presence of myoepithelial cells.
A
B Figure 19-11 A, Edge of a sclerosing papillary lesion. B, Staining with p63 confirms absence of invasion.
A
B
Figure 19-12 A, Cribriform growth pattern: in situ or invasive? B, Immunostain with smooth muscle myosin heavy chain confirms the diagnosis of ductal carcinoma in situ.
Immunohistochemistry of Papillary Lesions
A
717
B
Figure 19-13 A, Initial microscopic impression of tumor nest is that myoepithelial cells are present, manifested by the presence of smooth muscle myosin heavy chain. B, Closer inspection reveals staining of vessels (note lumina) around tumor nests. C, No immunostaining for p63 documents lack of myoepithelial cells in this case.
Immunohistochemistry of Papillary Lesions Papillary lesions range from benign papilloma to atypical papilloma to papillary carcinoma, both in situ and invasive. Several reports have evaluated the use of MEC markers to distinguish the different categories.12,32-34 A
C
papillary lesion could be classified as a papilloma if a uniform layer of MECs is evident in the proliferating intraluminal component of the lesion, whereas the absence of MECs would be suggestive of a papillary carcinoma.33 Some papillomas show the features of an atypical papilloma in that they contain areas in which ADH overgrows the papilloma.35 These atypical areas generally lack MECs by immunoperoxidase examination.36 However, the absence of staining for HMWCKs
TABLE 19-2 Differential Diagnosis of Tubular Carcinoma, Microglandular Adenosis, Tubular Adenosis, and Sclerosing Adenosis Diagnosis
Histology
MECs
Collagen IV
Other IHC
Tubular carcinoma
Invasive teardrop-shaped tubules, apical snouts, desmoplasia
Absent
Absent
Positive: EMA, ER/PR
Microglandular adenosis
Round glands in fat lined by flat to cuboidal epithelium; inspissated secretions within glands
Absent
Present
Positive: S-100 Negative: EMA, ER/PR, GCDFP-15
Tubular adenosis
Tubules sectioned longitudinally, lacks lobulocentric distribution
Present
Present
Negative: S-100 protein
Sclerosing adenosis
Lobular growth pattern, epithelial cell atrophy, and lobular fibrosis
Present (relative abundance)
Present
Negative: S-100 protein
EMA, Epithelial membrane antigen; ER, estrogen receptor; GCDFP-15, gross cystic disease fluid protein 15; IHC, immunohistochemistry; MECs, myoepithelial cells; PR, progesterone receptor.
718
Immunohistology of the Breast
such as 34βE12, CK5, and CK5/6 within the proliferative component is more helpful in classifying a lesion as ADH or in situ carcinoma.37 The distinction between a papilloma, atypical papilloma, and papillary DCIS—both de novo and DCIS involving papilloma—is not that problematic in the majority of cases, and diagnosis can be made by using morphology and IHC staining (Fig. 19-14). The more difficult and confusing area is the distinction between a large cystic in situ lesion and an invasive carcinoma. In situ papillary carcinoma has been referred by different names in the literature. The term intracystic papillary carcinoma has been used for a single, mass-forming cystic lesion with malignant papillary proliferation.38 The term papillary ductal carcinoma in situ has been used for more diffuse lesions.39 The use of MEC markers to assess invasion in these lesions have yielded variable results. In an IHC study of papillary breast lesions, Hill and Yeh32 found consistent staining patterns in cases originally diagnosed as papilloma or invasive papillary carcinoma but found variable staining in cases diagnosed as intraductal papillary carcinoma. Of the nine intraductal papillary carcinomas in their series, four cases showed unequivocal basal MECs by IHC, one case showed partial discontinuous staining, and four cases were predominantly negative for basal MECs. The authors found that lesions originally classified as intraductal papillary carcinoma that lacked basal MECs by IHC were uniformly large, expansile papillary lesions with pushing borders and a fibrotic rim. The authors hypothesized that such lesions form a part of the spectrum of progression intermediate between in situ and invasive disease and suggested that these lesions should be termed encapsulated papillary carcinoma.32 Collins and associates40 have also favored such designation. In some recent reviews of papillary lesions, an attempt has been made to classify the lesions in a uniform fashion using morphology and IHC. The papillary lesions are now classified as papilloma, papilloma with atypical ductal epithelial hyperplasia (atypical papilloma), papilloma with DCIS, papillary DCIS, intracystic papillary carcinoma (encysted or encapsulated papillary carcinoma), solid-papillary carcinoma, and invasive papillary carcinoma.36,41 The problem in diagnosis arises from the fact that intracystic and solid-papillary carcinomas have the morphology of an in situ lesion, but they lack the presence of MECs around the periphery (Fig. 19-15). However, almost all intracystic papillary carcinoma retains strong expression for collagen IV completely around the lesion (Fig. 19-16), which suggests that these are noninvasive lesions. It is true that collagen can also be laid down around the invasive front of carcinoma,42 but true invasive cancers generally show a weak and discontinuous type of staining.43,44 Nevertheless, we do not recommend using collagen IV in routine diagnostic testing because of interpretation issues, but presence of collagen IV around intracystic papillary carcinoma supports the in situ nature of the lesion. Moreover, the clinical behavior of these lesions is more akin to in situ disease,38,45-47 therefore intracystic papillary carcinomas and solid-papillary carcinomas, which by definition lack
A
B
C Figure 19-14 A, A low-power view of an intraductal papilloma with monomorphic cellular proliferation at the bottom right. B, High-power view demonstrates the presence of ductal carcinoma in situ in a papilloma. C, Lack of smooth muscle myosin heavy chain immunostaining in this morphologically abnormal area confirms the diagnosis of ductal carcinoma in situ involving papilloma.
Proliferative Ductal Epithelial Lesions and In Situ Carcinoma
719
A Figure 19-16 Another case of intracystic (encapsulated) papillary carcinoma that demonstrates strong, continuous collagen type IV staining.
KEY DIAGNOSTIC POINTS Myoepithelial Cell Antibodies in Papillary Lesions
B Figure 19-15 An intracystic (encapsulated) papillary carcinoma (A) with lack of p63 staining at the periphery of the lesion (B).
basal MECs around the periphery, should still be considered a variant of in situ carcinoma for practical purposes.48 However, it is extremely important to analyze the resection specimen on these lesions in its entirety by histologic evaluation because of the not so infrequent presence of frank invasion (into fat beyond the fibrotic rim) in the periphery of these lesions. We believe it is the presence of these minute foci of invasive carcinoma that is responsible for occasional metastatic disease reported with intracystic papillary carcinoma.49 The MEC staining pattern for each papillary lesion is summarized in Table 19-3.
• MECs and cytokeratin 5 are present in the proliferative cellular component of a papilloma but are absent in the areas of ADH or DCIS. • MECs are uniformly present around the periphery of the lesion in a papilloma, atypical papilloma, papilloma with DCIS, and papillary DCIS but are absent at the periphery of intracystic and solid-papillary carcinomas. • Caution is advised in diagnosing invasion based on MEC antibodies in a papillary lesion on a core biopsy; complete excision is recommended to assess invasion.
Proliferative Ductal Epithelial Lesions and In Situ Carcinoma Differences in cytokeratin expression have been described between hyperplasia and DCIS.50,51 The antibody 34βE12 recognizes cytokeratins 1, 5, 10, and 14, and these keratins are typically found in myoepithelium and squamous epithelium. Normal breast MECs and proliferative duct epithelium express 34βE12 (Fig. 19-17). The expression is lost in ADH.52 Low- to intermediate-grade DCIS is also largely negative for
TABLE 19-3 Papillary Lesions of the Breast Distribution of Myoepthelial Cells and Clinical Behavior Papillary Lesions
Myoepithelial Cells
Clinical Behavior
Papilloma
Present within and around ducts
Benign
Papilloma with ADH/DCIS or papillary DCIS
Reduced/absent within, present around ducts
Risk for invasive malignancy
Encapsulated papillary carcinoma
Absent within, absent/focal around ducts
Similar to DCIS, unless frankly invasive
Solid papillary carcinoma
Absent within, absent/focal around ducts
Similar to DCIS, unless frankly invasive
ADH, Atypical ductal hyperplasia; DCIS, ductal carcinoma in situ.
720
Immunohistology of the Breast
A
B
C
D
E
F
G
Figure 19-17 A, Florid duct hyperplasia. B, This case demonstrates strongly positive reactivity to keratin 903 (34βE12). In contrast to ductal hyperplasia, ductal carcinoma in situ (C) is negative with keratin 903 (D). A case of atypical ductal hyperplasia (E) is negative for cytokeratin 5 (F) and strongly expresses estrogen receptor (G).
Tumor Type Identification by Immunohistochemistry
34βE12 (see Fig. 19-17). Most low- to intermediategrade DCIS is uniformly positive for CAM5.2, reflecting a shift away from HMWCKs to the more simple keratins 8 and 18. The 34βE12 immunostaining profile for DCIS and ADH is very similar and cannot be used to help distinguish DCIS from ADH but can be an aid to histomorphology in separating DCIS from florid ductal epithelial hyperplasia (DEH) in difficult cases. CK5/6 antibody (clone D5/16B4) has also been shown to be largely negative in DCIS.52 Similar results have been obtained with CK5 antibody.53 This expression of HMWCKs (or basal keratins) in usual hyperplasia with loss in ADH and DCIS suggests that atypical lesions try to acquire a more “luminal” phenotype. Adding to the same theme, usual hyperplasia is generally negative or patchy positive for estrogen receptor (ER), but atypical hyperplasia and low- to intermediate-grade DCIS is often strongly and diffusely ER positive. So, in a lesion with ambiguous morphology for ADH, a combination of CK5 and ER may be helpful in rendering a more definitive diagnosis. A CK5-positive and ER-low or ER-negative immunophenotype of the proliferative component would favor usual hyperplasia, whereas the opposite profile—CK5 negative, ER strongly positive— would favor ADH/DCIS.53 A few pitfalls are worth mentioning in regard to using these IHC stains for making a diagnosis of ADH/ DCIS. First of all, this panel is not valid for columnar cell lesions, because even benign columnar cell changes strongly express ER. Secondly, apocrine DCIS and atypical apocrine lesions (atypical apocrine adenosis) are generally negative for ER and may express CK5. Finally, basal-like DCIS is almost always positive for CK5 and is ER negative. Another aspect to remember is that native luminal epithelium—that is, the normal epithelium that rests upon the myoepithelium—is almost always negative for CK5 and should not be interpreted as atypia. The results for CK5 should be interpreted in the intraductal proliferative component for atypia assessment. Therefore, CK5 and ER should be used in conjunction with defined morphologic criteria for diagnosing ADH/DCIS (see Fig. 19-17). The diagnosis of atypia in papillary lesions is also very challenging. Fortunately, the same cytokeratin patterns of immunostaining hold up for the differential of ADH/ DCIS in a papilloma versus florid hyperplasia in a papilloma.37 KEY DIAGNOSTIC POINTS Immunohistochemistry in Proliferative and in Situ Lesions • Cytokeratin antibody 34βE12 and cytokeratins 5 and 5/6 intensely stain florid ductal hyperplasia of breast, which may be useful in separating florid hyperplasia in ducts or papillomas from ADH and DCIS. • Both ADH and DCIS lack 34βE12 and cytokeratins 5 and 5/6 antibody staining and cannot be distinguished by IHC. • ER expression is diffuse and strong in ADH and low-grade DCIS, but it is either negative or patchy in ductal hyperplasia.
721
Tumor Type Identification by Immunohistochemistry Cell Adhesion: Ductal Versus Lobular Carcinoma Based on cell cohesiveness, the two broad categories of breast carcinoma, invasive and in situ, are ductal and lobular types. DCIS increases the risk of invasive malignancy at the local site, whereas lobular carcinoma in situ (LCIS) is considered a marker of generalized increased risk of invasive malignancy, although some recent data suggest precursor properties for LCIS.54-63 Invasive ductal carcinomas (IDCs) are often unifocal lesions, compared with invasive lobular carcinomas (ILCs), which are often multifocal and/or more extensive than what is estimated on clinical and mammographic examination.64-66 Distant metastases from ductal carcinoma preferentially involve lung and brain, whereas metastases from lobular carcinoma more often involve the peritoneum, bone, bone marrow, and visceral organs of the gastrointestinal (GI) and gynecologic tracts.67-70 In spite of the above differences, at present, with combined multimodality therapy, no difference in disease-free or overall survival is apparent between ductal and lobular carcinomas.65,71,72 However, the differences in patient preoperative evaluation and subsequent treatment are significant enough that an accurate diagnosis is warranted at the time of core biopsy. At some breast cancer centers, a preoperative (before lumpectomy or mastectomy) magnetic resonance image (MRI) of the breast is taken to evaluate the extent of disease with a core biopsy diagnosis of invasive lobular carcinoma,64,66,73,74 with the rationale that margins are difficult to obtain from the surgeon’s viewpoint. This approach probably has merit in selected patients, as it pertains to breast reconstruction. A core biopsy diagnosis of ductal versus lobular carcinoma is also important if the patient will be treated by neoadjuvant chemotherapy (NACT). Although therapy response is better predicted with tumor receptor status, rather than morphologic tumor type, a few studies do show response to NACT only in a subset of ductal cancers with minimal or no effect on lobular cancers.75-78 Therefore pathologists have to strive hard to give the best diagnosis possible for current management, and also for the future, as specific therapies become available. Strong E-cadherin (ECAD) membranous staining has been long used to define ductal carcinoma.79-81 Ductal carcinomas, both in situ and invasive, retain membranous ECAD, because they do not show homozygous mutation/silencing of the ECAD gene, CDH1.82-84 Mutation of this gene either leads to a mutant protein that loses its adhesive properties or to not have enough protein to function as an adhesive molecule. CDH1 is a large gene located on 16q22.1. The ECAD protein has an intracytoplasmic portion, an intramembranous portion, and an extracellular domain. Cell-to-cell adhesion through ECAD is also critically dependent on the subplasmalemmal cytoplasmic catenin complexes (alpha, beta, gamma, and p120 isoforms) that link
722
Immunohistology of the Breast
ECAD to the actin cytoskeleton of the cell. Abnormalities of the catenins or of CDH1 gene expression can result in a variety of IHC ECAD pathologies. Lobular carcinomas studied at the genetic level have often shown mutation that accounts for the loss of cohesiveness of the tumor cells.82,83 The majority of these mutations have been found in combination with loss of heterozygosity (LOH) of the wild-type ECAD locus (16q22.1), a hallmark of classic tumor suppressor genes. Immunohistochemically, this correlates with either a complete absence of the ECAD protein or abnormal localization (apical or perinuclear). This abnormal localization may be dependent on the type of mutation.85 Truncation mutations produce an ECAD product that is inept at binding to neighboring cells, resulting in a histologic pattern of widely dyshesive cells that are completely negative for ECAD by IHC (e.g., classic infiltrating lobular carcinoma; Fig. 19-18). Loss of membrane staining may be associated with a granular cytoplasmic immunostaining (Fig. 19-19, A-B) that represents cytoplasmic solubilization of a portion of the truncated protein. Proximal truncation mutations may result in the inability of ECAD to bind to the catenin complex, resulting in a short ECAD protein represented by focal or dotlike membrane immunostaining (see Fig. 19-19, C). Patients with focal staining of LCIS cells with ECAD may have an ipsilateral risk of carcinoma akin to DCIS.86 Mutations in the catenin complex can also lead to dysfunctional ECAD and loss of membrane staining.87,88 Although deletions of the CDH1 gene as a result of LOH are seen in ductal carcinomas, they are not early events and are not usually associated with the point mutations seen in lobular neoplasia. In the majority of cases, ECAD staining is unequivocal (positive or negative) and can be solely used in distinguishing ductal from lobular carcinomas. In a minority (~15%) of cases, the stain may be difficult to interpret. Another stain that could be used in such situations is p120. This stain represents p120 catenin, which binds with ECAD on the internal aspect of the cell membrane to form the cadherin-catenin complex (Fig. 19-20, A). This complex is essential for the formation of intercellular tight junctions and is composed of an external domain of calcium-dependent ECAD and an internal domain of ECAD to which are bound the alpha, beta, and p120 catenins.89-93 The alpha and beta catenin are complexed with the carboxy-terminal cytoplasmic tail of ECAD, whereas the p120 catenin is anchored to ECAD in a juxtamembranous site.94 The p120 catenin is actively involved in the status of cell motility, ECAD trafficking, ECAD turnover, promotion of cell junction formation, and regulation of the actin cytoskeleton.95 The binding of p120 to ECAD stabilizes the complex and increases the half-life of membrane ECAD by slowing the normal turnover of ECAD that normally occurs by cellular endocytosis.96 The p120 that is bound to ECAD exists in equilibrium with a small cytoplasmic pool of p120. When ECAD is absent, the cytoplasmic pool of p120 increases.97 Therefore in normal ducts and in ductal carcinomas, p120 shows a membranous pattern of staining (see Fig. 19-20, B-C). In contrast, lobular carcinomas, with absent or
A
B
C Figure 19-18 A, Classic invasive and in situ lobular carcinoma demonstrates complete lack of E-cadherin staining. Note the staining of myoepithelial cells. B, The growth pattern of in situ carcinoma is indeterminate for cell type. C, Positive membranous staining for E-cadherin indicates lobular involvement by ductal carcinoma in situ.
nonfunctional E-cadherin, show strong cytoplasmic p120 immunoreactivity (see Fig. 19-20, D-E). This positive cytoplasmic staining for lobular carcinoma is much easier to interpret than ECAD negative staining. A combination of ECAD and p120 drastically reduces the
Tumor Type Identification by Immunohistochemistry
A
B
723
cytoplasmic staining is observed in all morphologically characterized LCIS and atypical lobular hyperplasias (Fig. 19-21). Additionally, lack of ECAD within minimal epithelial proliferation in the breast terminal duct lobular unit defines atypical lobular hyperplasia (ALH) and distinguishes it from mild ductal hyperplasia. This distinction is important, because patients with ALH are typically referred to a risk clinic. Also, some studies have advocated surgical excision with core needle biopsy diagnosis of ALH.57,100 The IHC stains support the notion that the term lobular hyperplasia has no significance in breast pathology. IHC and molecular methods not only aid with the diagnostic issues, but to some extent they also demand that the pathologist look at the morphology, which has been learned in a new light. All invasive breast carcinomas that infiltrate in a single-file pattern with a low nuclear grade are not lobular carcinomas, because IDCs also have this pattern. The morphologic assessment of a ductal or lobular phenotype is not without controversy and has its limitations. The classic ILC is composed of small cells with bland cytology and some plasmacytoid features. The growth pattern is completely dyshesive. Breast tissue can grossly appear normal, as can the mammogram, yet it may show widespread dyshesive carcinoma of the classic type. These tumors are uniformly ECAD negative and are associated with specific patterns of systemic metastases.101 IDC can also show patterns seen in ILC, such as “single-filing” of tumor cells, targetoid patterns, and regional dyshesiveness. Such patterns may be confusing but are readily resolved with ECAD immunostaining. Subgroups of morphologically indeterminate lobular/ductal phenotypes also exist. ECAD separates these groups distinctly in most cases and demonstrates the existence of mixed lobular-ductal phenotypes in a minority of cases.86,102,103 ECAD stains MECs, a pitfall for misinterpretation of LCIS as DCIS (see Fig. 19-18, A). The morphologic reproducibility of distinguishing IDC from ILC and LCIS from DCIS is less than optimal, and variation can be substantial in the interpretation of ILC versus IDC and LCIS versus DCIS. For this reason alone, ECAD and similarly p120 IHC could be justified to aid in correctly classifying these lesions.
KEY DIAGNOSTIC POINTS C Figure 19-19 Aberrant staining with E-cadherin. A, Cytoplasmic staining in invasive lobular carcinoma. B, Lobular carcinoma in situ. C, Dotlike E-cadherin staining of invasive carcinoma.
number of ambiguous diagnoses. The stains also help correctly diagnose the category of mixed ductal and lobular carcinoma (see Fig. 19-20, F). These mixed carcinomas comprise no more than 10% of all breast carcinomas and probably arise because of “late” ECAD inactivation within a ductal carcinoma.98 In contrast, loss of ECAD protein occurs very early in lobular carcinogenesis.99 Lack of ECAD staining and strong p120
Ductal vs. Lobular Carcinoma • ECAD stain is a useful diagnostic adjunct in cases with indeterminate morphology. • Staining with p120 further enhances diagnostic accuracy by providing a “positive” stain for lobular carcinoma. • Lobular lesions are characteristically negative for ECAD and demonstrate intense cytoplasmic immunoreactivity for p120. • Aberrant ECAD immunostaining may be cytoplasmic or punctate/membranous, depending on the type of CDH1 mutation; in these instances, p120 catenin proves the lobular phenotype. • Normal ducts and ductal lesions demonstrate membranous staining for ECAD and p120.
724
Immunohistology of the Breast
Calcium ions
Extracellular domain 5
P
p120
N
P
Involved in strengthening cadherin-containing adhesion junctions
C N
P
β-catenin or plakoglobin Arm repeats
Extracellular domain 1
Zipper of E-cadherin dimers
N
α-catenin
N α-actinin
Adjoining cell Plasma membrane F-actin Intercellular space
Cytoplasm
A
B
C
D
E
Figure 19-20 A, Diagrammatic representation of the relationship between of E-cadherin (ECAD) and p120. B and C, The dynamic biology of ECAD/p120 can be illustrated by using a dual ECAD (brown)/p120 (red) stain. In this example of invasive ductal carcinoma, strong membranous reactivity (reddish brown) is identified for both ECAD and p120 (B). Similar reddish-brown membranous staining is identified in acinar cells within this lobule (C). An example of invasive and in situ lobular carcinoma (D) demonstrates strong cytoplasmic immunoreactivity for p120 by using a single-color stain (E).
Tumor Type Identification by Immunohistochemistry
F Figure 19-20, cont’d A dual ECAD/p120 stain demonstrates membranous ECAD and p120 (reddish brown) immunoreactivity in the ductal component with lack of ECAD (absence of brown staining) but strong cytoplasmic p120 (red) staining in this example of mixed ductal and lobular carcinoma (F).
Lobular Carcinoma Variants and Former Lobular Variants PLEOMORPHIC LOBULAR CARCINOMA
Described by Bassler in 1980104 and further detailed by Weidner and Semple,105 Eusebi and colleagues,106 and Reis-Filho and associates,107 the genetic, immunohistologic, and clinical features have been sufficiently detailed to recognize invasive pleomorphic lobular carcinoma (PLC) and pleomorphic lobular carcinoma in situ (PLCIS) as distinct clinicopathologic entities.108-111 Based on cell cohesiveness, PLC and PLCIS are basically a subtype of lobular carcinoma. The histologically recognizable PLC and PLCIS are almost always ECAD negative, or they show aberrant staining, and they demonstrate strong cytoplasmic immunoreactivity for p120.112 Histologically, these show grade 3 nuclei with a dyshesive pattern of growth in both in situ and infiltrating varieties (Fig. 19-22). The in situ component may be discovered on mammograms as calcifications. Core biopsies demonstrate in situ dyshesive grade 3 nuclei, and some cases show comedonecrosis and calcification. Recently, a comprehensive analysis of 26 PLCs revealed a closer association between PLC and classic lobular carcinoma than between PLC and ductal carcinoma.113 The authors analyzed 26 cases of PLC, 16 cases of classic lobular carcinoma, and 34 cases of IDC by IHC, array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and chromogenic in situ hybridization (CISH). Comparative analysis of aCGH data suggested the molecular features of PLC (ER/PR+, E-cadherin−, 1q+, 11q−, 16p+, and 16q−) were more closely related to those of classic ILC than IDC. However, PLCs also showed some molecular alterations more typical of high-grade IDC than ILC (p53 and HER2 positivity in some cases, 8q+, 17q24q25+, 13q−, and amplification of 8q24, 12q14, 17q12,
725
and 20q13). Some of these IDC-like alterations may be responsible for the aggressive biology of PLC. Sneige and colleagues114 studied 24 cases of PLCIS by IHC and found them to be universally positive for ER (100%). They also showed frequent p53 reactivity (25%) and moderate to high proliferative activity, and HER2 positivity was seen in 1/23 cases (4%). Of these 24 cases, 14 were associated with PLC, which showed a similar IHC profile. Our experience with PLC and PLCIS is also very similar. Although HER2 overexpression/amplification may be seen in PLCs, the HER2-positive rate is not very high, as previously reported,110 and PLCs are ER positive in the majority of cases, albeit expression levels may vary from case to case. Because the likelihood is high for invasive carcinoma in the vicinity of PLCIS, these lesions should be managed similar to DCIS. TUBULOLOBULAR CARCINOMA
Described originally by Fisher115 in 1977 as a lobular growth pattern with tiny tubules and single-filing characteristic of lobular carcinoma, the prognosis was described as intermediate between that of pure tubular carcinoma and ILC.116 This lesion had been categorized as a variant of ILC because of the small cells and characteristic ILC pattern of single-filing and targetoid infiltration. Both Esposito and colleagues117 and Wheeler and associates118 documented uniform membranous ECAD immunostaining in the tubules and lobular-appearing components (Fig. 19-23). The combination of small, rounded tubule profiles with infiltrating lobular-like patterns and ECAD positivity is a ductal immunoprofile. HISTIOCYTOID CARCINOMA
The term histiocytoid breast carcinoma (HBC) was coined by Hood and colleagues119 because of the tumor cells’ resemblance to histiocytes. In 1983, Filotico and associates120 described a case of lobular-appearing carcinoma with histiocytic features. Subsequent reports assumed that this variant was of lobular type by virtue of the characteristic infiltrating pattern. Only recently have immunohistologic studies been published on this rare entity. Gupta and associates121 found that 8 of 11 cases lacked ECAD, and 8 of 11 had LCIS. Three cases had ECAD, and the authors concluded that the histiocytic appearance and lack of distinct clinical features were insufficient to ascribe a distinct entity of histiocytoid carcinoma. The current evidence suggests that HBC is a morphologic pattern that can be observed in ductal, lobular, and apocrine tumors and that it is not a distinct entity by itself.122
Immunohistochemistry for Identifying Special Types of Breast Carcinoma INVASIVE MICROPAPILLARY CARCINOMA: USE OF EPITHELIAL MEMBRANE ANTIGEN
Tight clusters of neoplastic cells surrounded by clear spaces characterize invasive micropapillary
726
Immunohistology of the Breast
A
B
C
D
Figure 19-21 A, An example of lobular neoplasia. B, The cells of lobular neoplasia demonstrate strong cytoplasmic staining compared with membranous staining of normal duct cells with p120. C, Another example of lobular carcinoma in situ with pagetoid extension into ducts. D, A dual E-cadherin (ECAD) and p120 stain shows a thin layer of residual luminal cells staining with ECAD (brown) and the duct largely replaced by lobular carcinoma in situ cells that demonstrate strong cytoplasmic reactivity (red) for p120. E, Another example of lobular neoplasia stained with dual ECAD/p120 stain demonstrates intense red cytoplasmic staining with p120.
carcinoma.123,124 Unlike papillary carcinoma, the cell clusters are devoid of fibrovascular cores and often display tubular structures in the center. The stroma is typically described as “spongy” with little or no desmoplasia of the surrounding tissue.123,124 Some ductal carcinomas of “no special type” (NST) also show clear spaces around neoplastic cells, which are likely due to
E
retraction of the intervening fibrotic stroma and should not be confused with micropapillary morphology. Fortunately, the distinction between true micropapillary carcinoma and NST carcinoma with retraction artifact can be easily made by staining with epithelial membrane antigen (EMA, or MUC1).123,125,126 Ductal carcinoma of NST shows an apical or cytoplasmic staining
Tumor Type Identification by Immunohistochemistry
A
B
C
D
E
F
727
Figure 19-22 A-B, Pleomorphic lobular carcinoma in situ (PLCIS) in a lobular arrangement, showing nuclear grade 3 and prominent nucleoli. C, PLCIS is E-cadherin negative. D, Low magnification of PLCIS with comedonecrosis and calcification simulating ductal carcinoma in situ. E, Note the dyshesion and plasmacytoid cellular features characteristic of PLCIS. F, E-cadherin is negative in PLCIS and is positive in myoepithelial cells.
with EMA. In contrast, invasive micropapillary carcinomas show accentuation of the basal surface (stroma facing) of the neoplastic cells (Fig. 19-24). This “reverse polarity” of the neoplastic cells is a characteristic feature of invasive micropapillary carcinoma. The distinction between a ductal NST and micropapillary carcinoma
may not be clinically significant, because stage for stage, the difference between the two entities is insignificant. However, even when small, micropapillary morphology is highly predictive of lymph node metastases, and these tumors also more commonly tend to involve the skin and chest wall.127-131 The incidence of axillary lymph
728
Immunohistology of the Breast
A A
B
B Figure 19-24 A, Invasive micropapillary carcinoma of the breast. B, “Reverse polarity” of the neoplastic cells is demonstrated by epithelial membrane antigen. Note the intense staining at the stroma-facing side of the cells.
C Figure 19-23 A, The pattern of infiltration of tubulolobular carcinoma (TLC) is similar to that of lobular carcinoma. B, Tiny tubules populate the tumor, which otherwise simulates lobular carcinoma. C, E-cadherin is positive in tiny tubules and single cells.
node metastasis has been reported to be as high as 95%.132 However, we believe the actual incidence of axillary lymph node involvement in routine clinical testing is close to 60%. Recently, Acs and colleagues133 showed that even partial reverse cell polarity, defined as prominent linear EMA reactivity on at least part of the periphery of tumor cell clusters, had the same
implication as micropapillary differentiation and that these tumors may represent part of a spectrum of invasive micropapillary carcinoma. A confident diagnosis of a true micropapillary carcinoma on a core biopsy helps the surgeon plan appropriate management by way of special attention and clinical examination of the axilla, to perform fine needle aspiration if axillary nodes are slightly enlarged but not definitely suspicious, and to request an intraoperative frozen section even if the lymph node is grossly negative. BASAL-LIKE CARCINOMA: USE OF BASAL CYTOKERATINS
Use of routine hormone receptor (HR) protein and HER2 oncoprotein analysis on invasive breast carcinomas in the last decade has delineated clinically significant subgroups. One group is that of so-called triple-negative tumors, those negative for all three biomarkers: ER, PR, and HER2. These tumors have been
Tumor Type Identification by Immunohistochemistry
known to be clinically aggressive, and therapeutic options are limited, because they are not amenable to HR-based therapy or HER2 targeted therapy. The so-called basal-like breast carcinomas constitute at least 80% of the triple-negative tumors, and the basal-like subtype was initially recognized by gene-expression profiling studies.134,135 Basal-like carcinomas are histologically characterized by high Nottingham grade, geographic necrosis, good circumscription, and mild to moderate host lymphocytic response.136 These tumors generally show reduced (not absent) ECAD membranous expression and strong membranous p120 immunoreactivity (unpublished data); therefore we consider them as subtypes of ductal carcinoma. Basal-like carcinomas are characteristically triple negative and show expression of basal-type cytokeratin (CK5/6, CK14, CK17), epidermal growth factor receptor (EGFR), vimentin, and p53.136,137 Often a panel of basal-type cytokeratins and EGFR in triple-negative tumors is used to identify basal-like carcinomas (Fig. 19-25). The antibodies used to identify the basal-like variant are an example of genomic application of IHC. This details fundamental immunohistologic profiles, used as
A
Figure 19-25 A basal-like carcinoma (A) demonstrates immunoreactivity for cytokeratin 5/6 (B) and epidermal growth factor receptor (C).
729
surrogate markers, that reflect a genomic profile. Antibody to CK5 (clone XM26) is more sensitive than CK5/6 (clone D5/16B4) but is equally specific for identifying basal-like breast carcinomas.138 IHC studies have also confirmed the existence of in situ carcinoma of a basal phenotype.139,140 Gene-expression studies have consistently identified basal-like carcinomas to have poor prognosis.135,141,142 These tumors occur in both premenopausal and postmenopausal patients; however, identifying basal-like carcinoma in young premenopausal patients may suggest the presence of hereditary breast and ovarian carcinoma syndrome.143 Although no specific chemotherapeutic drugs are currently available to treat these patients, data are emerging, especially on poly ADP ribase polymerase (PARP) inhibitors,144 and it is important to recognize these tumors as therapies become more refined. METAPLASTIC CARCINOMA: USE OF KERATINS AND MELANOMA AND VASCULAR MARKERS
Metaplastic carcinoma comprises a group of heterogeneous neoplasms that exhibit pure epithelial or mixed
B
C
730
Immunohistology of the Breast
epithelial and mesenchymal phenotypes.145,146 Diagnosis is not problematic when there is a recognizable component of metaplastic carcinoma, such as an obvious adenocarcinoma, adenosquamous or squamous cell carcinoma, or osseous or chondroid differentiation. The most problematic cases are the ones that predominantly show spindle cell morphology without an obvious epithelial or DCIS component (Fig. 19-26). This is usually the issue on a core biopsy rather than on an excision specimen. IHC stains can be helpful in this situation.145 A panel composed of multiple keratin stains (CAM5.2, AE1/3, 34βE12, CK5, and CK7) and EMA is more useful than a single keratin.147 Another sensitive and specific marker for spindle cell metaplastic carcinoma is p63, and it should always be included in the panel.148,149 Vimentin expression in a tumor does not exclude a spindle cell carcinoma.147,150,151 Vimentin expression has been found in 50% of hormone-independent cell lines, and because metaplastic carcinomas are usually negative for receptors, vimentin expression is actually expected.152 If all the keratins, EMA, and p63 fail to show any immunoreactivity on a core biopsy, complete
excision of the lesion is recommended. In many cases, an epithelial component is present only focally. Although every effort should be made to prove an atypical spindle cell lesion to be a metaplastic carcinoma, it is important not to forget that melanomas and angiosarcomas can also occur in the breast; therefore at least two melanoma markers should be evaluated. S-100 protein is a very sensitive melanoma marker, but it has been reported to stain between 20% and 50% of metaplastic breast carcinomas and is therefore not the best stain for this differential diagnosis.153,154 Strong keratin reactivity or multiple-keratin positivity would also exclude melanoma. However, CAM5.2 positivity alone is not enough to exclude a melanoma, unless it is strong and diffuse.155,156 Another significant malignant lesion with which metaplastic carcinoma can be confused is angiosarcoma. These tumors may occur de novo or after radiation treatment. It is obvious to think about angiosarcoma in a malignant spindle or epithelioid lesion of the breast if there has been a prior history of radiation treatment. However, in the absence of such a clinical history, the
A
B
C
D
Figure 19-26 This predominantly spindled-cell neoplasm (A) showed only a focal area of epithelioid malignant cells (B). The tumor was completely negative for AE1/AE3 and CAM5.2 but demonstrated staining for basal cytokeratins (5/6, 14, 17) and p63 (C and D, respectively), supporting the diagnosis of spindle cell metaplastic carcinoma.
Tumor Type Identification by Immunohistochemistry
lesion should be extensively examined by available IHC stains. More than one vascular marker should be used because of the heterogeneous expression of vascular markers.157 Of the three commonly used vascular markers CD31, CD34, and factor VIII, CD31 is generally considered to be the most specific vascular endothelial marker, but occasional weak staining of carcinomas has been described.158 We have also seen staining of carcinoma cells with CD31, likely because of neovascularization within the tumor. This represents a diagnostic pitfall, especially in small samples. A diagnosis of de novo primary angiosarcoma of the breast should be made only if IHC staining for vascular markers is unequivocal, staining for p63 and HMWCKs is negative, and appropriate histology of the lesion has been ascertained. Other vascular markers that can be positive in angiosarcoma include D2-40 and FLI-1. In summary, a malignant spindle cell lesion should be considered a metaplastic carcinoma unless proved otherwise. A panel that comprises multiple keratins, EMA, p63, and melanoma and vascular markers is required in the workup of a malignant spindle cell lesion. OTHER SPINDLE CELL NEOPLASMS: MYOEPITHELIAL AND MESENCHYMAL TUMORS
Tumors of the breast in which MEC differentiation predominates include adenomyoepithelioma, myoepithelioma, and myoepithelial cell carcinoma (MECC).159-162 Although the majority of myoepithelial tumors are benign, occasional tumors may exhibit aggressive behavior in the form of carcinoma or myoepithelial carcinoma.163,164 The typical immunostaining pattern of the myoepithelial components of these tumors is strong cytoplasmic staining for 34βE12, CK5, and nuclear p63. Tumor cells are typically positive with S-100 protein (90%) and may be positive with muscle markers such as calponin (86%), MSA, desmin (14%), and α-SMA (36%).159,161 Occasional cells exhibit immunostaining with glial fibrillary acidic protein (GFAP). The presence of smooth muscle markers and immunostaining for GFAP is more in keeping with pure myoepithelial differentiation as opposed to metaplastic carcinomas, discussed previously, which are largely negative for these markers.165,166 Expression of SMA is
731
very nonspecific and is not a definitive marker for muscle differentiation. Metaplastic carcinomas of the breast (carcinosarcoma, spindle cell carcinoma, sarcomatoid carcinoma) have an immunoprofile very similar to myoepithelial differentiation; they regularly coexpress weak cytoplasmic CAM5.2 for low-molecularweight keratins (LMWKs), show strong cytoplasmic immunostaining for HMWCKs 34βE12, CK5/6, or CK5; and also express vimentin and show nuclear immunostaining for p63 (90%).148 However, GFAP and SMMHC are largely negative. Immunostaining with muscle markers is most indicative of a pure myoepithelial neoplasm as opposed to a metaplastic carcinoma. The immunoprofile of metaplastic carcinoma is shared to a great degree with myoepithelial neoplasms, and some investigators suggest that the MEC is the progenitor cell for metaplastic carcinomas.153,154,167,168 Leibl and colleagues154 also demonstrated that the experimental myoepithelial markers CD29 and 14-3-3 sigma stain metaplastic carcinomas, supplying further evidence of the myoepithelial nature of these tumors. The literature suggests that use of the term myoepithelial carcinoma instead of metaplastic carcinoma is a matter of semantics and may not have any clinical significance. Myoepithelial tumors need to be separated from the rare primary spindle cell sarcoma of the breast, which may include fibrosarcoma (vimentin+), leiomyosarcoma, and rhabdomyosarcoma (positive with muscle markers); synovial sarcoma (CK7+ and CK19+)169; malignant nerve sheath tumors (patchy S-100+ and vimentin+); and the so-called malignant fibrous histiocytomas (vimentin+). Although each of these tumors may have characteristic light-microscopic features, immunostaining patterns may be useful in the diagnostic distinction (Table 19-4). Primary liposarcomas (S-100+) of breast are rare tumors that may arise in a preexisting phyllodes tumor (CD34+ stroma). The rare myofibroblastoma of the breast (Fig. 19-27) is distinguished immunohistochemically from myoepithelial tumors by lack of immunostaining for keratins, S-100 protein, and SMMHC.170-172 The myofibroblastoma demonstrates CD34-positive cells. In contrast, fibromatosis that involves the breast is negative for CD34 but demonstrates abnormal (nuclear) localization of β-catenin.153,173
TABLE 19-4 Metaplastic Carcinoma Versus Spindle Cell Sarcoma of the Breast Diagnosis
CK5/6
K903
p63
GFAP
S-100
HHF-35
Metaplastic carcinoma
+
+
+
N
N
S
Myoepithelial carcinoma
+
S
S
S
+
+
Fibrosarcoma
−
−
−
−
−
R/−
Myosarcoma
−
−
R/−
−
−
+
MPNST
−
−
−
−
S
−
Synovial sarcoma
−
−
−
−
R/−
−
+, Almost always positive; S, sometimes positive; R, rare; −, negative. CK, Cytokeratin; GFAP, glial fibrillary acidic protein; MPNST, malignant peripheral nerve sheath tumor.
732
Immunohistology of the Breast
A
Figure 19-27 Myofibroblastoma of breast (A) typically shows the presence of desmin (B) and muscle-specific actin (HHF-35; C).
KEY DIAGNOSTIC POINTS Tumor Type Identification by Immunohistochemistry (Excluding Ductal vs. Lobular Differential Diagnosis) • Micropapillary carcinomas can be confidently identified by using epithelial membrane antigen (EMA, or MUC1) to demonstrate “reverse polarity” of the tumor cells. • Basal-like carcinomas are triple negative and show immunoreactivity for basal cytokeratins and epidermal growth factor receptor (EGFR). • Basal cytokeratins, especially cytokeratin 5, along with p63 are very sensitive markers for identifying spindle cell metaplastic carcinomas. • Although muscle differentiation in myoepithelial carcinomas separates them from spindle cell metaplastic carcinomas, they likely represent two different spectra of one entity. • Adenomyoepitheliomas are biphasic tumors that resemble, and are actually a variant of, intraductal papilloma at one extreme; at the other, they show pure spindle cell myoepithelioma. • Noncarcinomatous spindle cell proliferation in a breast core biopsy sample includes fibromatosis, myofibroblastoma, stromal component of phyllodes tumor, or rare primary sarcomas.
B
C
Paget Disease of the Breast The presence of malignant epithelium with breast cancer immunophenotype within the nipple epidermis is termed Paget disease of the breast. It is often the result of intraepidermal spread of malignant cells from underlying DCIS. The presence of an associated mass lesion indicates the presence of invasive carcinoma in addition to DCIS. Paget disease of the breast is manifested as CK7positive malignant cells that infiltrate the epidermis of the nipple. Tumor cells are conspicuous by their large size, infiltrative “shotgun” pattern, abundant cytoplasm, signet-ring forms, and sometimes mucin positivity (Fig. 19-28). The majority (>90%) of the underlying breast carcinomas are ductal in nature.174,175 In a nipple/areola biopsy, or in cases where underlying carcinoma could not be documented, the differential for Paget disease includes melanoma and squamous cell carcinoma in situ (Bowen disease). The single best stain for this differential diagnosis is CK7, which is positive in almost all cases of Paget disease (see Fig. 19-28). Cells of Paget disease are also positive for HER2 (in ~80% to 90%),176-179 and this correlates to the IHC expression of underlying breast carcinoma, which is often a HER2-positive, highgrade DCIS with apocrine differentiation and comedonecrosis.180 Additional stains that can be positive in Paget disease are gross cystic disease fluid protein 15
Paget Disease of the Breast
A
C
(GCDFP-15), mammaglobin (MGB), carcinoembryonic antigen (CEA), and HRs.181 However, it is important to remember that estrogen and progesterone receptors are not good markers of Paget disease. Although Paget disease is a manifestation of underlying breast carcinoma, most often the carcinoma is a DCIS with comedonecrosis, and these tumors are frequently negative for HRs.182 If the possibility of a melanoma is entertained, at least two melanoma markers should be used, because S-100 protein can be positive in about 18% of Paget disease.183 However, it should be noted that malignant melanoma on the nipple is extraordinarily rare. Pagetoid squamous carcinoma (Bowen disease) of the breast is rare and can be distinguished from Paget disease. Cells of Bowen disease are negative for CK7, and the squamous nature of the cells can be confirmed by CK5 or CK5/6 and p63 stains, whereas Paget disease shows reverse result for these antibodies. Toker cells are CK7 positive and may be present in the skin of the normal nipple,184 but generally they are inconspicuous compared with Paget cells and are cytologically bland, and they do not cause diagnostic problems. It has been suggested that Toker cells may be the origin of intraepithelial Paget cells, based on similarity of immunophenotypes.185 In cases of florid
733
B
Figure 19-28 Paget disease of the nipple (A), showing strong staining with antibodies to cytokeratin 7 (B) and HER2 (C).
papillomatosis of the nipple, some CK7-positive cells may be found in the epidermis, a pitfall to be aware of in diagnosing Paget disease of the nipple.186 In addition, the intraepidermal portion of nipple ducts can be a pitfall for intraepidermal CK7-positive cells.187 Pseudo-Paget disease may on occasion be seen in the major ducts. Large histiocytes infiltrate the epithelium and impart a picture that simulates Paget disease. These large cells are CK7 negative and are strongly positive for CD68 (Fig. 19-29).
KEY DIAGNOSTIC POINTS Mammary Paget Disease • Most often positive in Paget disease: CK7 and HER2. • Other positive but less helpful stains: GCDFP-15, MGB, CEA, ER, and PR. • Pitfall: CK7-positive cells in the epidermis in cases of florid nipple duct papillomatosis, Toker cells, or intraepithelial extension of lactiferous duct cells. • Pagetoid Bowen disease: CK5 and p63 positive, CK7 negative. • Melanoma: melanoma markers positive, keratin negative.
734
Immunohistology of the Breast
A
Figure 19-29 Duct ectasia with pseudopaget of the large ducts (A). Large clear cells intercalated in duct epithelium are CD68 positive (B) and cytokeratin 7 negative (C).
Detection of Lymphatic Space Invasion Lymphovascular space invasion in breast carcinoma is an independent predictor of axillary lymph node metastasis, which in turn is one of the most important prognostic factors in breast carcinoma.188-191 One recent study has shown that peritumoral lymphatic space invasion, not blood vessel invasion, was determinant of lymph node metastasis.192 In addition, identification of tumor emboli within dermal lymphatics is also important for correlation purposes in cases of inflammatory carcinoma.193-195 However, the pitfalls of interpretation of lymphatic channels in paraffin-embedded breast tissue are well known. Retraction artifacts, ducts with misplaced epithelium, and artifactual displacement of cells commonly complicate the interpretation of biopsy samples. D2-40 shows high sensitivity and specificity for normal lymphatic channels in a variety of tissues.196,197 It stains the lymphatic endothelium crisply and intensely but does not stain the normal vascular endothelium (Fig. 19-30).196,198 D2-40 is highly sensitive and specific in identifying lymphatic space invasion.196,198 In the breast, it stains lymphatic channels with a crisp, intense
B
C membrane staining of lymphatic endothelium and shows a smudgy immunostaining pattern with MECs and reactive stromal myofibroblasts. This is a pitfall, and this faint to occasionally moderate staining around the periphery of a small duct may be mistaken for lymphatic space invasion; however, it is important to remember that lymphatic vessels stain very intensely with D2-40 (see Fig. 19-30, C).
Sentinel Lymph Node Examination Historically, complete axillary lymph node dissection was performed with lumpectomy or mastectomy specimens, primarily for staging purposes, providing information that was used to determine adjuvant chemotherapy. Complete axillary lymph node dissection (CALND) may not change the course of the disease, although with removal of involved axillary nodes, the control of local recurrence in the axilla is easier. The morbidity associated with this procedure is substantial in terms of arm pain, limitation of arm motion, and chronic lymphedema. The concept of a sentinel lymph node (SLN) was spawned by Cabanas199 in his study of penile carcinoma. The pioneering studies of SLN metastasis (SLNM) originated with the study of melanoma patients with the
Sentinel Lymph Node Examination
A
C goal of sparing these patients the morbidity of large regional lymph node dissections. Patients with melanomas who had SLN surgery were found to have a relatively orderly progression of lymph node metastases, with the SLN receiving the initial deposits of metastatic cells, followed by metastases in more distal lymph node groups. The same rationale was subsequently used for breast cancer patients.200,201 The SLN is identified by injecting a radioisotope and blue dye before planned surgical excision. Identified by a combination of visual inspection for blue dye and intraoperative scanning for radioactivity, the SLN is harvested and submitted for pathologic study. The rationale for this approach is that for patients who are SLN negative, a further morbid procedure of axillary cleanout is unnecessary, but for SLN-positive patients, an axillary dissection is indicated for proper staging and possibly to provide better control for local recurrence. The controversy in this approach arises from several valid questions: 1. What is the natural history of micrometastatic (MM) disease in the axilla? 2. Is MM SLN disease an obligate pathway to clinically manifested local recurrence in the axilla? 3. Is MM SLN disease an indication for adjuvant chemotherapy?
735
B
Figure 19-30 Immunohistochemical stain for D2-40 demonstrates selective staining of lymphatic endothelium (A) compared with CD31, which stains both lymphatic and vascular endothelium (B). A side-by-side comparison of lymphatic channel and a breast duct shows intense reactivity of lymphatic endothelium but somewhat “smudgy” weak staining around the duct (C).
4. How should the excised SLN be examined pathologically? 5. Does MM SLN disease affect overall survival? 6. What are the biologic parameters of MM disease that can predict the behavior of the disease in an individual patient? 7. Is it possible to recognize “benign transport” of epithelial elements in an SLN? These are interesting and provocative questions. The American Joint Commission on Cancer defines micrometastasis as a cluster of cells no larger than 2 mm. One study with more than 10 years of follow-up concluded that micrometastases are associated with a small but statistically significant decrease in tumor-free survival and overall survival when compared with truly nodenegative cases,202 but they are not an independent prognostic factor. The size of the metastatic deposit, taken together with tumor size and other factors, may additionally stratify patients at risk for further disease. In most institutions, SLN biopsy with lumpectomy or mastectomy as indicated has become the standard of care.203 The vast majority of SLN metastases are found in the first three SLNs submitted.204 Additionally, with completion and reporting of American College of Surgeons Oncology Group (ACOSOG) Z0011 study data, axillary lymph node staging has further changed.205 This
736
Immunohistology of the Breast
study randomly assigned patients undergoing lumpectomy for invasive cancer to have either completion axillary dissection or no axillary dissection for up to two positive sentinel nodes. Follow-up on these two randomly assigned groups showed no statistical difference in disease-free and overall survival. The lack of worse prognosis in patients who did not receive complete axillary dissection is likely related to the beneficial effect of radiation therapy after lumpectomy and may also be due to the fact that in approximately 60% of cases of positive sentinel nodes, only the sentinel nodes are positive. In view of the Z0011 data, extensively sampling (i.e., on multiple levels of the lymph node) and use of highly sensitive methods for identifying metastatic tumor seems unnecessary.206,207
Sentinel Lymph Node Immunohistochemistry For the surgical pathologist, the appropriate triage and examination of the SLN is of utmost importance, but even here some controversy exists. When the SLN mapping procedure became the standard of care a few years ago, SLNs were histologically examined on multiple levels and by cytokeratin stains on at least two levels. Since then, more experience has been gained with the procedure and with the reporting of SLNs. It was soon realized that the majority of micrometastases (metastases between 0.2 to 2 mm) can be identified by
A
C
H&E alone, and IHC for cytokeratin stains generally highlights isolated tumor cells (aggregates ≤0.2 mm).208 Although the exact clinical significance of isolated tumor cells, and even micrometastases, remains uncertain, studies have shown that both are associated with non-SLN positivity in approximately 10% of cases, especially when the tumor size is larger than 1 cm (pathologic tumor [pT] stage 1C or higher).209-212 However, completion axillary dissection with one to two positive sentinel nodes in lumpectomy patients does not translate into improved disease-free or overall survival as shown in the Z0011 trial.205 If the primary breast carcinoma is of the ductal type, it would be difficult but not impossible to identify isolated tumor cells by H&E stain, and most pathologists would agree that they would be able to identify micrometastases (Fig. 19-31, A). Therefore cytokeratin stains on SLN do not add any significant information beyond H&E stain in a primary ductal cancer. However, differences are significant when the primary breast tumor shows a lobular morphology. Because of single-cell infiltration, small metastases (micrometastases) of lobular carcinoma of the classic type in a lymph node are extremely difficult to identify (see Fig. 19-31, B-C). Occasionally, cytokeratin stains would identify macrometastases not readily apparent on H&E stains.213 Cserni and colleagues213 reported that sentinel node positivity detected by IHC in lobular carcinomas was associated with further nodal metastases in 12 of 50 cases (24%). It is therefore not
B
Figure 19-31 A, Small foci of tumor deposits in the lymph node from a primary ductal-type breast carcinoma can be identified with relative ease on hematoxylin and eosin stain alone. Immunohistochemical stains are generally not required. B, In contrast to metastases from ductal carcinomas, a micrometastasis or even a macrometastasis from a primary lobular type carcinoma is sometimes difficult to identify on hematoxylin and eosin stain. C, These types of metastases are best identified by cytokeratin stains, which can also help in estimating the correct size of metastatic focus.
Sentinel Lymph Node Examination
unreasonable, although it is not required, to perform cytokeratin stains on SLNs in cases of lobular carcinoma. When performing cytokeratin immunostaining of SLNs, the pathologist should use a cocktail such as AE1/ AE3214; CAM5.2 is less desirable because of the manner in which it stains dendritic cells in the lymph nodes.215 Micrometastatic cells occur in small clusters less than 2 mm in diameter within the lymph node or subcapsular sinus, and they must be distinguished from the dendritic-appearing interstitial reticulum cells of the lymph node, which are also keratin positive.216 Studies with larger numbers of patients are needed to discern whether the site of lymph node micrometastasis (peripheral sinus vs. parenchyma of lymph node) is clinically significant. Aggregates of breast epithelial cells in the subcapsular sinus of axillary lymph nodes have been described by Carter and associates217 as occurring as a result of “mechanical transport” after a breast biopsy. Some impugn the core biopsy itself or the breast massage that follows isotope/dye injection as sources of mechanical displacement of cells into the SLN.218-220 Solitary keratinpositive cells may be transported to the SLN, and the histologic feature often associated with true benign transport is the association of CK-positive cells with altered red blood cells, hemosiderin, and macrophages (Fig. 19-32). Diaz and colleagues218 described benign
A
C
737
epithelial tissue in skin dermal lymphatics and SLNs from patients with pure DCIS, which lends morphologic documentation to the concept of “benign mechanical transport.” The distinction between benign transport and true metastasis is easy, if the cells in lymph node appear benign; but no objective way is available to distinguish benign transport from true metastasis when the cells appear cytologically malignant.
Intraoperative Molecular Testing of Sentinel Lymph Node In the past few years, a few studies have shown the usefulness of intraoperative molecular tests in determining metastatic disease.221,222 These are reverse transcription polymerase chain reaction (RT-PCR) assays, which use a completely closed system and are fully automated, from RNA extraction to final interpretation. One such assay was the GeneSearch Breast Lymph Node (BLN) assay (Veridex LLC, Warren, NJ), which was approved by the U.S. Food and Drug Administration (FDA) for axillary lymph node testing. The GeneSearch BLN assay was composed of a sample preparation kit, all reagents required for performing RT-PCR, and protocol software to be used with the SmartCycler System (Cepheid, Sunnyvale, CA). According to the company, the test was optimized for detecting metastatic disease larger than 0.2 mm. The test analyzed the expression of CK19 and
B
Figure 19-32 Possible “benign transport.” A, Negative sentinel lymph node sinus with giant cells, macrophages, broken red blood cells, and epithelioid cells. B, Low magnification of keratinpositive cells involving the lymph node sinus. C, Higher magnification shows a mixture of keratin-positive cells with debris of macrophages and degenerated red blood cells.
738
Immunohistology of the Breast
mammaglobin genes. The studies showed high sensitivity, specificity, and positive and negative predictive values for the test.222-225 Overall, this molecular assay was very much comparable to examination of frozen and permanent sections and even IHC. However, just like any other test, the pathologist should be aware of the false-positive and false-negative test results in addition to the usefulness and pitfalls of a particular test. Because molecular tests are not morphologic assays, extreme care must be taken with any sources of contamination. A cutting-bench metastasis, or “floater,” can be easily recognized on an H&E stained slide as such, but it will give a false-positive result by RT-PCR with no definite way to identify this as an error. The SLNs identified in the axillary tail may contain minute amounts of breast tissue in the surrounding adipose tissue, which may also give a false-positive result. Therefore lymph nodes should be completely trimmed of the adipose tissue before being sectioned for the molecular analysis. Moreover, fat interferes with the assay itself, which could be an issue when the SLN is diffusely replaced by adipose tissue. Occasionally, a benign epithelial inclusion (>0.2 mm) within the lymph node could also be a source of a false-positive result (Fig. 19-33). Given the significance of the treatment decision based on a positive SLN result (complete axillary lymph node dissection, which cannot be undone) and several sources of false-positive results with molecular tests, we believe insufficient data are available to justify replacing the morphologic methods with a molecular assay. At present, we suggest that a positive molecular result should be confirmed by morphology, either by frozen or permanent sections, before a final decision is made. In contrast, a negative result is highly valuable given the very high negative predictive value of the molecular tests. Notably, the commercial assay mentioned earlier, the GeneSearch assay, was taken off the market for a number of reasons, including workflow problems and lack of interest in identifying micrometastasis and isolated tumor cells not seen with routine testing.
Figure 19-33 A benign epithelial glandular inclusion in an axillary lymph node, as shown here, may result in a false-positive molecular test for assessing micrometastatic disease.
KEY DIAGNOSTIC POINTS Sentinel Lymph Node Micrometastatic Disease • Section the lymph node perpendicular to the long axis at 2-mm intervals, examine with H&E (no levels required). • For primary ductal carcinomas, AE1/AE3 keratin stain should be avoided. • AE1/AE3 staining can be performed for lobular carcinomas, because even large tumor aggregates may be missed on H&E examination alone. • The SLN procedure has been adopted as a standard of care in many institutions. • Ninety-seven percent of all SLN metastases will be found in the first three SLNs when multiple SLNs are submitted. • Intraoperative molecular tests are comparable to morphologic examination, but potential sources of falsepositive results must be acknowledged. • Molecular tests to identify micrometastatic tumor in the SLN seem unnecessary for individual patient management.
Systemic Metastasis of Breast Carcinoma The diagnosis of breast carcinoma at a metastatic site requires a careful histologic examination, review of all prior case material, and immunohistologic evaluation of tumor cells. If a patient has a prior history of breast cancer, it is valuable to know whether the tumor showed ductal or lobular morphology. Comparison to a prior tumor is helpful in making the correct diagnosis in a majority of cases. Immunohistologic evaluation is mainly required in cases of carcinoma of unknown origin, and CK7 and CK20 have been generally used in this evaluation to narrow the differential diagnosis.226,227 Breast carcinomas are generally CK7 positive and CK20 negative; however, a similar cytokeratin profile is seen in a number of other carcinomas, including those from the lung and gynecologic tract. Gross cystic disease fluid protein 15 (GCDFP-15) has been used for several years as the most specific marker of breast carcinoma,228,229 although its sensitivity in formalin-fixed paraffin-embedded (FFPE) tissue is less than optimal.228 Originally described by Pearlman and colleagues230 and Haagensen and associates,231 the prolactin-inducing protein identified by Murphy and coworkers232 has the same amino acid sequence as GCDFP-15 and is found in abundance in breast cystic fluid and in any cell type that has apocrine features.229,233 The latter, in addition to breast, includes acinar structures in salivary glands, apocrine glands, and sweat glands and in Paget disease of skin, vulva, and prostate.229,234-237 Homologous-appearing carcinomas of the breast, skin adnexa, and salivary glands demonstrate a great deal of overlap immunostaining with GCDFP15.238 Aside from these immunoreactivities, most other carcinomas show no appreciable immunostaining. Breast carcinoma metastatic to the skin (or locally recurrent) may be difficult to distinguish from skin
Fibroepithelial Tumors
adnexal tumors.233 Wick and associates,239 in a study of the overlapping morphologic features of breast, salivary gland, and skin adnexal tumors, found that GCDFP-15 was infrequently found in eccrine sweat gland carcinomas, a paucity of CEA was found in breast carcinomas, and estrogen receptors were largely absent in salivary duct carcinomas. The positive predictive value and specificity for detection of breast carcinoma with GCDFP15 have been reported to be as high as 99%.229 The sensitivity for the GCDFP-15 antibodies has been reported to be as high as 75% for tumors with apocrine differentiation,229,233 but the overall sensitivity is 55%, and it is only 23% for tumors without apocrine differentiation.233 The sensitivity is even worse when it comes to core biopsy, because the pattern of staining for GCDFP-15 is often patchy. Because the specificity of GCDFP-15 antibodies for breast carcinoma is so high, this antibody is often used in a screening panel in the appropriate clinical situation, which often turns out to be when a woman with a history of breast cancer comes to medical attention with metastasis of unknown primary or a new lung mass. Others have demonstrated the utility and specificity of GCDFP-15 antibodies in the distinction of breast carcinoma metastatic in the lung.240-242 However, Striebel and colleagues243 demonstrated GCDFP-15 immunoreactivity in 11 of 211 (5.2%) lung adenocarcinomas. This study again stresses the importance of a panel, rather than an individual stain, in determining site of origin of a metastatic tumor. On a similar note, a specific marker of ovarian serous carcinoma, WT1, shows nuclear expression in a subset of breast carcinomas that demonstrate mucinous differentiation. However, the expression is generally weak to moderate in contrast to ovarian serous carcinoma, in which the expression is generally strong and diffuse.244 Estrogen and progesterone receptors may be helpful in patients with a history of receptorpositive breast cancer; however, a large proportion of gynecologic tumors are positive for HRs, which have also been reported to be positive in nonbreast and nongynecologic sites.245-247 Recently, mammaglobin (MGB) has been described to be a more sensitive marker than GCDFP-15 for diagnosis of breast carcinoma.248,249 The mammaglobin gene is a member of the uteroglobin family that encodes a glycoprotein associated with breast epithelial cells. The immunostaining pattern is cytoplasmic, analogous to GCDFP-15. If the weak equivocal staining is disregarded, because it is not helpful in determining site of origin in actual clinical situations, the sensitivity of MGB is between 50% and 60% compared with less than 30% for GCDFP-15. We have seen that even in cases positive for both GCDFP-15 and MGB, the percentage of cells and intensity of staining is much higher with MGB than with GCDFP-15 (Fig. 19-34).250 Some initial studies have suggested association of MGB staining with HR positivity, but we have not found such an association, therefore MGB may be useful in identifying breast tumors that are negative or low (patchy) positive for receptors. The drawback of using MGB is its lack of specificity. It is noteworthy, however, that MGB stains a substantial number (~40%) of endometrioid
739
carcinomas and also stains occasional melanomas.250 With respect to the distinction of breast carcinoma from skin adnexal tumor or ductal salivary gland tumor, both MGB and GCDFP-15 are unreliable, because these tumors have a similar IHC profile.239,247 In spite of some nonspecificity, it appears that a combination of GCDFP15 and MGB is better than GCDFP-15 alone in diagnosis of metastatic breast cancer. Another positive breast marker that can be used is NYBR1, the reactivity for which correlates with HRs. NYBR1 is generally negative in müllerian tumors and in other CK7-positive, CK20-negative tumors that can mimic breast carcinomas.251-254 A useful negative marker in the workup of tumor suspected to be of breast origin is Pax-8. This antibody is almost always negative in breast carcinoma but is positive in a substantial proportion of tumors of müllerian origin.254,255 In contrast to WT1, the Pax-8 reactivity in müllerian tumors is not type specific. Pax-8 is also positive in thyroid tumors, renal tumors, GI carcinoid tumors, and thymic carcinomas.254-258 KEY DIAGNOSTIC POINTS Metastatic Breast Carcinoma • Diagnostic confirmation requires use of a panel. • The usual breast carcinoma immunoprofile is positive for CK7, GATA3, GCDFP-15, MGB, ER, and NYBR1; it is negative for CK20, thyroid transcription factor 1 (TTF-1), Wilms tumor 1 (WT1), and Pax-8. • GCDFP-15 is the most specific marker of breast carcinoma; however, weak or equivocal staining is not helpful in the workup of a tumor of unknown origin. • MGB is a more sensitive marker of breast carcinoma than is GCDFP-15. • MGB also stains endometrioid adenocarcinomas (up to 40% of cases) and rare melanomas. • Salivary gland carcinomas and skin adnexal carcinomas have overlap staining with GCDFP-15 and MGB. • As many as 30% of breast carcinomas may be negative for both GCDFP-15 and MGB.
Fibroepithelial Tumors Fibroepithelial tumor is a term used for biphasic tumors that contain both an epithelial and stromal component. Fibroadenomas and phyllodes tumors make up the majority of fibroepithelial tumors. No IHC stains are required for the diagnosis of fibroadenoma; however, the intracanalicular variant and cellular subtypes must be distinguished from benign phyllodes tumor.
Fibroadenoma and Phyllodes Tumor Phyllodes tumors are biphasic neoplasms distinguished from fibroadenomas mainly on morphologic grounds. Unlike fibroadenoma, phyllodes tumors are relatively large, heterogeneous neoplasms that histologically show a leaflike architecture and periglandular stromal condensation. The variably cellular spindled-cell stroma
740
Immunohistology of the Breast
A
B
C
D
Figure 19-34 A, Adenocarcinoma involving abdominal wall. The tumor cells were strongly and diffusely positive for cytokeratin (B), patchy positive for gross cystic disease fluid protein 15 (C), and showed diffuse strong staining for mammaglobin (D). In spite of negative receptor status, the morphology and immunohistochemical profile was consistent with the patient’s known history of breast carcinoma from several years prior.
has been proven by clonal analysis to be the neoplastic compartment.259 IHC reports have shown the majority, regardless of grade, to express CD34 in the stromal compartment, consistent with fibroblastic and/ or myofibroblastic differentiation, which is supported by ultrastructural studies.260-262 The World Health Organization (WHO) has adopted a three-tiered classification of benign, borderline, and malignant phyllodes tumors. Benign and borderline tumors can recur, especially with positive or close margins, but metastases are generally seen only with malignant phyllodes tumor. Benign phyllodes tumors are histologically akin to cellular intracanalicular fibroadenomas but with stromal heterogeneity, leaflike architecture, periglandular stromal condensation, and/or 1 to 3 mitoses per 10 hpf. Because of this low proliferation activity, benign phyllodes tumors often must be distinguished from fibroadenomas. A great deal of morphologic overlap is found between cellular fibroadenomas and benign phyllodes tumor; unfortunately, IHC stains are also of limited value in this differential diagnosis. Nevertheless, investigators have tried proliferation markers for this distinction.
Jacobs and colleagues262a found significantly higher stromal proliferation indexes—such as Ki-67, a marker of all phases of the cell cycle, and topoisomerase II-alpha (topo 2-α), a marker of the G2-M phase—in phyllodes tumors compared with fibroadenomas on core needle biopsy. In this report, however, Ki-67 index ranged from 0.4% to 4.4% (average 1.6%) in fibroadenomas and from 0% to 18% (average 6%) in benign phyllodes tumors. Thus the margin of error in determination of the proliferation index is relatively small, and given the subjectivity involved in its estimation, it may not be entirely reliable to distinguish between the two lesions (Fig. 19-35). Malignant phyllodes tumors are akin to sarcomas with stromal expansion, pleomorphism, and mitotic rates of greater than 5 per 10 HPF, and borderline tumors lie somewhere in between benign and malignant phyllodes tumor. Numerous IHC studies have shown correlation of biomarker expression with tumor grade. Studies that have targeted Ki-67 with the monoclonal antibody MIB-1 have shown progressively increased expression with tumor grade. Ki-67 labeling indexes range from 1% to 5% in benign tumors, 6% to 16% in
Fibroepithelial Tumors
borderline tumors, and 12% to 50% in malignant tumors in published reports (Fig. 19-36).263-266 P53 expression often parallels Ki-67 expression in phyllodes tumors.266-269 More recently, the expression of proteins with targeted therapy implications in phyllodes tumors have been explored. In 2000, Chen and colleagues270 first reported c-Kit expression in the stroma of phyllodes tumors and found c-Kit to be preferentially expressed in histologically malignant phyllodes tumors. Since then, several additional studies have reported increased c-Kit expression in malignant phyllodes tumors compared with benign and/or borderline tumors.271-273 However, whether c-Kit expression in these tumors implies susceptibility to the KIT receptor tyrosine kinase inhibitor imatinib mesylate is doubtful, because activating KIT mutations have yet to be reported. Of interest is the study by Djordjevic and Hanna,274 which suggests that c-Kit expression in fibroepithelial tumors may be related to the presence of mast cells. The authors have argued against any appreciable true stromal cell c-Kit staining in fibroepithelial tumors. Epidermal growth factor receptor (EGFR) has also been studied in phyllodes tumors, and most reports correlate increased
741
stromal expression with tumor grade and chromosome 7 polysomy (Fig. 19-37).268,275,276 However, prognostication solely based on histologic categories has proved problematic in some cases, because histologically benign phyllodes tumors may recur as higher grade tumors with associated metastases, and many histologically malignant tumors neither recur nor metastasize.277-279 The primary aim of most IHC studies has thus been to correlate biomarker expression with recurrence and/ or patient outcome rather than diagnostic category. Unfortunately, most reports have failed to do so and are conflicting. For example, one study reported an inverse relationship between Ki-67 expression and overall survival in multivariate analysis of 117 phyllodes tumors, but others have not corroborated this finding.263,265,271,280 Similarly, whereas p53 expression appears to correlate with tumor grade as noted above, it has failed to consistently predict tumor recurrence.266,269,281 The potential prognostic value of EGFR has yet to be determined given the modest body of literature that exists on the subject to date. Overall, studies aimed at developing better prognostic markers in phyllodes tumors have suffered from low sample size, lack of patient follow-up data, and lack
A
B
C
D
Figure 19-35 Proliferative activity in fibroadenomas vs. benign phyllodes tumors. The fibroadenoma depicted in A and B shows no Ki-67– positive stromal cells, whereas that depicted in C and D shows focal (~1%) Ki-67 stromal cells immunoreactive.
742
Immunohistology of the Breast
E
F
G
H
Figure 19-35, cont’d Note the numerous positive ductal epithelial cells. Similarly, benign phyllodes tumor may show minimal to no proliferative activity with Ki-67, as shown in E through H. Courtesy Dr. Nicole N. Esposito, Tampa, FL.
of reproducibility. The most significant consistently reported variables in prediction of phyllodes tumor behavior remain histologic characteristics, particularly the presence of stromal overgrowth, and adequate surgical resection margins.265,282-288
variant of fibroadenoma or as adenosis tumor with myoid metaplasia, which may be regarded as a variant of mammary hamartoma. These tumors show positive stromal immunoreactivity for SMA, desmin, myosin heavy chain, and vimentin (Fig. 19-38) and are negative for S-100.290,291
Periductal Stromal Tumor Periductal stromal tumor was initially described by Burga and Tavassoli289 as an entity distinct from phyllodes tumors, although histologically identical, except that periductal stromal tumor lacks the intracanalicular or leaflike pattern. As with phyllodes tumors, however, the stromal cells express CD34, and thus some have proposed that these are best regarded as a phyllodes tumor variant that lacks the classic leaflike architecture rather than a distinct entity.173,289
Other Fibroepithelial Lesions The so-called myoid hamartoma can also be considered a fibroepithelial lesion, because it has both stromal and epithelial components. Some authors consider it a
KEY DIAGNOSTIC POINTS Fibroepithelial Tumors • Phyllodes tumor stroma is CD34 positive, a finding that is useful in the workup of spindle cell lesion in a core biopsy. • Ki-67 may supplement grading of phyllodes tumor in addition to morphology and counting of mitotic figures. • Ki-67 proliferation index does not reliably distinguish between fibroadenoma and benign phyllodes tumor. • Molecular analyses so far have also been inconclusive in distinguishing fibroadenoma from phyllodes tumor. • Periductal stromal tumor is likely a variant of phyllodes tumor and also has CD34-positive stroma.
Fibroepithelial Tumors
A
B
C
D
E
F
743
Figure 19-36 Ki-67 expression in benign (A and B), borderline (C and D), and malignant (E and F) phyllodes tumors of the breast. Labeling indexes as demonstrated by Ki-67 expression generally correlate linearly with tumor grade. Courtesy Dr. Nicole N. Esposito, Tampa, FL.
744
Immunohistology of the Breast
A
B
Figure 19-37 A and B, Epidermal growth factor receptor (EGFR) expression in two malignant phyllodes tumors. EGFR expression has been shown to be more common in malignant phyllodes tumors and usually corresponds to polysomy 7 rather than EGFR amplification. Courtesy Dr. Nicole N. Esposito, Tampa, FL.
Theranostic Applications Paraffin-embedded tissue, used for the primary morphologic diagnosis of breast carcinoma, also lends itself to a variety of antibody tests that not only shed a great deal of light on the biology of the disease but also serve as a cutting-edge medium for the development of tests that may have an impact on how the disease is treated. Pathologic features of breast carcinoma that have prognostic value include 1) tumor size, grade, and histologic type; 2) lymph node status; 3) HER2 expression; and 4) expression of HRs. These parameters, which should be included in each pathologist’s surgical pathology report, have been thoroughly studied and were found to have significant prognostic value in regard to clinical course and response to therapy. IHC stains for estrogen receptors (ERs), progesterone receptors (PRs), and HER2 are the most common stains performed for prognostic and predictive information. We have come a long way from ligandbinding assays to IHC for analyzing ER and PR. ER and PR ligand-binding assays have been validated by longterm follow-up for clinical use, with established cutoffs for positive results. Although several studies have shown excellent correlation between the two tests,292-294 over the years concerns have been raised about the quality, reproducibility, and accuracy of IHC to study markers that predict response to targeted therapy.295-297 The preanalytical and analytical factors that affect IHC results should be carefully taken into account when interpreting these results.298-300 In this section, we discuss the IHC tests that are performed routinely, because they may have direct, immediate therapeutic implications. Needle biopsy of the breast and fine needle aspiration (FNA) cytologic techniques are the most common methods used for making the diagnosis of breast carcinoma. All of the diagnostic and prognostic tests—for SMMHC, MSA, ER, PR, Ki-67, p53, HER2, and so on—can be applied to these small core biopsies and yield reliable results.301-306 Of all these tests on core
biopsy specimens, only progesterone gives a substantial number of false-negative results because of the greater heterogeneity of immunoperoxidase staining in tissue.306 However, caution is advised when performing prognostic assays (ER, PR, HER2) on FNA material. Apart from preanalytical factors related to fixation, the pathologist cannot be absolutely sure about the presence of invasive carcinoma in the cytology material and may erroneously report ER, PR, or HER2 on in situ carcinoma.
Hormone Receptors Recognition that estrogen ablation had an impact on groups of patients with breast cancer307 and recognition that clinical responsiveness correlated with the expression of ER were seminal events in the treatment of patients with breast cancer. ER and PR bind hormones that exert their effects in the nucleus. Nuclear immunostaining for both receptor proteins can be demonstrated in normal breast acini, which serve as internal controls for the testing procedure. In general, approximately 15% to 20% of the luminal epithelial cells in a duct or lobule stain with ER and PR. However, nuclear staining in normal breast tissue is heterogeneous and may vary with the menstrual cycle.308 One of the effects of estrogen is to induce the PR, and thus the coordinate expression of both hormones in the same cell reflects the fidelity of the ER/PR axis in the cell. In carcinomas of the breast, most PR-positive tumors are also ER positive, and ER-negative, PR-positive tumors account for fewer than 1% of all breast cancers. In general, patients with positive PRs have a significantly longer disease-free survival than patients who are PR negative.309-316 Since the early 1990s, the IHC assay determination of ER/PR levels has replaced the dextran-coated charcoal (DCC) method, also referred to as the ligandbinding assay (LBA). The DCC method, the gold standard for many years, suffered significant drawbacks; namely, 1) tumor sampling error; 2) heavy reliance on obtaining tissue immediately on termination of the blood supply to the tumor, usually in the operating
Theranostic Applications
A
B
C
D
E
room; 3) normal tissue contamination; and 4) analytic error. Some of the advantages of the IHC method include 1) histologic documentation of the tumor tissue to be assayed; 2) appreciation of the heterogeneity of ERs and PRs in tumor cell nuclei; 3) rapid assessment of the tissue for ERs and PRs by the semiquantitative method; 4) rapid turn-around time; 5) lower cost; and 6) the ability to use minute quantities of tissue. Despite the widespread use of IHC for HR determination, the lack of standardization of preanalytic and
745
Figure 19-38 A well-circumscribed biphasic lesion with stromal smooth muscle metaplasia is consistent with the diagnosis of myoid hamartoma (A and B). The stromal smooth muscle metaplasia is positive for actin (C), h-caldesmon (D), and smooth muscle myosin heavy chain (E). Note that actin and myosin heavy chain also stain the blood vessels and myoepithelial cells around the ducts.
analytic variables, scoring schemes, and threshold for determining HR positivity remain a concern. PREANALYTIC VARIABLES
Preanalytic variables mainly relate to tissue fixation: cold ischemic time, time in fixative, and type of fixative used. Cold ischemic time is defined as the time taken to place the specimen in fixative after it has been removed from the patient’s body. The College of American
746
Immunohistology of the Breast
Pathologists (CAP) and the American Society of Clinical Oncology (ASCO) recommend that the specimen have a cold ischemic time of 1 hour or less if used for receptor testing in breast cancer. However, only a few studies exist on this subject. Neumeister and colleagues317 found no evidence for loss of antigenicity with time to fixation for ER, PR, HER2, or Ki-67 in a 4-hour time window. However, with a bootstrapping analysis, the authors did observe a trend toward loss of ER and PR. This particular study was mainly done on tissue specimens represented on tissue microarray by using automated quantitative analysis (AQUA) technology, and it assessed the effect of ischemia for a relatively short time beyond the 1-hour limit recommended by ASCO. In a study of 97 patients with paired core biopsy and resection specimens, Li and colleagues318 studied the impact of cold ischemic time (ranging from 64 to 357 min) on ER IHC staining. Although the difference in the percentage of ER staining between core biopsy and resection was not statistically significant, they did identify a trend of decreased ER staining with a cold ischemic time of less than 2 hours. In our own study of cold ischemic time, nonrefrigerated samples were affected more by prolonged cold ischemic time than refrigerated samples. Significant reduction in IHC staining for HRs did not result until 4 hours for refrigerated samples and 2 hours for nonrefrigerated samples.319 Because most studies with clinical outcome have been performed by using FFPE tissue, the current CAP/ ASCO recommendation is to use 10% neutral phosphate buffered (NPB) formalin and to fix the tissue for 6 to 48 hours. If an alternative fixative or fixation method is used, it has to be validated with standard fixation before it is implemented in clinical testing. Yildiz-Aktas320 showed that formalin fixation for as long as 96 hours does not affect semiquantitative receptor results. Most laboratories perform hormone receptor analysis on core biopsy samples, and some misconception has resulted regarding formalin exposure time on smaller samples. Tissue permeation and fixation are not synonymous: fixation is a chemical reaction that takes time, and therefore both smaller and larger tissue samples have to be fixed for a similar amount of time. However, larger resection specimens must be sectioned for efficient permeation of formalin. Impact of formalin fixation time on ER staining has been elegantly shown by Goldstein and associates.321 The authors studied tissue sections from 24 known, strongly ER-positive tumors that were fixed for 3, 6, 8, and 12 hours for 1, 2, and 7 days. They used a semiquantitative score (Q score with a range of 0 to 7) to determine ER expression. With 40 minutes of antigen retrieval, the mean Q score at 3 hours was 2.46, and it reached a plateau of 6.70 at 8 hours. The mean Q score at 7 days was 6.60. However, with 25 minutes of antigen retrieval, the mean Q score at 3 hours was 1.75; it progressively increased with time to 6.62 at 10 hours, and then declined with time to 3.79 at 7 days. This classic example shows that optimum formalin exposure time for ER determination is 8 hours and that antigen can be
retrieved with increasing retrieval times for overexposed tissue, but underfixed tissue is completely useless for biomarker study. ANALYTIC VARIABLES
Another issue of concern for HRs and other biomarker testing is the use of rapid or alternate processors. It is strongly recommended that breast cancer specimens be processed in conventional processors. If alternate processors or alternate solutions are being used, the procedures must be validated for ER results against similar samples processed in conventional processors. Documentation of the type of fixative and the formalin exposure time on pathology reports is also needed. Availability of the fixation time for each breast tissue sample might prove valuable for interpreting and troubleshooting aberrant and/or unexpected ER results. Another important issue is the choice of antibody. Current available literature mostly describes three antibody clones: 1D5, 6F11, and SP1 for ER. Most studies suggest a high degree of concordance among these antibodies; however, subtle differences are present that depend on the cut-off value for a positive result. All the commercially available antibodies for ER assessment in breast carcinoma target only the ER-α isoform. Although another isoform, ER-β, exists, its role in breast cancer is not well defined.322 Conflicting data have been reported that concern its potential role as a prognostic or predictive factor.323-326 Unlike ER-α, ER-β expression is seen in variety of tissues other than breast and female pelvic organs.327,328 Additionally, similar to HER2 or any other biomarker assay, controls should ideally be placed on each test slide. POSTANALYTIC INTERPRETATION
An equally important component for HR assays is the postanalytic portion of the tests, although scoring schemes and the threshold for determining HR positivity remain a concern. A critically important statement from the National Institutes of Health (NIH) Consensus Conference of December 2000 stated that “any nuclear expression of HRs should be regarded as a positive result and render a patient eligible for hormonal therapy.”329 This statement was supported by the studies performed by Cheang and coworkers330 and Pertschuk and colleagues,331 both of whom documented a cutoff of greater than 1% of cells for the 1D5 and SP1 ER antibody clones respectively. The current CAP/ASCO guidelines on HR testing also recommend 1% of positive cells as a positive result for ER and PR.332 The guidelines also recommend that the pathologist quantify the HR test results using one of several methods. Quantitative results of the IHC method correlate closely with biochemical results and are predictive of prognosis.333-336 In a study that used ER1D5, Veronese and colleagues337 found that immunostaining was predictive of response to tamoxifen in 65 homogeneously treated patients, and it was a discriminator for relapsefree and overall survival. Barnes and coworkers338 and Goulding and colleagues339 confirmed that the ER
Theranostic Applications
747
ALLRED SCORE Proportional Score (PS)
Intensity Score (IS)
0: No staining
0: No staining
1: Staining of <1% of tumor cells
1: Average weak intensity
2: Staining between 1% and 10% of tumor cells Figure 19-39 Semiquantitative scoring method for hormone receptors, known as the Allred score. Modified from Harvey JM, Clark GM, Osborne CK, Allred DC: Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 1999;17:1474-1481.
3: Staining between 1/10 and 1/3 of tumor cells 4: Staining between 1/3 and 2/3 of tumor cells 5: Staining of 2/3 of tumor cells
finding by IHC correlated better than the DCC method, and results from both studies related strongly to patient outcome regardless of the method of immunoscoring. The effect of quantitation and establishing that ER is a continuous variable, and not bimodal, was clearly shown by Harvey and associates293 in 1999 using the Allred Score (Fig. 19-39) in their series of 1982 primary breast cancer cases. Allred scores of 0, 2, 3, 4, 5, 6, 7, and 8 were seen in 26%, 3%, 6%, 10%, 16%, 19%, 16%, and 4% cases, respectively. The authors demonstrated a linear correlation between Allred score and ER content as measured by LBA. This study also showed differences in disease-free survival based on the Allred score. Even the current RT-PCR assay for HR has shown a broad dynamic range of HRs present in tumor cells.340,341 These tests do not demonstrate a bimodal distribution of HR results as suggested by a few studies.342,343 Although the data from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B14 clinical trial showed that the level of ER expression as measured by RT-PCR has little prognostic significance in the absence of endocrine therapy, the expression level has clinical significance when patients are treated with tamoxifen. When the tamoxifen-treated cohort was examined, patients in the top two tertiles showed benefit, whereas the lower tertile showed the same outcome as the placebo control group. The results suggest that the benefit from tamoxifen is limited to patients with the higher level of expression.344 The degree of ER and PR staining has consistently been shown to identify groups of patients with significantly different risks of overall survival, time to recurrence, and treatment response to hormonal therapy.293,345 Moreover, combination of standard histologic and semiquantitative IHC results for prognostic/predictive markers in breast cancer can help prognosticate individual patient risk and also has the potential to predict standard chemotherapy benefit.346-348 Therefore ER and PR results should be reported by using a semiquantitative method. At our institution, we prefer the histochemical score (H score) method of reporting, because it has a broad dynamic range compared with the Allred method. H score is given as the sum of the percent staining multiplied by an ordinal value that corresponds to the
2: Average moderate intensity 3: Average strong intensity
Allred score (range, 0 to 8) PS IS Possible Allred scores are 0 (negative), 2, 3, 4, 5, 6, 7, 8 (diffusely & strongly positive tumor)
Figure 19-40 In this example of an estrogen receptor–positive case, 10% of the cells are completely negative, 50% stain with 1+ intensity, 35% stain with 2+ intensity, and 5% stain with 3+ intensity. The H score is calculated as follows: (0 × 10) + (1 × 50) + (2 × 35) + (3 × 5) = 135.
intensity level (0, none; 1, weak; 2, moderate; 3, strong). With four intensity levels, the resulting score ranges from 0, no staining in the tumor, to 300, diffuse intense tumor staining. We also recently showed excellent interobserver concordance for H score among pathologists.349 The reason for good concordance is likely because of the way the H score is calculated, and because slightly more time is spent by the pathologist evaluating the slide to calculate the score (Fig. 19-40). Because the likelihood of benefit correlates with the amount of HR protein in tumor cells, we recommend using a semiquantitative criterion in addition to a positive or negative result that clearly states the percentage and intensity of ER and PR staining within tumor cells. Routine H scoring for HRs definitely has advantages but also poses some challenges in terms of pathologist training and performance review. Automated imageanalysis systems have been suggested as an alternative to human scoring, but more performance studies are still needed. The image-analysis systems currently used for analyzing IHC ER/PR slides are incapable of
748
Immunohistology of the Breast
reporting H-score results on the entire tumor section, although most systems provide quite accurate assessment of the percentage of positive cells in the field of view, which is generally a very small area of the tumor, unless the whole slide is scanned and interpreted. Whole-slide scanning takes time, and even if it is performed, the final onus is on the pathologist to make sure the invasive tumor cells are counted by the computer for reporting. Improvement in image-analysis technology may overcome some of these aspects. Image-analysis systems that use fluorescent probes have also been described for identifying immunoreactive tumor cells with an increased interobserver reproducibility. However, the challenge of correctly identifying invasive carcinoma for reporting under dark field microscopy and the increased time required to do such analysis can further impede incorporation into diagnostic pathology. Furthermore, efforts to use mRNA expression levels of ER/PR through quantitative RT-PCR have been advocated as an alternative to IHC. The increased use of oncotype DX (Genomic Health, Redwood City, CA) and MammaPrint (Agendia, Irvine, CA) diagnostic testing as ordered by clinicians may increase the popularity of these methods. However, as noted by the CAP/ ASCO committee, a paucity of data exists on the concordance between mRNA-based assays and IHC-based clinical validation studies. In regard to this issue, we recently reported good agreement between IHC semiquantitative H-score results and oncotype DX quantitative RT-PCR results, with IHC being slightly more sensitive than PCR for both ER and PR.350 No reason exists to replace IHC with PCR, and moreover, because of the speed, higher sensitivity, preservation of morphology, and widespread use in every pathology laboratory, IHC is more desirable. As far as PR IHC is concerned, Press and colleagues351 compared 12 different PR antibodies and found that PgR636 and PgR1294 stained the highest percentage of breast carcinomas known to be positive by the biochemical assay (95% to 98%), and they exhibited the highest concordance with the biochemical assay (88% to 90%). Later, Mohsin and coworkers345 performed clinical validation of the PR by IHC. Using a positive result of 1% of cells with PR clone 1294 and the Allred scoring system, the authors demonstrated that PR was better than the ligand-binding assay at predicting disease-free and overall survival. Progesterone nuclear staining by the IHC method is more heterogeneous than ER and may be a cause of false-negative results, especially in core biopsies306 or needle aspirates. We have seen similar results on some core biopsies; as a result, if a negative ER and PR result on a core biopsy specimen is obtained, the test is repeated on the excised breast specimen, particularly when the HRs are negative in tumors expected to be positive, such as lobular, tubular, and mucinous types. The sensitivity and specificity of other PR antibodies, namely 1A6 and the more recently available rabbit monoclonal antibodies, such as 1E2, are similar to the previously used antibodies. Two isoforms for PR, PRA and PRB, are recognized by the commercially available antibodies. Our preliminary data indicate that far
fewer cases are ER negative and PR positive with the 1E2 clone (1% of cases) compared with clone 1A6 (5% to 6%). Although less importance is given to PR compared with ER in the clinical literature, it is increasingly recognized that the amount of PR expression in ER-positive tumors provides significant prognostic and predictive information.352 In a comparative/correlative study by Clark and colleagues,353 semiquantitative PR expression had a huge impact on oncotype DX recurrence score independent of grade. In addition, Prat and colleagues354 showed the significance of PR and even proposed to modify the definition of luminal A tumors based on the amount of PR expression. Therefore it is critical that laboratories perform and interpret PR testing in a manner similar to that of ER testing. KEY DIAGNOSTIC POINTS Hormone Receptors • Preanalytic, analytic, and postanalytic factors can significantly alter hormone receptor (HR) results. • Clinically validated studies have been done for ER clones SP1, 1D5, and 6F11, all of which demonstrate a positive cut-off value of greater than 1% of cells staining linked to response to tamoxifen. • Some form of semiquantitation (H score, Q score, Allred score) should be performed on all positive cases, because semiquantitation is therapeutically important. • HR should be repeated on resection specimen if the tumor was double negative on core biopsy sample and the tumor histology suggests a positive result.
ERBB2 (HER-2/neu) The ERBB2 (formerly HER2) gene was originally called NEU because it was first derived from rat neuroblastoma/ glioblastoma cell lines. Coussens and colleagues355 named it HER2 because its primary sequence was very similar to human epidermal growth factor receptor (EGFR, ERBB, or ERBB1). Semba and associates356 independently identified an ERBB-related but distinct gene, which they named ERBB2. Di Fiore and colleagues357 indicated that both NEU and HER2 were the same as ERBB2, and Akiyama and associates358 precipitated the ERBB2 gene product from adenocarcinoma cells and demonstrated it to be a 185-kD glycoprotein with tyrosine kinase activity. In 1987, 2 years after its discovery, the clinical significance of HER2 gene amplification was shown in breast cancer.359 We now know that approximately 15% to 20% of breast cancers demonstrate HER2 gene amplification and/or protein overexpression.360,361 In the absence of adjuvant systemic therapy, HER2-positive breast cancer patients have a worse prognosis, that is, they have higher rates of recurrence and mortality, which clearly demonstrates the prognostic significance. An even more important aspect of determining HER2 status is its role as a predictive factor. HER2 positivity is predictive of response to anthracycline- and taxane-based therapies, although the benefits derived
Theranostic Applications
from non-anthracyclines and non-taxane therapies may be inferior.362-366 It is also important to note that HER2positive tumors generally show relative resistance to all endocrine therapies; however, this effect may be more toward selective endocrine receptor modulators, such as tamoxifen, and less likely toward estrogen-depletion agents, such as aromatase inhibitors.367,368 Most importantly, the availability of HER2-targeted therapy brought this biomarker to the forefront of theranostic testing for breast cancer. Trastuzumab is a humanized monoclonal antibody to HER2 that was approved by the FDA in 1998 for use in metastatic breast cancer. Trastuzumab improves response rates, time to progression, and survival when used alone or in combination with chemotherapy in treatment of metastatic breast cancer. Although approved for use in metastatic cancer, several prospective randomized clinical trials have shown significant therapeutic benefits from trastuzumab in early stage breast cancers.369-372 The same paradigm has also shifted to neoadjuvant chemotherapy using trastuzumab in HER2-positive tumors. Given the enormous therapeutic benefit derived from trastuzumab in HER2-positive tumors, it is absolutely critical that an accurate determination of HER2 status be made in each case. Because of its prognostic and predictive value, HER2 status should be determined on all newly diagnosed invasive breast cancers, which is now also recommended in the 2007 CAP/ASCO guidelines,373 which provide a detailed review of the literature and offer recommendations for optimal HER2 testing. The issues that ensure reliable HER2 testing can be divided into three categories: 1) preanalytic, 2) analytic, and 3) postanalytic issues. All three are equally important, and just like HR testing, they require a commitment to continuous quality improvement. PREANALYTIC ISSUES
The current CAP/ASCO recommendation is to use 10% NPB formalin and to fix the tissue for 6 to 48 hours. If an alternative fixative or fixation method is used, it must be validated with standard fixation before it is implemented in clinical testing. Although the guidelines focus more on the pitfalls of overfixation, we believe underfixation to be the real problem with HER2 testing. The antigen can be retrieved by various methodologies, and the enzymatic digestion times for in situ hybridization (ISH) can be altered if the tissue is overfixed, but nothing can be done if the tissue is underfixed. Overfixation may become an issue with alcohol fixation, which can lead to antigen diffusion, but it is generally not an issue with formalin fixation. We have validated tissue fixation times up to 96 hours for performing HRs and HER2 testing on breast carcinoma at our institution.320 The effect of underfixation on biomarker testing has been nicely shown by Goldstein and colleagues,321 as discussed previously, using ER as an example. In our own study of impact of cold ischemic time on breast cancer prognostic/ predictive factors, we319 reported that significant reduction in IHC staining generally does not result for up to 4 hours for refrigerated samples and for up to 2 hours for
749
nonrefrigerated samples. Additionally, HER2 staining is less commonly impacted compared with HR staining. Portier and colleagues374 also studied the impact of cold ischemic time on tumors subjected to varying cold ischemic times (45 cases with <1 hour of cold ischemia, 27 cases with 1 to 2 hours, 6 cases with 2 to 3 hours, and 6 cases with >3 hours). The tumors were assessed for HER2 gene and protein via ISH assay and IHC, and the authors concluded that cold ischemic time of up to 3 hours has no deleterious effect on the detection of HER2 via ISH or IHC. It should also be noted that the CAP/ ASCO guidelines for fixation times were addressed in regard to resection specimens, but there is no reason to believe that these cannot be applied to needle core biopsies. As a matter of fact, the guidelines should remain the same irrespective of the size of the specimen. This is because tissue permeation (~1 mm/h) is not equal to fixation. It is true that formalin will permeate core biopsy samples faster and make it harder for sectioning, but actual fixation or chemical reaction of aldehyde cross-linking takes time and is independent of specimen size. ANALYTIC ISSUES
The term analytic issues refers to the actual testing protocol; this includes IHC equipment, reagents, competency of the staff performing IHC, use of appropriate controls, and finally the type of antibody used, which deserves special mention. The very first clinical trial assay for assessing the effect of trastuzumab on metastatic breast cancer used CB11 and 4D5 antibodies for determining HER2 status. In these studies, only patients with 2+ or 3+ scores were eligible to receive trastuzumab. Retrospective analyses have revealed therapeutic benefit in cases with either a 3+ score or HER2 amplification by FISH.375 Only 24% of the 2+ cases showed amplification by FISH. At the time of FDA approval of trastuzumab, a polyclonal antibody (HercepTest) was compared with the clinical trial assay antibody CB11 by using the same scoring criteria. HercepTest received FDA approval based on its 79% concordance with CB11. However, it was soon realized and shown by studies that HercepTest had a slightly higher falsepositive rate than other monoclonal antibodies (CB11, TAB250) when compared with FISH.376,377 Even to this day, several different antibodies are being used, but all IHC 2+ cases are sent for reflex FISH testing, which in a majority of cases resolves the clinical dilemma about HER2 status. A more reliable rabbit monoclonal antibody, 4B5, has become available. In one study, Powell and associates378 showed that rabbit monoclonal 4B5 demonstrates sharper membrane staining with less cytoplasmic and stromal background staining than CB11. The major advantage of 4B5 was its excellent interlaboratory reproducibility (kappa of 1.0). POSTANALYTIC ISSUES
Postanalytic issues involve interpretation criteria, reporting methods, and quality assurance measures that
750
Immunohistology of the Breast
include competency of the interpreting pathologist. The literature regarding HER2 IHC testing suggests that a 2+ score is the most problematic379-381; however, we believe that the 1+ and 3+ scores are often misinterpreted, which has grave clinical consequences. Almost all laboratories would do FISH for HER2 gene copynumber assessment when the IHC score is 2+ but would skip HER2 FISH testing for scores of 0, 1+, or 3+.382-384 Ample data support the fact that HER2 FISH has great correlation with response to trastuzumab treatment,385,386 therefore, a 2+ HER2 IHC score coupled with HER2 FISH has no adverse clinical consequences. In contrast, a false-positive HER2 IHC score of 3+ would result in inappropriate (ineffective, expensive, and potentially harmful) therapy. Similarly, a falsenegative HER2 IHC score of 1+ would not be tested by FISH. We urge pathologists to exercise the utmost care in interpreting HER2 IHC results. The best strategy is to keep a low threshold for scoring a case as 2+ and a high threshold for scoring a case 3+. Some laboratories perform only FISH testing, but one of the drawbacks of performing FISH as the only test is that assessment of heterogeneity is better appreciated at low power on bright-field microscopy, rather than at high power, such as the examination required for FISH. The 2007 CAP/ASCO guidelines also modified the criteria by changing the number of positive cells for a 3+ score from 10% to 30%.373 Although the numerical change appears to be significant, this has had negligible practical effect. HER2 IHC heterogeneity definitely exists, but it is not common in strongly positive cases. Occasionally, a small area of clustered overexpression or amplification is present, which should be reported regardless of the percentage of positive cells. With respect to IHC, HER2 heterogeneity (with scattered positive cells) is much more common in weakly positive cases, in which the score ranges from 1+ to 2+. The change in criteria is an attempt to reduce the number of 3+ false positives, because a small percentage of cells may show intense staining as a result of edge artifacts. Moreover, scoring cases with 11% to 30% of strongly staining cells as 2+, which is considered equivocal per the 2007 guidelines, will result in additional confirmation by FISH if they are true positives. Image-analysis systems could be further used to achieve consistency in interpretation, but these instruments should be calibrated and must undergo regular maintenance just like any other laboratory equipment. Apart from judging the HER2 score, it is also important that it is effectively communicated to the treating physician. A standardized template could be used that states the time tissue was fixed, controls used, antibody used, and the HER2 IHC score with a description of the staining. Last but not least, a quality assurance program should be in place for laboratories that perform HER2 testing. Quality control procedures for HER2 IHC should include the laboratory statistics of percentage of positive cases and the percentage of IHC cases amplified by FISH. Periodic laboratory assessment of these correlations is essential for quality reporting. Rigorous adherence to quality, tissue fixation times, control tissues/ cell lines, and improved interobserver interpretation
agreement or image-assisted analysis is preferable.387-389 In addition, the CAP/ASCO guidelines recommend participation in a proficiency testing program specific to each method used.
KEY DIAGNOSTIC POINTS HER2 Immunohistochemistry • Because of its predictive value, HER2 IHC is currently the most important theranostic test for breast cancer. • Accurate assessment of HER2 status is critical, and the lessons learned from HER2 testing will be applied for assessment of future biomarkers. • Tissues should be fixed in 10% NBF for at least 6 hours (preferably 8 hours) for accurate assessment. • Choice of antibody may vary, but this should be mentioned in the report. • Scoring criteria should be rigidly followed so that there are no 3+ false positives or 1+ false negatives. • All 2+ cases should go for reflex FISH testing. • Continuous quality measures should be in place for any laboratory that performs HER2 testing.
HER2 Fluorescence In Situ Hybridization and Other In Situ Hybridization Assays Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes to detect specific DNA sequences on the chromosome. In case of HER2 FISH, a probe is used to identify HER2 gene amplification. The probe could be a single-color HER2 probe or a dual-color probe with one sequence labeled for the HER2 gene and another for the chromosome 17 centromere (chromosome enumeration probe 17 [CEP17]), on which the HER2 gene resides. For a single-color probe, an absolute HER2 gene copy number determines amplification, whereas for a dual-color probe, a ratio of HER2 to CEP17 is used to define amplification. DNA is a more robust molecule than protein, therefore HER2 gene amplification studies could be performed on a wide variety of samples. However, because of the significance of this test result, and to avoid any variability, the CAP/ASCO guidelines recommend preanalytic conditions similar to those required for HER2 IHC. In the available literature, HER2 FISH has a better track record than HER2 IHC in predicting response to trastuzumab. This may be due to several factors, including tissue fixation, criteria used to define positivity, number of antibodies used, and subjectivity in interpreting the HER2 IHC test result compared with the result of FISH. However, after years of experience with both HER2 IHC and FISH, the current CAP/ASCO guidelines demand 95% concordance (of negative and unequivocal positive results) between the two methods.373 This seems like a high number, but theoretically it is not unreasonable, because HER2 gene amplification almost always results in HER2 protein overexpression. For many genes, alternative ways of
Theranostic Applications
protein overexpression exist, but the HER2 gene is unique in the sense that its gene amplification is very tightly coupled with protein overexpression. In the past few years, it has also been realized that, just like IHC, a FISH assay may give equivocal results. Previously, HER2 gene amplification was defined as a HER2/CEP17 ratio of 2 or higher, and lack of amplification was defined as a ratio less than 2. This was also the cutoff used for the clinical trial assay. Using 2 as the cutoff makes sense, but over the years, the variability in interpretation when the value is around 2 has become apparent.390 Therefore the 2007 CAP/ASCO guidelines recommended that a ratio of 1.8 to 2.2 should be considered equivocal for HER2 gene amplification.373 A ratio less than 1.8 is negative for amplification, and a ratio greater than 2.2 is positive for amplification. If a laboratory is using a single-color probe to define gene amplification, a positive result is an average HER2 gene copy number greater than 6, a negative result is an average HER2 copy number less than 4, and an equivocal result is a HER2 gene copy number of 4 to 6. Apart from its better prediction value than IHC, FISH is also very useful when the HER2 IHC test result is equivocal; that is, when the IHC score is 2+.391 A 2+ score is seen in approximately 25% of all breast cancers,392 and it is now standard of care to study these 2+ cases by FISH to determine whether they show gene amplification. We reviewed FISH results on 186 IHC 2+ cases at our institution and found that a majority (~80%) do not show amplification (i.e., the HER2/ CEP17 ratio was <1.8). An equal number of cases (10% in each category) demonstrated equivocal (ratio of 1.8 to 2.2) or true amplification (ratio >2.2); however, it was extremely unusual, even in cases with a ratio above 2.2, for IHC 2+ cases to show large HER2 gene clusters— a characteristic of IHC 3+ cases. Although FISH is useful for clinical decision making in these cases, it appears
751
that HER2 IHC 2+ cases may be biologically different from HER2 IHC 3+ cases. Our review also showed that some of these HER2 nonamplified IHC 2+ cases contained more than three copies of HER2 and CEP17, which is indicative of polyploidy/aneuploidy for chromosome 17 as reported previously.381,393 Another caveat applies when a HER2-equivocal FISH result is obtained from FISH performed on a core biopsy or resection specimen. Striebel and colleagues394 showed that evaluating HER2 status by FISH on a larger tumor sample (resection specimen) may affect patient management if the core biopsy shows an equivocal FISH result, indicating genetic heterogeneity in tumors that show low-level HER2 gene copy numbers. In spite of its usefulness, FISH assay has some limitations, mainly related to dark field fluorescence microscopy and lack of morphologic details (Table 19-5). To overcome some of these limitations, the chromogenic in situ hybridization (CISH) method has gained popularity. A range of studies has compared FISH and CISH, showing 84% to 100% concordance.395-398 CISH uses diaminobenzidine (DAB) as the chromogen and therefore results in brown signals. It is a method that combines the expertise of a cytogenetic laboratory with IHC. This may be the reason for lack of wide acceptance for CISH, which we believe can improve with automation. Some pathologists also feel uncomfortable interpreting HER2 CISH slides when between 2 and 8 gene signals per nucleus are evident, because the signals may not be very discrete, especially when the pathologist is looking under a ×40 objective using bright-field microscopy. Silver in situ hybridization (SISH) has been developed specifically to overcome this problem. SISH uses an enzyme-linked probe to deposit silver ions from the solution to the target site, which provides a dense, punctate, high-resolution black stain that can be readily
TABLE 19-5 Benefits and Limitations of Immunohistochemistry and in Situ Hybridization Assays Immunohistochemistry
FISH
CISH/SISH
Availability of the test
Widely available
Available at major labs
Available at major labs
Microscopy
Bright field
Fluorescent
Bright field
Training for interpretation
No special training required
Special training required
Minimal training, but experience required
Amount of tumor analyzed with ease
Large tumor area can be analyzed
Generally, analysis of a small tumor area
Large tumor area can be analyzed
Morphology
Morphology well preserved
Morphology not well preserved
Morphology well preserved
Turnaround time
4 to 6 h
3 days
2 days with CISH; 1 day with SISH
Average time for interpretation
<1 min
20 min
5 to 10 min
Number of equivocal results
Approximately 25%
<5%
<5% (but limited experience)
Cost
Relatively inexpensive
Expensive
Intermediate
Automation
Possible
Possible
Possible; available with SISH
FISH, Fluorescence in situ hybridization; CISH, chromogenic (generally diaminobenzidine as chromogen) in situ hybridization; SISH, silver in situ hybridization.
752
Immunohistology of the Breast
distinguished from other commonly used stains. This SISH methodology is combined with another probe for chromosome 17 in a test recently approved by the FDA, called the INFORM Dual In Situ Hybridization (DISH) assay from Ventana (Tucson, AZ). The availability of DISH, in which the ISH signals for both chromosome 17 and HER2 are visualized on a single slide, has made this technique a more practical alternative to FISH, and it offers several advantages. These include the ability to use a standard bright-field microscope and the preservation of signals for archival review; but perhaps most importantly, it allows more reliable identification of invasive tumor cells for assessment. Bright-field techniques may also be superior in assessment of heterogeneity, especially when a clustered area of amplification is present within a tumor; this is an increasingly important issue in HER2 testing. The test performs extremely well in unequivocally positive and unequivocally negative cases (those at the extremes).399-401 However, data on difficult-to-score cases, such as those with a 2+ IHC score and cases that show aneuploidy or duplication by FISH, are somewhat limited. Therefore each laboratory should perform an internal quality assurance audit before adopting DISH for these cases. Additionally, more studies are needed on difficult-to-score cases to evaluate the performance of DISH assay in routine clinical practice. Alternative HER2 techniques have also become available and are being used in clinical testing without appropriate prospective data. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for HER2 mRNA is an attractive technique, because it is more quantitative than IHC, and mRNA measurement using expression microarrays is also possible. Both these techniques are currently utilized in multigene expression assays for prognostic and predictive purposes in breast cancer. Genomic Health (Redwood City, CA) recently started reporting ER, PR, and HER2 expression levels as a separate report to the oncotype DX test, and Agendia BV (Amsterdam, The Netherlands) offers the same with their TargetPrint assay. A corporate study from Genomic Health suggests excellent concordance between HER2 FISH and the HER2 qRT-PCR oncotype DX test.402 However, a multiinstitutional study indicates otherwise.403 Although we have found excellent concordance on IHC/FISH-negative cases, the oncotype DX HER2 assay fails to identify more than half of unequivocally HER2-positive cases. A critical review of these cases suggests either suboptimal microdissection or no microdissection at all on the tissue sections, resulting in a false-negative HER2 qRT-PCR assay. Most breast cancers generally contain an admixture of nonneoplastic tissue—lymphocytes, fibrous breast stroma, adipose breast tissue, normal breast tissue, necrosis, benign cellular proliferations, or even biopsy cavities—either admixed within invasive tumor or in the immediate vicinity. Therefore most cases will have some degree of contamination with nonneoplastic tissue that can proportionately reduce HER2 mRNA levels and result in an equivocal or negative result by RT-PCR on an unequivocally positive case (Fig. 19-41). In addition, substantial overlap of mRNA levels of HER2 can
occur in the oncotype DX test that probably result in the plethora of false-negative HER2 results (Dr. Soonmyung Paik, personal communication with D. Dabbs). In other independent study of HER2 FISH and HER2 assay by oncotype DX, Dvorak and colleagues404 found an overall percent agreement of 96% and percent negative agreement of 100%, but percent positive agreement was only 50%. Of the 194 cases in total, they found 8 FISH-positive cases not identified by oncotype DX. Of these 8 cases, three showed heterogeneous amplification, but the remaining 5 were homogeneously amplified. Most importantly, 7 of 8 cases had less than 50% invasive tumor in the tissue block sent for oncotype DX testing. The authors concluded that multiple factors contribute to this discrepancy, including a suboptimal microdissection and possibly heterogeneous amplification of HER2 gene in some cases. Pathologists and clinicians are strongly cautioned not to use oncotype DX HER2 results for clinical decision making.405,406 Others have also expressed similar opinions.407,408 KEY DIAGNOSTIC POINTS HER2 Fluorescence in Situ Hybridization • All IHC 2+ cases should go for reflex FISH testing. • The preanalytic variables are similar to IHC testing. • FISH interpretation also has an equivocal category: HER2/ CEP17 ratio of 1.8 to 2.2 or average HER2 gene copy number between 4 and 6. • Alternate ISH assays, especially silver in situ hybridization (SISH) and dual in situ hybridization (DISH), will become more widely available and acceptable. • Continuous quality measures should be in place for any laboratory that performs HER2 FISH or alternative ISH testing.
Genomic Applications of Immunohistochemistry: Breast Cancer Molecular Classification and Immunogenomics Advances in molecular genetic techniques in the new millennium have transformed the way breast cancer is studied. Gene microarray techniques that first analyzed hematologic malignancies409 are now applied to several solid cancers. The breakthrough for breast cancer microarray studies occurred in 2000, when Perou and colleagues134 reported molecular classification of breast cancer using gene-expression analysis on DNA microarrays.
Molecular Classification: “Intrinsic” Gene Set Perou and associates134 used a complementary DNA (cDNA) microarray to study 8102 human genes on 65 surgical specimens (36 IDCs, 2 lobular carcinomas, 1 DCIS, 1 fibroadenoma, 3 normal breast tissues). Of the 36 IDCs, 20 tumors had samples obtained before and
Genomic Applications of Immunohistochemistry: Breast Cancer Molecular Classification and Immunogenomics
A
C
after doxorubicin chemotherapy, and two additional tumors were paired with lymph nodes. These paired samples were a unique feature of their study design, because in spite of chemotherapy and a time interval of 16 weeks between them, these paired samples clustered together in the hierarchical clustering algorithm. These findings implied that each tumor is unique and has a distinct gene-expression signature. It also implied that the type and number of nonepithelial cells in carcinoma remains fairly constant and does not interfere in expression analysis. Therefore the authors selected a subset of 496 genes, which showed significantly greater variation in expression among different tumors than between paired samples of the same tumor. This subset was called the “intrinsic” gene set, because it consisted of genes whose expression patterns were characteristic of an individual tumor, as opposed to those that vary as a function of tissue sampling. The same investigative group further extended the analyses to 115 carcinoma samples to reveal five distinct classes of breast carcinoma with prognostic significance.135,143 These were termed luminal A, luminal B (which likely also includes the initially described luminal C), ERBB2, basal-like, and normal breast–like. The luminal tumors were named as such because of the high expression of genes
753
B
Figure 19-41 This invasive ductal carcinoma with abundant intratumoral lymphocytic infiltrate (A) showed unequivocal protein overexpression by immunohistochemistry (B) and unequivocal gene amplification by fluorescence in situ hybridization (C). It was reported to be equivocal for HER2 by oncotype DX assay.
normally expressed by luminal epithelium of the breast. These luminal tumors also expressed ER and ER-related genes (SLC39A6 [formerly LIV1], GATA3, FOXA1 [formerly HNF3A], XBP1), and luminal A tumors showed the highest expression of ER. Luminal B tumors express ER cluster genes at a lower level but also express some unique genes (GGH, LAPTM4B, YBX1 [formerly NSEP1] CCNE1) the coordinated function of which is unknown. The other three subtypes constitute the ER-negative group. The ERBB2 tumors, or HER2enriched tumors, as the name implies, are characterized by expression of genes in the ERBB2 or HER2 amplicon at 17q22.24. The basal like tumors are named as such because they express genes expressed by the basal cells (MECs) of the breast. Therefore basal-like tumors are characterized by expression of keratin genes KRT5 and KRT17. The normal breast like group has been described to express genes known to be expressed by adipose tissue and other nonepithelial cell types. These tumors were also shown to express basal epithelial genes. However, it is now considered to be an artificial category because of poorly sampled tumor tissue.410 Over the years, the validity of this classification system has been tested with respect to overall and relapse-free survival.143 The basal-like and ERBB2 tumors have shown
754
Immunohistology of the Breast
the worst outcome, luminal A has shown the best outcome, and luminal B is intermediate. Given the simplicity and the clinical relevance of the molecular classification, IHC markers have been used to identify molecular subtypes in routine diagnostic use. A few studies have detailed the use of IHC in identifying a particular molecular subtype.136,137,141 It is true that analyzing a few biomarkers by IHC cannot be directly compared with a classification that analyzes expression of hundreds and thousands of genes. But it is also true that we have gained enough experience over a number of years in analyzing some key biomarkers that we can predict a molecular class quite accurately. A tumor that demonstrates 3+ IHC staining for HER2 and is completely negative for ER and PR very likely belongs to the HER2-enriched or ERBB2 molecular class. However, we also realize that a 100% concordance cannot be achieved between the molecular classification (using the intrinsic gene set) and IHC criteria, but a working formulation for use in routine pathology practice is feasible. This correlation is further discussed below.
Luminal Tumors Based on the published literature, it appears that ER-positive tumors, those with at least 10% of ER-positive cells, are luminal tumors.411-413 A majority of these tumors are slow growing, have good prognosis, and respond quite effectively to hormonal therapies; these tumors constitute the group known as luminal A tumors. The remainder of the ER-positive tumors are comparatively faster growing and still benefit from hormonal therapies, but a certain proportion may derive huge benefit from additional chemotherapy. These tumors are generally termed luminal B. Some investigators have proposed pure IHC criteria (Table 19-6) to distinguish luminal A and luminal B tumors.354 Instead of these rigid criteria, a more pragmatic approach is to consider all factors together; that is, if an ER-positive/HER2-negative tumor is well differentiated (Nottingham grade I), shows diffuse strong ER and Bcl-2 positivity with a low (preferably <10%) Ki-67 labeling index (LI), and expresses PR, FOXA1,
TABLE 19-6 Molecular Classes and Corresponding Immunohistochemistry Categories* Molecular Class
Immunohistochemistry Criteria as per Current Literature
Luminal A
ER/PR+ (at least 20% of cells +), HER2−, Ki-67 LI <14%
Luminal B
ER+, PR− (or PR+ with <20% cells +), HER2− or +, Ki-67 LI ≥14%
HER2 enriched
HER2+, ER/PR−
Basal-like
ER/PR−, HER2−; majority are positive for basal markers
*Simplified criteria. ER, Estrogen receptor; LI, labeling index; PR, progesterone receptor.
and GATA3, it is certainly a luminal A tumor (see the discussion of FOXA1, GATA3, and BCL2 later in the chapter). In contrast, if an ER-positive, HER2-negative tumor is poorly differentiated (Nottingham grade III), shows weak ER expression, is Bcl-2 negative, has a high Ki-67 LI, and shows lack of expression for PR, FOXA1 and GATA3, it is certainly a luminal B tumor. However, this distinct separation will not be seen in all of the ER-positive/HER2-negative tumors. For these equivocal cases, using all the available information to make a clinical judgment would be more helpful than relying on one criterion to classify a tumor as luminal A or B. Regarding tumors that coexpress HRs and HER2, they do cluster together with luminal B tumors but they should be classified as luminal positive, HER2 positive for treatment purposes. At the current time, it is difficult to justify using PAM50 or other multigene prediction assays for all of these cases.
Triple-Negative or Basal-Like Tumors The initial attempts to extrapolate molecular findings to morphologic and IHC criteria focused mainly on basal-like breast carcinomas. Morphologically, basal-like breast carcinomas are often, but not always, fairly wellcircumscribed, high-grade tumors with abundant lymphoplasmacytic infiltration and geographic necrosis.136 Immunohistochemically, they often lack HR expression and are also negative for HER2. They are considered a basal-like carcinoma because they express basal-phenotype markers such as high-molecular-weight cytokeratins 5, 5/6, 14, and 17 and also express EGFR. Based on patient demographics, association with BRCA1 mutants, and morphologic and IHC features, it appears that basal-like breast carcinoma comprises a majority of medullary and atypical medullary carcinomas.414-419 In spite of this almost perfect correlation, one area of concern is the poor prognosis of basal-like carcinomas as reported by gene-expression studies compared with the relatively good prognosis of pure medullary carcinomas in the past. However, if strict criteria are applied for the diagnosis of medullary carcinoma, it is a rather rare entity (<1% of all breast tumors) compared with the entire group of basal-like breast carcinoma, which comprises almost 15% of all breast tumors. It is conceivable that medullary carcinoma is a rare subtype of basallike breast carcinoma with a fairly good prognosis. Although a vast majority of so-called triple-negative tumors—those negative for ER, PR, and HER2— demonstrate expression of basal-phenotype markers, current debate centers on whether these additional markers have any prognostic value and whether they should be performed in routine practice. In an IHC study by Jumppanen and colleagues,420 the basal-like phenotype was not associated with patient survival in ER-negative breast cancer. However, the number of tumors that express basal-like markers in triple-negative tumors was much lower than expected in this study. In contrast, Cheang and associates421 showed the prognostic significance of basal-phenotype markers in triplenegative tumors. The 10-year breast cancer–specific survival was worse for triple-negative tumors that
Genomic Applications of Immunohistochemistry: Breast Cancer Molecular Classification and Immunogenomics
expressed basal-phenotype markers compared with triple-negative tumors negative for basal markers (62% vs. 67%). It appears that evaluating basal phenotype markers by IHC may provide some prognostic information, but at the current time, no specific therapy is available based on the presence or absence of these markers.
ERBB2 (or HER2-Enriched) Tumors As the name suggests, these tumors are HR negative and HER2 positive and should be easy to identify, because all primary breast cancers are examined for HRs and HER2. However, concern has been raised that IHCbased categorization does not definitively identify the molecular classes defined by intrinsic gene set–based expression analysis. Specifically, it has been mentioned that the ERBB2 tumor class consists of some tumors that are clinically HER2 negative. There could be different reasons for this discrepancy, such as heterogeneity for HER2 overexpression or amplification or some truly HER2-negative apocrine tumors that are generally negative for ER and PR but express androgen receptor (AR).422 These triple-negative apocrine tumors likely cluster with the ERBB2 (HER2-enriched) class. Moreover, the triple-negative apocrine tumors appear morphologically more similar to ERBB2 tumors (ER/ PR− and HER2+) than to usual triple-negative basal-like tumors. Farmer and colleagues423 described the molecular apocrine tumors and suggested a simple IHC classification, based on their expression-profiling experiments, in which they considered luminal tumors to be AR and ER positive, basal tumors to be AR and ER negative, and molecular apocrine tumors to be AR positive and ER negative. Other molecular and IHC studies also suggest that molecular apocrine tumors are either ER/PR negative and HER2/AR positive or ER/PR and HER2 negative and AR positive, and they show significant overlap with intrinsic molecular class ERBB2 tumors.135,143 These findings likely explain the inclusion of some clinically HER2-negative tumors within the ERBB2 intrinsic molecular class. Conversely, Parker and colleagues424 also showed that 11% of ER-positive tumors clustered within the ERBB2 molecular class. The exact reason is not known, but differences in the clinical cut points to define ER positivity (i.e., tumors that express very low ER but are considered clinically ER positive) is likely the most reasonable explanation for this discrepancy. Although distinct molecular classes are identified by gene-expression profiling, some tumors always form a continuum between two distinct classes. The molecular classification using the intrinsic gene set was derived mainly by analyzing IDC of no special type, but we have no reason to believe that it cannot be applied to other morphologic tumor types. As a matter of fact, Weigelt and colleagues425 reported molecular characterization of the various histologic subtypes of breast cancer. In this study, the molecular classes— luminal, ERBB2, and basal-like—correlated with IHC surrogate markers of the ER, PR, HER2 protein–negative expression profile; the triple-negative adenoid cystic, medullary, and metaplastic tumors clustered with the
755
usual type of basal-like carcinomas; and the ER-positive, HER2-negative tumors such as tubular, mucinous, and classic lobular carcinomas clustered together as luminal tumors. One important use and validation of IHC-based molecular classification is exemplified in predicting response to NACT. Gene-expression–based studies have shown that pathologic complete response (pCR) is seen mainly in basal-like and ERBB2 (HER2-enriched) molecular classes; only rare tumors in the luminal categories show pCR.426 In a single-institution study of 359 cases, we showed that an IHC-based classification using semiquantitative ER, PR, and HER2 results provides similar information.346 In this study, pCR was identified in 33% (19/57) of ERBB2 tumors; 30% (24/79) of triple-negative tumors; 8% (2/24) of weak ER-positive, HER2-positive tumors; and in 1.5% (3/198) of the other ER-positive tumors. The mean tumor size reduction was also higher in ERBB2 and triple-negative tumors compared with other classes. The data suggest that pCR and average tumor size reduction may be related to the amount of ER expression and presence of HER2 overexpression. The usefulness of these IHCderived classes has also been documented with respect to targeted therapy. In a study of over 100 cases of HER2-positive tumors predominantly treated with docetaxel, carboplatin, and trastuzumab NACT, or TCH, we have shown that the amount of trastuzumab benefit varies with tumor HR content.347 In contrast to our prior study, pCR and percentage tumor-volume reduction was significantly improved in all HER2positive tumors. The pCR rates in ER negative, HER2 positive; weakly to moderately ER and HER2 positive; and strongly ER and HER2 positive tumors were 52%, 33%, and 11% respectively—an increase of approximately 10% to 20% in all categories. Similar changes were observed with respect to percentage tumor-volume reduction, indicating that response to the current chemotherapeutic regimen is inversely related to tumor HR content. As a matter of fact, a metaanalyses based on more than 30 studies on the subject showed a pCR rate of 8%, 19%, 39%, and 31% for ER+/ HER2−, ER+/HER2+, ER−/HER2+, and triple-negative tumors, respectively.427 In summary, these studies suggest that carefully conducted IHC analyses can provide useful clinical and predictive information in a majority of cases. Apart from the intrinsic gene set–based expression analysis of breast cancer, several other expression studies that used different models were published in the last decade. These included 70-gene profile, wound response, Rotterdam signature, recurrence score, and two-gene ratio, and most of them have been commercialized. Mammostrat (Clarient/GE, Aliso Viejo, CA; discussed below under Other Tumor Markers) is an IHC-based model developed to distinguish between luminal A and luminal B tumors.
70-Gene Profile (MammaPrint) The 70-gene, good-outcome versus poor-outcome model was developed by van de Vijver and colleagues428
756
Immunohistology of the Breast
and van’t Veer and associates.429 The authors used oligonucleotide array to identify genes that predict prognosis in breast cancer and estimated that an odds ratio for metastasis among tumors with a “good prognosis” gene signature as compared with a “poor prognosis” gene signature was approximately 15 using a crossvalidation procedure. The poor-prognosis signature consisted of genes that regulate cell cycle, invasion, metastasis, and angiogenesis. They further studied 295 cases of breast cancer from young patients, including cases graded on the tumor/node/metastasis scale as pT1 and pT2 with (n = 144) or without (n = 151) lymph node metastasis. Of the 295 cases, 180 showed poor and 115 showed good prognosis profiles, and the mean (standard error) overall 10-year survival rates were 54.6% (±4.4%) and 94.5% (±2.6%) respectively. The estimated hazard ratio (HR) for distant metastases with the poor-prognosis signature as compared with the group with the good-prognosis signature was 5.1. This ratio remained significant when the groups were analyzed with respect to the lymph node status. This assay has now formed the basis of a commercial test called MammaPrint (Agendia BV).430 The test has been cleared by the FDA for clinical use, however, the ASCO guidelines committee for tumor markers in breast cancer judged that more evidence is required for advocating use in clinical practice.431 Previously, the test required fresh-frozen tissue containing at least 30% of the invasive tumor, but now the test can be performed on FFPE tissue material. To compete with other commercial assays, the company also offers TargetPrint, by which ER, PR, and HER2 gene expression levels are reported. The company additionally offers the BluePrint assay, which uses an 80-gene signature to classify breast cancer into Basal, Luminal, and ERBB2 molecular classes.
Wound Response Gene Set The wound response model for predicting breast cancer prognosis was described by Chang and associates.432 The authors used the same set of 295 cases used for validating the 70-gene profile. Breast cancer samples showed predominant expression of either serum-induced or serum-repressed genes, which allowed the investigators to assign each sample to an activated or quiescent wound-response signature. Patients with the activated wound-response signature (126/295, or 42.7%) had a significantly decreased distant metastasis–free probability and overall survival in univariate analysis. Even when the analysis was extended to different pathologic subsets by way of pT1 tumors, lymph node–positive tumors, and lymph node–negative tumors, the results remained the same: patients with tumors that showed an activated wound-response signature had significantly worse distant metastasis–free probability and overall survival compared with those who had a quiescent wound signature. Based on this study and the authors’ prior studies on other epithelial tumors, they concluded that physiologic response to a wound is frequently activated in common human epithelial tumors, and it confers increased risk of metastasis and cancer progression.
76–Gene Profile (Rotterdam Assay) The Rotterdam signature is also known as the 76-gene profile/assay and was developed at the Erasmus University Medical Center in Rotterdam, The Netherlands, in collaboration with Veridex (Warren, NJ). Using the Affymetrix Human U133a GeneChips, Wang and colleagues433 analyzed the expression of 22,000 transcripts in a series of 286 lymph node–negative patients who had not received adjuvant systemic treatment. Of these 286 tumors, 115 were used as a training set to identify 76 genes (60 ER+, 16 ER−) with expression levels that correlated with distant metastasis within 5 years. Although genes involved in cell death, cell proliferation, and transcriptional regulation were found in both groups of patients stratified by ER status, the 60 genes selected for the ER-positive group and the 16 selected for the ER-negative group had no overlap. The remaining 171 tumors were used as the testing set. The 76-gene profile was highly informative in identifying patients who developed distant metastases within 5 years (HR 5.67; 95% confidence interval [CI] 2.59 to 12.4). This signature showed 93% sensitivity and 48% specificity. When this testing set of 171 patients was divided into 84 premenopausal and 87 postmenopausal patients, the 76-gene profile was still a strong prognostic factor for the development of metastasis. Similar results were obtained when 79 patients with pT1C tumors were analyzed. The Rotterdam assay was further validated in a multicenter study of lymph node–negative patients.434 The developmental history of the 76-gene profile is very similar to that of the 70-gene profile, but the two tests are directed at a different patient population. The 76-gene profile was developed for lymph node–negative patients irrespective of the hormone status and patient age. However, similar to the 70-gene profile, the Rotterdam assay is an oligonucleotide array–based test that requires fresh-frozen tissue for analysis. It is also interesting to note that there is only a three-gene overlap between the MammaPrint and the Rotterdam assays. At present, a commercial assay based on the 76-gene profile is not available.
Recurrence Score Model (Oncotype DX) The recurrence score model is better known as oncotype DX, a commercially available RT-PCR–based assay that provides a recurrence score (RS) and has been shown to provide prognostic and predictive information in ER-positive lymph node–negative breast cancers.435 The test analyzes the expression of 21 genes, 16 cancerrelated and 5 control genes, to give a distant-disease RS that ranges from 0 to 100. The RS was created by using training sets and a proprietary analytic method. The oncotype DX RS was originally validated in 668 lymph node–negative, ER-positive breast cancer patients receiving tamoxifen in NSABP trial B-14, in which a multivariate analysis of patient age, tumor size, tumor grade, HER2 status, HR status, and RS demonstrated that only tumor grade and RS were significant predictors of distant recurrence; RS also correlated significantly with the relapse-free interval and overall survival. The RS was
Genomic Applications of Immunohistochemistry: Breast Cancer Molecular Classification and Immunogenomics
subsequently validated as a predictive marker for response to chemotherapy and tamoxifen in 651 patients in NSABP trial B-20 and 645 patients in B-14.436 The 16 genes analyzed by the test can be categorized into five groups: 1) the estrogen group comprises ESR1, PGR, BCL2, and SCUBE2; 2) the HER2 group includes GRB7 and, of course, HER2; 3) the proliferation group comprises Mki67, AURKA (formerly STK15), BIRC5 (formerly Survivin), CCNB1, and MYBL2; 4) the invasion group includes MMP11 and CTSL2; and 5) a group of others includes GSTM1, CD68, and BAG1. Because ESR1, PGR, and HER2 genes are already analyzed by either protein expression or gene amplification, and pathologists analyze the morphologic expression of the proliferation genes by the mitotic count, we examined the relationship between traditional histopathologic variables and the RS in our pilot series of 42 cases. We found that RS significantly correlated with tubule formation, nuclear grade, mitotic count, ER IHC score, PR IHC score, and ERBB2 status. We also found that the equation RS = 13.424 + 5.420 (nuclear grade) + 5.538 (mitotic count) − 0.045 (E ER IHC score) − 0.030 (PR IHC score) + 9.486 (ERBB2)
predicts the RS with an R2 of 0.66, indicating that the full model accounts for 66% of the data variability.437 Using a database of more than 800 cases with oncotype DX test results, we developed three more equations—new Magee equations (nMEs)—that can predict oncotype DX recurrence score.438 These are represented below. nME 1: Recurrence Score = 15.31385 + (Nottingham score × 1.4055) + [ER IHC × ( −0.01924)] + [PR IHC × ( −0.02925)] + (0 for HER2 negative, 0.77681 for equivocal, 11.58134 for HER2 positive) + ( Tumor size × 0.78677) + (Ki-67 index × 0.13269) nME 2: Recurrence Score = 18.8042 + (Nottingham score × 2.34123) + [ER IHC × ( −0.03749)] + [PR IHC × ( −0.03065)] + (0 for HER2 negative, 1.82921 for equivocal, 11.51378 for HER2 positive) + ( Tumor size × 0.04267) nME 3: Recurrence Score = 24.30812 + [ER IHC × ( −0.02177)] + [PR IHC × ( −0.02884)] + (0 for HER2 negative, 1.46495 for equivocal, 12.75525 for HER2 positive) + (Ki-67 × 0.18649)
These four equations, the original and the three new equations, have been validated in 255 separate cases. The concordance between actual oncotype DX RS and the estimated recurrence score calculated from Magee equations with respect to categorization ranged from 54.3% to 59.4%, and the highest concordance was obtained with nME 2. The Pearson correlation coefficient between estimated and actual RS was similar for each of the equations (0.60404, 0.61661, 0.60386, and 0.59407 for the original Magee equation and nMEs 1, 2, and 3, respectively). With exclusion of the intermediate risk categories for both the actual recurrence score and estimated recurrence score, the concordance for each equation increased to more than 95%, reflecting
757
the very low two-step discordance (concordance 96.9% [95/98], 100% [76/76], 98.7% [75/76], and 98.8% [79/80] for the original Magee equation and nMEs 1, 2, and 3 respectively). Even when the estimated RS fell in the intermediate category with any of the equations, the actual RS was either intermediate or low in more than 80% of the cases. These results suggest that if an estimated RS falls clearly in the high-risk or low-risk category, then oncologists should not expect a dramatically different result from oncotype DX. Moreover, any unusual/unexpected result from oncotype DX should be thoroughly investigated by the pathologist.
Two-Gene Ratio, HOXB13:IL17RB Index, and Molecular Grade Index The two-gene ratio model analyzes the expression of HOXB13 and IL17RB genes. In the initial cohort of 60 tamoxifen-treated patients, Ma and colleagues439 identified HOXB13, a homeodomain–containing protein, and IL17RB, interleukin 17 receptor B, which were significantly associated with clinical outcome. The authors hypothesized that a two-gene expression index (HOXB13:IL17RB index, or H/I index) might be a novel biomarker for predicting treatment outcome in tamoxifen monotherapy. They further tested their hypothesis on 852 FFPE primary breast cancers from 566 untreated and 286 tamoxifen-treated breast cancer patients using a qRT-PCR technique.440 They found that expression of HOXB13 was associated with shorter recurrence-free survival (P = .008), and expression of IL17RB was associated with longer recurrence-free survival (P < .0001). In ER-positive patients, the H/I index predicted clinical outcome independently of treatment but predicted it more strongly in nodenegative patients. In spite of these validation assays, a comparative study of five-gene expression–based assays, Fan and colleagues441 found concordance among four tests— intrinsic gene set, RS, 70-gene profile, and woundresponse gene set—but not with the two-gene ratio. Therefore to improvise on the two-gene ratio test, the same group of investigators then described what they referred to as a molecular grade index (MGI) by selecting five cell-cycle related genes to be used concurrently with the H/I index to improve risk stratification.442 Using their previously published gene-expression database on preinvasive and invasive lesions that showed differential gene expression within higher grade and lower grade lesions,443 the investigators selected five genes—BUB1B, CENPA, NEK2, RACGAP1, and RRM2—based on functional annotation, tumor grade, and clinical outcome. A numerical score (MGI) was created by using these five genes, and the prognostic performance of the MGI was tested by using two independent, publicly available microarray datasets to predict clinical outcome. The five-gene MGI was also compared with the previously described 97-gene genomic grade index (GGI) by Sotiriou and colleagues444 and was found to be equivalent. Interestingly, the five
758
Immunohistology of the Breast
genes of the MGI were part of the 97-gene GGI. Ma and associates442 developed an RT-PCR–based assay for calculating MGI so that the assay could be easily applied to FFPE tissues and further described the complementary value of MGI and H/I to stratify patients into risk groups (similar to oncotype DX) to determine 10-year distant metastasis–free survival probability. One primary reason MGI and H/I are complementary is due to the fact that a high proliferation rate (high MGI) and decreased cell death (high H/I index) promote aggressive tumor growth in a synergistic manner. The combined MGI and H/I assay is now commercially offered as the Breast Cancer Index (BCI) by bioTheranostics (San Diego, CA). Gene-expression studies have improved our understanding of breast carcinoma, but it is difficult to integrate all this data into current practice. The intrinsic gene set–based classification is simple to follow, and key biomarkers assessment by IHC can predict the molecular class with high confidence. Other gene expression– based assays may be used in special circumstances, when clinical decision making is not so straightforward using conventional parameters. These gene-expression models are summarized in Table 19-7.
Other Tumor Markers TP53 TP53 is a tumor suppressor gene commonly mutated in several human cancers. In routine diagnostic surgical pathology, mutation status is assessed based on its expression by IHC. In some tumor types, such as bladder carcinomas, it has been shown that diffuse, strong p53 expression by IHC correlates with TP53 mutation by molecular techniques.445,446 The commonly used antibody clone DO7 recognizes both wild-type and mutant p53 proteins. However, because the mutant protein has a longer half-life than the wild-type protein, the mutant protein is more diffusely and intensely stained on IHC. Therefore weak or patchy staining with p53 antibodies should be considered a negative result (i.e., wild-type p53). Some of the confusion regarding p53 staining in breast and other cancers stems from the interpretation. In any event, p53 staining has been associated with poor prognosis in breast carcinoma. With our continued improved understanding of breast carcinoma, we now know that a large majority of tumors that demonstrate
TABLE 19-7 Gene Expression Models Described for Use in Breast Carcinoma Expression Models
Test Purpose
Method
Classes
Test Name
Intrinsic subtype
Molecular classification for all breast carcinomas
cDNA microarray, commercial assay uses RT-PCR for 50 genes
Luminal A, luminal B, HER2-enriched, basal-like
PAM50 (ARUP Laboratories, Salt Lake City, UT)
70-gene profile
Prognostic for young patients, pT1 or pT2 lymph node– negative tumors, also predictive use
Oligonucleotide microarray
Good prognosis vs. poor prognosis
MammaPrint (Agendia BV, The Netherlands)
Wound response
Prognostic; improved risk stratification
Oligonucleotide microarray
Quiescent vs. activated
NA
76-gene profile
Prognostic for lymph node– negative tumors irrespective of HR status
Oligonucleotide microarray
Good prognosis vs. poor prognosis
Rotterdam Assay (not commercially available)
Recurrence score (RS)
Prognostic use in ER-positive, lymph node–negative tumors; predictive for chemotherapy use
qRT-PCR
Low risk (RS <18) Intermediate risk (RS 18 to 30) High risk (RS ≥31)
oncotype DX (Genomic Health, Redwood City, CA)
2-gene ratio*
Prognostic use in ER-positive, lymph node–negative tumors
qRT-PCR
Low vs. high ratio
Theros H/I (bioTheranostics, San Diego, CA)
5-gene index*
Objective measurement of tumor grade and chemotherapy prediction in ER-positive tumors
qRT-PCR
High or low
Theros MGI (bioTheranostics)
*2-gene ratio (Theros H/I) is now tested in combination with the 5-gene index (Theros MGI) as a combined assay, bioTheranostics’ breast cancer index (BCI), to stratify patients into low (score <5), intermediate (≥5 to <6.4), and high (≥6.4) categories for risk of recurrence. cDNA, Complementary DNA; ER, estrogen receptor; FFPE, formalin-fixed paraffin-embedded; HR, hormone receptor; MGI, molecular grade index; NA, not available; pT, pathologic tumor stage; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; RS, recurrence score.
Other Tumor Markers
strong p53 immunoreactivity or show TP53 mutations belong to the basal subtype.143 The clinical usefulness of TP53 mutation analysis or diffuse strong immunoreactivity in nonbasal tumors has not been well studied. However, a recent study showed that TP53 gene abnormalities, as defined by sequencing, were associated with worse prognosis and that TP53 mutations and deletions were particularly prognostic in node-negative, ER-positive patients.447 However, the present data are insufficient to recommend use of p53 measurements for management of patients with breast cancer as per the 2007 ASCO tumor guidelines for breast cancer.431
Mammostrat Panel Mammostrat is a commercial assay that comprises a fivemarker IHC panel that includes TP53, NDRG1, CEACAM5, SLC7A5, and TRMT2A (formerly HTF9C); developed to assess prognosis in patients with early stage ER-expressing breast cancer, it was tested further in several institutional cohorts, largely in the setting of adjuvant tamoxifen administration.448-450 The markers used in the panel were purposely selected to be distinct from conventional breast cancer markers. They represent components of DNA damage and cell cycle (TP53, TRMT2A), stress response (NDRG1), and nutritional (SLC7A5) and differentiation (CEACAM5) pathways linked to cell growth and differentiation. Their expression is qualitatively assessed and then combined into a quantitative risk index for recurrence by using a defined mathematic algorithm to identify individuals with low (≤0), moderate (>0 and ≤7), or high (>7) risk of recurrence. This algorithm is very similar to the more commonly used 21-gene test, the oncotype DX assay, and the less well-known seven-gene test, bioTheranostics’ BCI. However, a direct comparison between these assays has not been performed, and it remains to be seen as to how Mammostrat will perform in routine clinical practice.451
Ki-67 Numerous studies have been published regarding proliferation activity of breast carcinomas, many of which date back to the era before expression profiling. Investigators have used either flow cytometry to determine S-phase fraction or IHC to study expression of proliferating cell nuclear antigen (PCNA) or Ki-67.452,453 Good correlation has been found among different methodologies. Many studies that analyzed Ki-67 labeling index (LI) have shown high LI to be a poor prognostic factor in breast cancer.454,455 However, different cut-off points have been used to define a high proliferation index. In addition, various techniques have been used to determine the LI. Because of these factors, it is somewhat difficult to compare these studies, which likely explains the reluctance to universally accept Ki-67 LI as a prognostic marker in breast cancer. In a thorough review of 132 articles that included data on 159,516 patients in regard to the prognostic and predictive value of Ki-67 and other proliferation markers (cyclin D, cyclin E, p27, p21, tyrosine kinase [TK], and topo 2-α), Colozza and colleagues456 appropriately
759
pointed out that all studies concerning these markers represent level IV or III evidence at best (level I or II evidence is required for use in clinical practice), and they demonstrate the difficulty in interpreting the literature as a result of lack of standardization of assay reagents, procedures, and scoring. Therefore the authors recommended not using these markers in routine clinical practice, a view also endorsed by the 2007 ASCO tumor guidelines for breast cancer.431 The scoring of Ki-67 LI is also compounded by the lack of consensus as to which area of the tumor should be counted: should it be the entire tumor section, the advancing edge of the tumor, or the area within the field of highest proliferative activity? Depending on where the pathologist chooses to count, and regardless of the manual or image-analysis method, variability in Ki-67 will be expected. In spite of the above argument, it is interesting to note that the very first gene-expression profiling study not only revealed “molecular portraits” but also identified genes responsible for the biologic differences among the tumor types.134,457 One of the largest distinct gene clusters identified by expression profiling was of the proliferation genes and included both PCNA and Ki-67. Subsequently, a few studies have primarily focused on the issue of Ki-67 LI and its correlation to all the molecular classes. We examined the Ki-67 LI using image analysis in approximately 200 consecutive breast carcinomas divided into molecular class using IHC criteria.411 It was interesting to note that the average Ki-67 LI was highest in triple-negative tumors, and most tumors showed an index greater than 50%. The ER-negative, HER2-positive tumors were a distant second, followed by HR-positive tumors. Although the mean Ki-67 LI was low in HR-positive tumors, not all tumors had low Ki-67 LI and showed a wide range. This difference in proliferation activity coupled with quantitative differences in ER expression has been exploited in the development of a commercial assay (oncotype DX) for predicting breast cancer prognosis and treatment.435 Cheang and colleagues412 have also used Ki-67 proliferation index (cut off at 14%) to distinguish between luminal A and luminal B tumors. Although not the most robust prognostic or predictive marker, Ki-67 LI is an additional piece of information that can be used in clinical decision making, provided the physician understands the limitations of the test and the test result. To achieve a uniform methodology and create greater interlaboratory and interstudy comparability, the international Ki-67 in Breast Cancer working group met on March 12, 2010, and published their recommendations in 2011.458 Although this is a work in progress, the current recommendation is to calculate Ki-67 LI within tumor fields representative of the initial overview of the whole section. The panel has further recommended counting 500 to 1000 tumor cells, which is not practical in routine practice. However, we believe that better image-analysis systems that can perform whole-slide imaging and provide automated counts on a large number of tumor cells will be helpful in standardizing some of these issues.
760
Immunohistology of the Breast
Epidermal Growth Factor Receptor The epidermal growth factor receptor (EGFR; HER-1, c-erbB-1) is one of the four transmembrane growth factor receptor proteins that share similarities in structure and function. Using a criterion similar to that for assessing HER2 IHC expression, EGFR overexpression in breast carcinoma is seen in less than 10% of all tumors.459 The best correlation of EGFR increased genecopy number is with a 3+ IHC score. Breast tumors that show EGFR expression or overexpression are generally negative for steroid HRs. With our current understanding of breast carcinoma molecular classification, EGFR expression would be predominantly seen in basal-like breast carcinomas, therefore it has been proposed to use EGFR along with CK5/6 in identifying basal-like carcinomas.137,421 It seems as though EGFR does have a diagnostic use in breast pathology. As far as prognostic and predictive value are concerned, the role of EGFR IHC is unknown. As per our understanding from lung carcinoma studies, the tumors that are responsive to small molecule tyrosine kinase inhibitors demonstrate mutations in exon 19 and 21 of EGFR. Such mutations have not been identified in breast carcinomas.459,460 In colon carcinoma, use of an EGFR inhibitor (cetuximab) was initially based on EGFR expression. However, subsequent studies have found no correlation between EGFR expression and response to cetuximab. Whether cetuximab therapy would have a role in breast cancer, especially basal-like cancer, remains to be seen.461,462 Until further clinical trials and additional studies have been done, the role of EGFR is limited to diagnostic use only.
Urokinase Plasminogen Activator and Plasminogen Activator Inhibitor 1 Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) are part of the plasminogen activating system, which includes the receptor for uPA and other inhibitors (PAI-2 and PAI-3). This system has been shown experimentally to be associated with invasion, angiogenesis, and metastasis.463 Low levels of both markers are associated with a sufficiently low risk of recurrence, especially in HR-positive women who will receive adjuvant endocrine therapy, such that chemotherapy will only contribute minimal additional benefit. Furthermore, cytoxan, methotrexate, and 5fluorouracil–based adjuvant chemotherapy provide substantial benefit, compared with observation alone, in patients with high risk of recurrence as determined by high levels of uPA and PAI-1. Although any technique— IHC, RT-PCR, or enzyme-linked immunosorbent assay (ELISA)—could be used to determine levels of uPA and PAI-1, the outcome is best correlated with ELISA.464-466 Unfortunately, IHC results do not reliably predict outcomes, and the prognostic value of ELISA by using smaller tissue specimens, such as tissue collected by core biopsy, has not been validated.467 We believe the clinical utility of this test is limited, because the requirement for availability of 300 mg of fresh or frozen breast tumor would be a severe
impediment in this era of mammographically or MRIdetected cancers.
Insulin-Like Growth Factor Receptor 1 Insulin-like growth factor receptor 1 (IGF-1R) is an integral part of the IGF system that plays an important role in neoplastic processes.468 The IGF family includes the two ligands, IGF-1 and IGF-2; two cell-surface receptors, IGF-1R and IGF-2R; and a family of six IGF binding proteins (IGFBPs) that regulate free IGF levels. Of the entire IGF system, IGF-1R appears to be the most critical molecule that can be analyzed in tumor samples. Moreover, with the advent of IGF-1R–targeted therapies, it may be useful to analyze IGF-1R expression levels in the tumor tissue. Studies to evaluate tissue expression of IGF-1R have shown expression in a significant number of breast carcinomas.469-471 In our own study of IGF-1R expression in normal breast tissue, proliferative breast lesions, and breast carcinomas, we found that even normal breast ducts and lobules also express membranous IGF-1R to a moderate degree.472 Defining underexpression, normal expression, and overexpression in comparison to normal breast tissue, IGF-1R overexpression was predominantly seen in ER-positive tumors. The tumor group that consistently showed reduced expression was the ERBB2 group (ER−/PR−/HER2+). The expression was somewhat heterogeneous in the triple-negative group. IGF-1R expression was not predictive of pCR or tumor volume reduction in ER-negative tumors, but reduced IGF-1R was associated with pCR and significant tumor-volume reduction in ER-positive tumors.472 Therefore we believe therapies that target IGF-1R should first be tried on ER-positive tumors and on a subset of triple-negative tumors that overexpress IGF-1R.
BCL2 BCL2 is an antiapoptotic gene expressed in approximately 75% of breast carcinoma, and expression level correlates with ER expression.473-476 An additional role for BCL2 in breast tumor prognostication has been described. Abdel-Fateh and colleagues477 proposed a modified grading system that combines mitotic index and BCL2 reactivity that can be applied to both ER-positive and ER-negative tumors and would be helpful in eliminating or reducing the number of tumors classified as Nottingham grade 2, in which clinical decision making is often difficult. The investigators divided the tumors into low-risk and high-risk categories based on mitotic activity score (M1 = low, <10 mitoses; M2 = medium, 10 to 18 mitoses; M3 = high, >18 mitoses per 10 hpf, field diameter 0.56 mm) and BCL2 reactivity (cutoff of 10%). The low-risk tumors were described as M1/BCL2 equivocal and M2/BCL2 positive, and the high-risk tumors included M2 to M3/BCL2-negative and M3/BCL2-positive tumors. The results showed that 87% of Nottingham grade 2 tumors, a clinically ambiguous category, were reclassified as either good prognosis (M1 to M2/BCL2+; 74%) or poor prognosis (M2 to M3/BCL2−; 13%), and only 13% (69/531) of
Summary
Nottingham grade 2 tumors were allocated to an intermediate prognosis (M1/BCL2−). In further subset analysis of ER-positive patients treated with hormonal therapy alone, those that were M2 to M3/BCL2 negative and M3/BCL2 positive showed a 2.5-fold to fourfold increase in risk of death, recurrence, and distant metastasis after 10 years compared with patients with M1 to M2/BCL2-positive and M1/BCL2-negative phenotypes. These findings suggest that ER-positive, HER2negative tumors can be classified as luminal A (good prognosis tumors) if they are Nottingham grade 1 or 2 and positive for BCL2, and all other such tumors could be considered luminal B (poor prognosis) tumors.
FOXA1 FOXA1 is a forkhead family transcription factor that segregates with genes that characterize the luminal subtypes in DNA microarray analyses.478 Using genomewide analysis, Laganiere and colleagues479 identified 153 promoters bound by ER-α in the breast cancer cell line MCF-7 in the presence of estradiol. One of the promoters identified was for FOXA1, whose expression correlated with expression of ER-α. Laganiere and colleagues479 further found that ablation of FOXA1 expression in MCF-7 cells suppressed ER-α binding to the prototypic trefoil factor 1 (TFF1) promoter (which contains a FOXA1 binding site), hindered the induction of TFF1 expression by estradiol, and prevented hormoneinduced reentry into the cell cycle. The practical utility of FOXA1 was assessed by Badve and associates,478 who showed positive correlation between FOXA1 expression and ER/PR expression by IHC. Another IHC study by Thorat and coworkers480 demonstrated a positive correlation between FOXA1 expression and ER-α (P < .0001), PR (P < .0001), and luminal subtype (P < .0001) and a negative correlation with basal subtype (P < .0001), proliferation markers, and high histologic grade (P = .0327). Although FOXA1 was a significant predictor of overall survival in univariate analysis in this study, only nodal status and ER expression were significant predictors of overall survival on multivariate analyses. FOXA1 has also been proposed as a clinical/IHC marker to identify the luminal A molecular subtype,481 but data are currently limited on this subject.
GATA3 GATA binding protein 3 (GATA3) is a transcriptional activator highly expressed by the luminal epithelial cells in the breast, and it is involved in growth and differentiation. Gene-expression profiling has shown that GATA3 is highly expressed in the luminal A subtype of breast cancer.143,410 In an IHC study of 139 breast
761
cancers, Mehra and associates482 showed that low GATA3 expression was associated with higher histologic grade (P < .001), positive nodes (P = .002), larger tumor size (P = .03), negative ER and PR (P < .001 for both), and ERBB2 overexpression (P = .03). Patients whose tumors expressed low GATA3 had significantly shorter overall and disease-free survival when compared with those whose tumors had high GATA3 levels.482 In a much larger series of 3119 breast cancer cases, Voduc and colleagues483 showed somewhat similar findings; however, they also clarified some of the issues. In their study, GATA3 was almost exclusively expressed in ER-positive patients and was also associated with lower tumor grade, older age at diagnosis, and the absence of HER2 overexpression. GATA3 was a marker of good prognosis and predicted superior breast cancer–specific survival, relapse-free survival, and overall survival in univariate analysis.483 However, in multivariate models that included patient age, tumor size, histologic grade, nodal status, ER status, and HER2 status, GATA3 was not independently prognostic for these same outcomes. Furthermore, in the subgroups of ER-positive patients treated with or without tamoxifen, GATA3 was again nonprognostic for all outcomes.483 Both FOXA1 and GATA3 are molecular markers that are highly associated with ER expression, but they do not seem to have prognostic value independent of ER. Therefore additional clinical validation studies are required before their use can be recommended in routine practice. Several other prognostic/predictive markers published in the literature—microvascular density, nm23, cathepsin D, PS2, p-Glycoprotein, fibroblast growth factor, transforming growth factor beta (TGF-β), androgen receptor, matrix metalloproteinase, and so on—are not discussed here because of their limited clinical utility.
Summary IHC is a critical tool for diagnostic, theranostic, and genomic applications in breast pathology. We believe as more and more targeted therapy is applied in breast cancer, pathologists will be under pressure to analyze additional biomarkers. We also predict that pathologists will have to reconcile not only with morphology and IHC but also with additional molecular tests that clinicians will use in the future. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Joshi MG, Lee AK, Pedersen CA, et al: The role of immunocytochemical markers in the differential diagnosis of proliferative and neoplastic lesions of the breast. Mod Pathol. 9:57–62, 1996. 2. Rudland PS, Leinster SJ, Winstanley J, et al: Immunocytochemical identification of cell types in benign and malignant breast diseases: variations in cell markers accompany the malignant state. J Histochem Cytochem. 41:543–553, 1993. 3. Ahmed A: The myoepithelium in human breast carcinoma. J Pathol. 113:129–135, 1974. 4. Bussolati G: Actin-rich (myoepithelial) cells in lobular carcinoma in situ of the breast. Virchows Arch B Cell Pathol Incl Mol Pathol. 32:165–176, 1980. 5. Bussolati G, Botta G, Gugliotta P: Actin-rich (myoepithelial) cells in ductal carcinoma-in-situ of the breast. Virchows Arch B Cell Pathol Incl Mol Pathol. 34:251–259, 1980. 6. Bussolati G, Botto Micca FB, Eusebi V, et al: Myoepithelial cells in lobular carcinoma in situ of the breast: a parallel immunocytochemical and ultrastructural study. Ultrastruct Pathol. 2:219– 230, 1981. 7. Gould VE, Jao W, Battifora H: Ultrastructural analysis in the differential diagnosis of breast tumors. The significance of myoepithelial cells, basal lamina, intracytoplasmic lumina and secretory granules. Pathol Res Pract. 167:45–70, 1980. 8. Gusterson BA, Warburton MJ, Mitchell D, et al: Distribution of myoepithelial cells and basement membrane proteins in the normal breast and in benign and malignant breast diseases. Cancer Res. 42:4763–4770, 1982. 9. Dwarakanath S, Lee AK, Delellis RA, et al: S-100 protein positivity in breast carcinomas: a potential pitfall in diagnostic immunohistochemistry. Hum Pathol. 18:1144–1148, 1987. 10. Jarasch ED, Nagle RB, Kaufmann M, et al: Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins. Hum Pathol. 19:276–289, 1988. 11. Nagle RB, Bocker W, Davis JR, et al: Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells. J Histochem Cytochem. 34:869–881, 1986. 12. Raju UB, Lee MW, Zarbo RJ, et al: Papillary neoplasia of the breast: immunohistochemically defined myoepithelial cells in the diagnosis of benign and malignant papillary breast neoplasms. Mod Pathol. 2:569–576, 1989. 13. Lele SM, Graves K, Gatalica Z: Immunohistochemical detection of maspin is a useful adjunct in distinguishing radial sclerosing lesion from tubular carcinoma of the breast. Appl Immunohistochem Mol Morphol. 8:32–36, 2000. 14. Mohsin SK, Zhang M, Clark GM, et al: Maspin expression in invasive breast cancer: association with other prognostic factors. J Pathol. 199:432–435, 2003. 15. Navarro Rde L, Martins MT, de Araujo VC: Maspin expression in normal and neoplastic salivary gland. J Oral Pathol Med. 33:435–440, 2004. 16. Umekita Y, Yoshida H: Expression of maspin is up-regulated during the progression of mammary ductal carcinoma. Histopathology. 42:541–545, 2003. 17. Bocker W, Bier B, Freytag G, et al: An immunohistochemical study of the breast using antibodies to basal and luminal keratins, alpha-smooth muscle actin, vimentin, collagen IV and laminin. Part II: Epitheliosis and ductal carcinoma in situ. Virchows Arch A Pathol Anat Histopathol. 421:323–330, 1992. 18. Bose S, Derosa CM, Ozzello L: Immunostaining of Type IV Collagen and Smooth Muscle Actin as an Aid in the Diagnosis of Breast Lesions. Breast J. 5:194–201, 1999. 19. Gottlieb C, Raju U, Greenwald KA: Myoepithelial cells in the differential diagnosis of complex benign and malignant breast lesions: an immunohistochemical study. Mod Pathol. 3:135–140, 1990. 20. Gugliotta P, Sapino A, Macri L, et al: Specific demonstration of myoepithelial cells by anti-alpha smooth muscle actin antibody. J Histochem Cytochem. 36:659–663, 1988. 21. Raymond WA, Leong AS: Assessment of invasion in breast lesions using antibodies to basement membrane components and myoepithelial cells. Pathology. 23:291–297, 1991.
761.e1
22. Gimona M, Herzog M, Vandekerckhove J, et al: Smooth muscle specific expression of calponin. FEBS Lett. 274:159–162, 1990. 23. Werling RW, Hwang H, Yaziji H, et al: Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain. Am J Surg Pathol. 27:82–90, 2003. 24. Winder SJ, Walsh MP: Calponin: thin filament-linked regulation of smooth muscle contraction. Cell Signal. 5:677–686, 1993. 25. Titus MA: Myosins. Curr Opin Cell Biol. 5:77–81, 1993. 26. Strasser P, Gimona M, Moessler H, et al: Mammalian calponin. Identification and expression of genetic variants. FEBS Lett. 330:13–18, 1993. 27. Barbareschi M, Pecciarini L, Cangi MG, et al: p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol. 25:1054–1060, 2001. 28. Kaufmann O, Fietze E, Mengs J, et al: Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol. 116:823–830, 2001. 29. Bhargava R, Dabbs DJ: Use of immunohistochemistry in diagnosis of breast epithelial lesions. Adv Anat Pathol. 14:93–107, 2007. 30. Eusebi V, Foschini MP, Betts CM, et al: Microglandular adenosis, apocrine adenosis, and tubular carcinoma of the breast. An immunohistochemical comparison. Am J Surg Pathol. 17:99– 109, 1993. 31. Lee KC, Chan JK, Gwi E: Tubular adenosis of the breast. A distinctive benign lesion mimicking invasive carcinoma. Am J Surg Pathol. 20:46–54, 1996. 32. Hill CB, Yeh IT: Myoepithelial cell staining patterns of papillary breast lesions: from intraductal papillomas to invasive papillary carcinomas. Am J Clin Pathol. 123:36–44, 2005. 33. Papotti M, Eusebi V, Gugliotta P, et al: Immunohistochemical analysis of benign and malignant papillary lesions of the breast. Am J Surg Pathol. 7:451–461, 1983. 34. Saddik M, Lai R: CD44s as a surrogate marker for distinguishing intraductal papilloma from papillary carcinoma of the breast. J Clin Pathol. 52:862–864, 1999. 35. Raju U, Vertes D: Breast papillomas with atypical ductal hyperplasia: a clinicopathologic study. Hum Pathol. 27:1231–1238, 1996. 36. Collins LC, Schnitt SJ: Papillary lesions of the breast: selected diagnostic and management issues. Histopathology. 52:20–29, 2008. 37. Rabban JT, Koerner FC, Lerwill MF: Solid papillary ductal carcinoma in situ versus usual ductal hyperplasia in the breast: a potentially difficult distinction resolved by cytokeratin 5/6. Hum Pathol. 37:787–793, 2006. 38. Carter D, Orr SL, Merino MJ: Intracystic papillary carcinoma of the breast. After mastectomy, radiotherapy or excisional biopsy alone. Cancer. 52:14–19, 1983. 39. Carter D: Intraductal papillary tumors of the breast: a study of 78 cases. Cancer. 39:1689–1692, 1977. 40. Collins LC, Carlo VP, Hwang H, et al: Intracystic papillary carcinomas of the breast: a reevaluation using a panel of myoepithelial cell markers. Am J Surg Pathol. 30:1002–1007, 2006. 41. Mulligan AM, O’Malley FP: Papillary lesions of the breast: a review. Adv Anat Pathol. 14:108–119, 2007. 42. Wynveen CA, Nehhozina T, Akram M, et al: Intracystic papillary carcinoma of the breast: An in situ or invasive tumor? Results of immunohistochemical analysis and clinical follow-up. Am J Surg Pathol. 35:1–14, 2011. 43. Barsky SH, Siegal GP, Jannotta F, et al: Loss of basement membrane components by invasive tumors but not by their benign counterparts. Lab Invest. 49:140–147, 1983. 44. Esposito NN, Dabbs DJ, Bhargava R: Are encapsulated papillary carcinomas of the breast in-situ or invasive? A basement membrane study of 27 cases. Am J Clin Pathol. In Press. 45. Leal C, Costa I, Fonseca D, et al: Intracystic (encysted) papillary carcinoma of the breast: a clinical, pathological, and immunohistochemical study. Hum Pathol. 29:1097–1104, 1998. 46. Lefkowitz M, Lefkowitz W, Wargotz ES: Intraductal (intracystic) papillary carcinoma of the breast and its variants: a clinicopathological study of 77 cases. Hum Pathol. 25:802–809, 1994.
761.e2
Immunohistology of the Breast
47. Solorzano CC, Middleton LP, Hunt KK, et al: Treatment and outcome of patients with intracystic papillary carcinoma of the breast. Am J Surg. 184:364–368, 2002. 48. Bhargava R, Esposito NN, Dabbs DJ: Intracystic papillary carcinomas of the breast are more similar to in situ carcinomas than to invasive carcinoma. Am J Surg Pathol. 35:778–779; author reply 779–781, 2011. 49. Mulligan AM, O’Malley FP: Metastatic potential of encapsulated (intracystic) papillary carcinoma of the breast: a report of 2 cases with axillary lymph node micrometastases. Int J Surg Pathol. 15:143–147, 2007. 50. Masood S, Sim SJ, Lu L: Immunohistochemical differentiation of atypical hyperplasia vs. carcinoma in situ of the breast. Cancer Detect Prev. 16:225–235, 1992. 51. Moinfar F, Man YG, Lininger RA, et al: Use of keratin 35betaE12 as an adjunct in the diagnosis of mammary intraepithelial neoplasia-ductal type–benign and malignant intraductal proliferations. Am J Surg Pathol. 23:1048–1058, 1999. 52. Lacroix-Triki M, Mery E, Voigt JJ, et al: Value of cytokeratin 5/6 immunostaining using D5/16 B4 antibody in the spectrum of proliferative intraepithelial lesions of the breast. A comparative study with 34betaE12 antibody. Virchows Arch. 442:548–554, 2003. 53. Grin A, O’Malley FP, Mulligan AM: Cytokeratin 5 and estrogen receptor immunohistochemistry as a useful adjunct in identifying atypical papillary lesions on breast needle core biopsy. Am J Surg Pathol. 33:1615–1623, 2009. 54. Cangiarella J, Guth A, Axelrod D, et al: Is surgical excision necessary for the management of atypical lobular hyperplasia and lobular carcinoma in situ diagnosed on core needle biopsy?: a report of 38 cases and review of the literature. Arch Pathol Lab Med. 132:979–983, 2008. 55. Chuba PJ, Hamre MR, Yap J, et al: Bilateral risk for subsequent breast cancer after lobular carcinoma-in-situ: analysis of surveillance, epidemiology, and end results data. J Clin Oncol. 23:5534– 5541, 2005. 56. Crisi GM, Mandavilli S, Cronin E, et al: Invasive mammary carcinoma after immediate and short-term follow-up for lobular neoplasia on core biopsy. Am J Surg Pathol. 27:325–333, 2003. 57. Elsheikh TM, Silverman JF: Follow-up surgical excision is indicated when breast core needle biopsies show atypical lobular hyperplasia or lobular carcinoma in situ: a correlative study of 33 patients with review of the literature. Am J Surg Pathol. 29:534–543, 2005. 58. Fisher ER, Costantino J, Fisher B, et al: Pathologic findings from the National Surgical Adjuvant Breast Project (NSABP) Protocol B-17. Five-year observations concerning lobular carcinoma in situ. Cancer. 78:1403–1416, 1996. 59. Fisher ER, Land SR, Fisher B, et al: Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelveyear observations concerning lobular carcinoma in situ. Cancer. 100:238–244, 2004. 60. Leonard GD, Swain SM: Ductal carcinoma in situ, complexities and challenges. J Natl Cancer Inst. 96:906–920, 2004. 61. Li CI, Malone KE, Saltzman BS, et al: Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1988-2001. Cancer. 106:2104–2112, 2006. 62. Maluf H, Koerner F: Lobular carcinoma in situ and infiltrating ductal carcinoma: frequent presence of DCIS as a precursor lesion. Int J Surg Pathol. 9:127–131, 2001. 63. Winchester DP, Jeske JM, Goldschmidt RA: The diagnosis and management of ductal carcinoma in-situ of the breast. CA Cancer J Clin. 50:184–200, 2000. 64. Bedrosian I, Mick R, Orel SG, et al: Changes in the surgical management of patients with breast carcinoma based on preoperative magnetic resonance imaging. Cancer. 98:468–473, 2003. 65. Molland JG, Donnellan M, Janu NC, et al: Infiltrating lobular carcinoma—a comparison of diagnosis, management and outcome with infiltrating duct carcinoma. Breast. 13:389–396, 2004. 66. Munot K, Dall B, Achuthan R, et al: Role of magnetic resonance imaging in the diagnosis and single-stage surgical resection of invasive lobular carcinoma of the breast. Br J Surg. 89:1296– 1301, 2002.
67. Borst MJ, Ingold JA: Metastatic patterns of invasive lobular versus invasive ductal carcinoma of the breast. Surgery. 114:637– 641; discussion 641–632, 1993. 68. Harris M, Howell A, Chrissohou M, et al: A comparison of the metastatic pattern of infiltrating lobular carcinoma and infiltrating duct carcinoma of the breast. Br J Cancer. 50:23–30, 1984. 69. Jain S, Fisher C, Smith P, et al: Patterns of metastatic breast cancer in relation to histological type. Eur J Cancer. 29A:2155– 2157, 1993. 70. Tham YL, Sexton K, Kramer R, et al: Primary breast cancer phenotypes associated with propensity for central nervous system metastases. Cancer. 107:696–704, 2006. 71. Arpino G, Bardou VJ, Clark GM, et al: Infiltrating lobular carcinoma of the breast: tumor characteristics and clinical outcome. Breast Cancer Res. 6:R149–R156, 2004. 72. Mersin H, Yildirim E, Gulben K, et al: Is invasive lobular carcinoma different from invasive ductal carcinoma? Eur J Surg Oncol. 29:390–395, 2003. 73. Kneeshaw PJ, Turnbull LW, Smith A, et al: Dynamic contrast enhanced magnetic resonance imaging aids the surgical management of invasive lobular breast cancer. Eur J Surg Oncol. 29:32– 37, 2003. 74. Schelfout K, Van Goethem M, Kersschot E, et al: Preoperative breast MRI in patients with invasive lobular breast cancer. Eur Radiol. 14:1209–1216, 2004. 75. Cocquyt VF, Blondeel PN, Depypere HT, et al: Different responses to preoperative chemotherapy for invasive lobular and invasive ductal breast carcinoma. Eur J Surg Oncol. 29:361–367, 2003. 76. Cristofanilli M, Gonzalez-Angulo A, Sneige N, et al: Invasive lobular carcinoma classic type: response to primary chemotherapy and survival outcomes. J Clin Oncol. 23:41–48, 2005. 77. Mathieu MC, Rouzier R, Llombart-Cussac A, et al: The poor responsiveness of infiltrating lobular breast carcinomas to neoadjuvant chemotherapy can be explained by their biological profile. Eur J Cancer. 40:342–351, 2004. 78. Tubiana-Hulin M, Stevens D, Lasry S, et al: Response to neoadjuvant chemotherapy in lobular and ductal breast carcinomas: a retrospective study on 860 patients from one institution. Ann Oncol. 17:1228–1233, 2006. 79. Acs G, Lawton TJ, Rebbeck TR, et al: Differential expression of E-cadherin in lobular and ductal neoplasms of the breast and its biologic and diagnostic implications. Am J Clin Pathol. 115:85– 98, 2001. 80. Gamallo C, Palacios J, Suarez A, et al: Correlation of E-cadherin expression with differentiation grade and histological type in breast carcinoma. Am J Pathol. 142:987–993, 1993. 81. Moll R, Mitze M, Frixen UH, et al: Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas. Am J Pathol. 143:1731–1742, 1993. 82. Berx G, Cleton-Jansen AM, Nollet F, et al: E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. Embo J. 14:6107–6115, 1995. 83. Berx G, Cleton-Jansen AM, Strumane K, et al: E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene. 13:1919–1925, 1996. 84. Vos CB, Cleton-Jansen AM, Berx G, et al: E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis. Br J Cancer. 76:1131–1133, 1997. 85. Handschuh G, Candidus S, Luber B, et al: Tumour-associated E-cadherin mutations alter cellular morphology, decrease cellular adhesion and increase cellular motility. Oncogene. 18:4301– 4312, 1999. 86. Goldstein NS, Bassi D, Watts JC, et al: E-cadherin reactivity of 95 noninvasive ductal and lobular lesions of the breast. Implications for the interpretation of problematic lesions. Am J Clin Pathol. 115:534–542, 2001. 87. De Leeuw WJ, Berx G, Vos CB, et al: Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ. J Pathol. 183:404–411, 1997. 88. Gonzalez MA, Pinder SE, Wencyk PM, et al: An immunohistochemical examination of the expression of E-cadherin, alphaand beta/gamma-catenins, and alpha2- and beta1-integrins in invasive breast cancer. J Pathol. 187:523–529, 1999.
References 89. Aberle H, Schwartz H, Kemler R: Cadherin-catenin complex: protein interactions and their implications for cadherin function. J Cell Biochem. 61:514–523, 1996. 90. Aghib DF, McCrea PD: The E-cadherin complex contains the src substrate p120. Exp Cell Res. 218:359–369, 1995. 91. Gooding JM, Yap KL, Ikura M: The cadherin-catenin complex as a focal point of cell adhesion and signalling: new insights from three-dimensional structures. Bioessays. 26:497–511, 2004. 92. Piepenhagen PA, Nelson WJ: Defining E-cadherin-associated protein complexes in epithelial cells: plakoglobin, beta- and gamma-catenin are distinct components. J Cell Sci. 104(Pt 3):751–762, 1993. 93. Reynolds AB, Daniel J, McCrea PD, et al: Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes. Mol Cell Biol. 14:8333–8342, 1994. 94. Yap AS, Niessen CM, Gumbiner BM: The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn. J Cell Biol. 141:779–789, 1998. 95. Shibamoto S, Hayakawa M, Takeuchi K, et al: Association of p120, a tyrosine kinase substrate, with E-cadherin/catenin complexes. J Cell Biol. 128:949–957, 1995. 96. Davis MA, Ireton RC, Reynolds AB: A core function for p120catenin in cadherin turnover. J Cell Biol. 163:525–534, 2003. 97. Noren NK, Liu BP, Burridge K, et al: p120 catenin regulates the actin cytoskeleton via Rho family GTPases. J Cell Biol. 150:567– 580, 2000. 98. Berx G, Van Roy F: The E-cadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res. 3:289–293, 2001. 99. Berx G, Nollet F, van Roy F: Dysregulation of the E-cadherin/ catenin complex by irreversible mutations in human carcinomas. Cell Adhes Commun. 6:171–184, 1998. 100. Karabakhtsian RG, Johnson R, Sumkin J, et al: The clinical significance of lobular neoplasia on breast core biopsy. Am J Surg Pathol. 31:717–723, 2007. 101. Goldstein NS: Does the level of E-cadherin expression correlate with the primary breast carcinoma infiltration pattern and type of systemic metastases? Am J Clin Pathol. 118:425–434, 2002. 102. Jacobs TW, Pliss N, Kouria G, et al: Carcinomas in situ of the breast with indeterminate features: role of E-cadherin staining in categorization. Am J Surg Pathol. 25:229–236, 2001. 103. Lehr HA, Folpe A, Yaziji H, et al: Cytokeratin 8 immunostaining pattern and E-cadherin expression distinguish lobular from ductal breast carcinoma. Am J Clin Pathol. 114:190–196, 2000. 104. Bassler R, Kronsbein H: Disseminated lobular carcinoma—a predominantly pleomorphic lobular carcinoma of the whole breast. Pathol Res Pract. 166:456–470, 1980. 105. Weidner N, Semple JP: Pleomorphic variant of invasive lobular carcinoma of the breast. Hum Pathol. 23:1167–1171, 1992. 106. Eusebi V, Magalhaes F, Azzopardi JG: Pleomorphic lobular carcinoma of the breast: an aggressive tumor showing apocrine differentiation. Hum Pathol. 23:655–662, 1992. 107. Reis-Filho JS, Simpson PT, Jones C, et al: Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity. J Pathol. 207:1–13, 2005. 108. Bentz JS, Yassa N, Clayton F: Pleomorphic lobular carcinoma of the breast: clinicopathologic features of 12 cases. Mod Pathol. 11:814–822, 1998. 109. Frolik D, Caduff R, Varga Z: Pleomorphic lobular carcinoma of the breast: its cell kinetics, expression of oncogenes and tumour suppressor genes compared with invasive ductal carcinomas and classical infiltrating lobular carcinomas. Histopathology. 39:503– 513, 2001. 110. Middleton LP, Palacios DM, Bryant BR, et al: Pleomorphic lobular carcinoma: morphology, immunohistochemistry, and molecular analysis. Am J Surg Pathol. 24:1650–1656, 2000. 111. Radhi JM: Immunohistochemical analysis of pleomorphic lobular carcinoma: higher expression of p53 and chromogranin and lower expression of ER and PgR. Histopathology. 36:156– 160, 2000. 112. Dabbs DJ, Kaplai M, Chivukula M, et al: The spectrum of morphomolecular abnormalities of the E-cadherin/catenin complex
761.e3
in pleomorphic lobular carcinoma of the breast. Appl Immunohistochem Mol Morphol. 15:260–266, 2007. 113. Simpson PT, Reis-Filho JS, Lambros MB, et al: Molecular profiling pleomorphic lobular carcinomas of the breast: evidence for a common molecular genetic pathway with classic lobular carcinomas. J Pathol. 215:231–244, 2008. 114. Sneige N, Wang J, Baker BA, et al: Clinical, histopathologic, and biologic features of pleomorphic lobular (ductal-lobular) carcinoma in situ of the breast: a report of 24 cases. Mod Pathol. 15:1044–1050, 2002. 115. Fisher ER, Gregorio RM, Redmond C, et al: Tubulolobular invasive breast cancer: a variant of lobular invasive cancer. Hum Pathol. 8:679–683, 1977. 116. Green I, McCormick B, Cranor M, et al: A comparative study of pure tubular and tubulolobular carcinoma of the breast. Am J Surg Pathol. 21:653–657, 1997. 117. Esposito NN, Chivukula M, Dabbs DJ: The ductal phenotypic expression of the E-cadherin/catenin complex in tubulolobular carcinoma of the breast: an immunohistochemical and clinicopathologic study. Mod Pathol. 20:130–138, 2007. 118. Wheeler DT, Tai LH, Bratthauer GL, et al: Tubulolobular carcinoma of the breast: an analysis of 27 cases of a tumor with a hybrid morphology and immunoprofile. Am J Surg Pathol. 28:1587–1593, 2004. 119. Hood CI, Font RL, Zimmerman LE: Metastatic mammary carcinoma in the eyelid with histiocytoid appearance. Cancer. 31:793–800, 1973. 120. Filotico M, Trabucco M, Gallone D, et al: Histiocytoid carcinoma of the breast. A problem of differential diagnosis for the pathologist. Report of a case. Pathologica. 75:429–433, 1983. 121. Gupta D, Croitoru CM, Ayala AG, et al: E-cadherin immunohistochemical analysis of histiocytoid carcinoma of the breast. Ann Diagn Pathol. 6:141–147, 2002. 122. Reis-Filho JS, Fulford LG, Freeman A, et al: Pathologic quiz case: a 93-year-old woman with an enlarged and tender left breast. Histiocytoid variant of lobular breast carcinoma. Arch Pathol Lab Med. 127:1626–1628, 2003. 123. Luna-More S, Gonzalez B, Acedo C, et al: Invasive micropapillary carcinoma of the breast. A new special type of invasive mammary carcinoma. Pathol Res Pract. 190:668–674, 1994. 124. Siriaunkgul S, Tavassoli FA: Invasive micropapillary carcinoma of the breast. Mod Pathol. 6:660–662, 1993. 125. Li YS, Kaneko M, Sakamoto DG, et al: The reversed apical pattern of MUC1 expression is characteristics of invasive micropapillary carcinoma of the breast. Breast Cancer. 13:58–63, 2006. 126. Nassar H, Pansare V, Zhang H, et al: Pathogenesis of invasive micropapillary carcinoma: role of MUC1 glycoprotein. Mod Pathol. 17:1045–1050, 2004. 127. Guo X, Chen L, Lang R, et al: Invasive micropapillary carcinoma of the breast : association of pathologic features with lymph node metastasis. Am J Clin Pathol. 126:740–746, 2006. 128. Nassar H: Carcinomas with micropapillary morphology: clinical significance and current concepts. Adv Anat Pathol. 11:297–303, 2004. 129. Nassar H, Wallis T, Andea A, et al: Clinicopathologic analysis of invasive micropapillary differentiation in breast carcinoma. Mod Pathol. 14:836–841, 2001. 130. Pettinato G, Manivel CJ, Panico L, et al: Invasive micropapillary carcinoma of the breast: clinicopathologic study of 62 cases of a poorly recognized variant with highly aggressive behavior. Am J Clin Pathol. 121:857–866, 2004. 131. Walsh MM, Bleiweiss IJ: Invasive micropapillary carcinoma of the breast: eighty cases of an underrecognized entity. Hum Pathol. 32:583–589, 2001. 132. Paterakos M, Watkin WG, Edgerton SM, et al: Invasive micropapillary carcinoma of the breast: a prognostic study. Hum Pathol. 30:1459–1463, 1999. 133. Acs G, Esposito NN, Rakosy Z, et al: Invasive ductal carcinomas of the breast showing partial reversed cell polarity are associated with lymphatic tumor spread and may represent part of a spectrum of invasive micropapillary carcinoma. Am J Surg Pathol. 34:1637–1646, 2010. 134. Perou CM, Sorlie T, Eisen MB, et al: Molecular portraits of human breast tumours. Nature. 406:747–752, 2000.
761.e4
Immunohistology of the Breast
135. Sorlie T, Perou CM, Tibshirani R, et al: Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 98:10869–10874, 2001. 136. Livasy CA, Karaca G, Nanda R, et al: Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol. 19:264–271, 2006. 137. Nielsen TO, Hsu FD, Jensen K, et al: Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 10:5367–5374, 2004. 138. Bhargava R, Beriwal S, McManus K, et al: CK5 is more sensitive than CK5/6 in identifying “basal-like” phenotype of breast carcinoma. Am J Clin Pathol. 2008, In Press. 139. Bryan BB, Schnitt SJ, Collins LC: Ductal carcinoma in situ with basal-like phenotype: a possible precursor to invasive basal-like breast cancer. Mod Pathol. 19:617–621, 2006. 140. Dabbs DJ, Chivukula M, Carter G, et al: Basal phenotype of ductal carcinoma in situ: recognition and immunohistologic profile. Mod Pathol. 19:1506–1511, 2006. 141. Carey LA, Perou CM, Livasy CA, et al: Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA. 295:2492–2502, 2006. 142. van de Rijn M, Perou CM, Tibshirani R, et al: Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol. 161:1991–1996, 2002. 143. Sorlie T, Tibshirani R, Parker J, et al: Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 100:8418–8423, 2003. 144. Park SR, Chen A: Poly(Adenosine diphosphate-ribose) polymerase inhibitors in cancer treatment. Hematol Oncol Clin North Am. 26:649–670, ix, 2012. 145. Carter MR, Hornick JL, Lester S, et al: Spindle cell (sarcomatoid) carcinoma of the breast: a clinicopathologic and immunohistochemical analysis of 29 cases. Am J Surg Pathol. 30:300–309, 2006. 146. Davis WG, Hennessy B, Babiera G, et al: Metaplastic sarcomatoid carcinoma of the breast with absent or minimal overt invasive carcinomatous component: a misnomer. Am J Surg Pathol. 29:1456–1463, 2005. 147. Adem C, Reynolds C, Adlakha H, et al: Wide spectrum screening keratin as a marker of metaplastic spindle cell carcinoma of the breast: an immunohistochemical study of 24 patients. Histopathology. 40:556–562, 2002. 148. Koker MM, Kleer CG: p63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma. Am J Surg Pathol. 28:1506–1512, 2004. 149. Tse GM, Tan PH, Chaiwun B, et al: p63 is useful in the diagnosis of mammary metaplastic carcinomas. Pathology. 38:16–20, 2006. 150. Ellis IO, Bell J, Ronan JE, et al: Immunocytochemical investigation of intermediate filament proteins and epithelial membrane antigen in spindle cell tumours of the breast. J Pathol. 154:157– 165, 1988. 151. Wargotz ES, Norris HJ: Metaplastic carcinomas of the breast. III. Carcinosarcoma. Cancer. 64:1490–1499, 1989. 152. Sommers CL, Walker-Jones D, Heckford SE, et al: Vimentin rather than keratin expression in some hormone-independent breast cancer cell lines and in oncogene-transformed mammary epithelial cells. Cancer Res. 49:4258–4263, 1989. 153. Dunne B, Lee AH, Pinder SE, et al: An immunohistochemical study of metaplastic spindle cell carcinoma, phyllodes tumor and fibromatosis of the breast. Hum Pathol. 34:1009–1015, 2003. 154. Leibl S, Gogg-Kammerer M, Sommersacher A, et al: Metaplastic breast carcinomas: are they of myoepithelial differentiation?: immunohistochemical profile of the sarcomatoid subtype using novel myoepithelial markers. Am J Surg Pathol. 29:347–353, 2005. 155. Miettinen M, Franssila K: Immunohistochemical spectrum of malignant melanoma. The common presence of keratins. Lab Invest. 61:623–628, 1989. 156. Zarbo RJ, Gown AM, Nagle RB, et al: Anomalous cytokeratin expression in malignant melanoma: one- and two-dimensional western blot analysis and immunohistochemical survey of 100 melanomas. Mod Pathol. 3:494–501, 1990. 157. Pusztaszeri MP, Seelentag W, Bosman FT: Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand
factor, and Fli-1 in normal human tissues. J Histochem Cytochem. 54:385–395, 2006. 158. Miettinen M, Lindenmayer AE, Chaubal A: Endothelial cell markers CD31, CD34, and BNH9 antibody to H- and Y-antigens–evaluation of their specificity and sensitivity in the diagnosis of vascular tumors and comparison with von Willebrand factor. Mod Pathol. 7:82–90, 1994. 159. Chen PC, Chen CK, Nicastri AD, et al: Myoepithelial carcinoma of the breast with distant metastasis and accompanied by adenomyoepitheliomas. Histopathology. 24:543–548, 1994. 160. Foschini MP, Eusebi V: Carcinomas of the breast showing myoepithelial cell differentiation. A review of the literature. Virchows Arch. 432:303–310, 1998. 161. Thorner PS, Kahn HJ, Baumal R, et al: Malignant myoepithelioma of the breast. An immunohistochemical study by light and electron microscopy. Cancer. 57:745–750, 1986. 162. Young RH, Clement PB: Adenomyoepithelioma of the breast. A report of three cases and review of the literature. Am J Clin Pathol. 89:308–314, 1988. 163. Schurch W, Potvin C, Seemayer TA: Malignant myoepithelioma (myoepithelial carcinoma) of the breast: an ultrastructural and immunocytochemical study. Ultrastruct Pathol. 8:1–11, 1985. 164. Tavassoli FA: Myoepithelial lesions of the breast. Myoepitheliosis, adenomyoepithelioma, and myoepithelial carcinoma. Am J Surg Pathol. 15:554–568, 1991. 165. Hornick JL, Fletcher CD: Myoepithelial tumors of soft tissue: a clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters. Am J Surg Pathol. 27:1183–1196, 2003. 166. Hornick JL, Fletcher CD: Cutaneous myoepithelioma: a clinicopathologic and immunohistochemical study of 14 cases. Hum Pathol. 35:14–24, 2004. 167. Popnikolov NK, Ayala AG, Graves K, et al: Benign myoepithelial tumors of the breast have immunophenotypic characteristics similar to metaplastic matrix-producing and spindle cell carcinomas. Am J Clin Pathol. 120:161–167, 2003. 168. Reis-Filho JS, Milanezi F, Paredes J, et al: Novel and classic myoepithelial/stem cell markers in metaplastic carcinomas of the breast. Appl Immunohistochem Mol Morphol. 11:1–8, 2003. 169. Smith TA, Machen SK, Fisher C, et al: Usefulness of cytokeratin subsets for distinguishing monophasic synovial sarcoma from malignant peripheral nerve sheath tumor. Am J Clin Pathol. 112:641–648, 1999. 170. Damiani S, Miettinen M, Peterse JL, et al: Solitary fibrous tumour (myofibroblastoma) of the breast. Virchows Arch. 425:89–92, 1994. 171. Julien M, Trojani M, Coindre JM: [Myofibroblastoma of the breast. Report of 8 cases]. Ann Pathol. 14:143–147, 1994. 172. Wargotz ES, Weiss SW, Norris HJ: Myofibroblastoma of the breast. Sixteen cases of a distinctive benign mesenchymal tumor. Am J Surg Pathol. 11:493–502, 1987. 173. Lee AH: Recent developments in the histological diagnosis of spindle cell carcinoma, fibromatosis and phyllodes tumour of the breast. Histopathology. 52:45–57, 2008. 174. Chaudary MA, Millis RR, Lane EB, et al: Paget’s disease of the nipple: a ten year review including clinical, pathological, and immunohistochemical findings. Breast Cancer Res Treat. 8:139– 146, 1986. 175. Yim JH, Wick MR, Philpott GW, et al: Underlying pathology in mammary Paget’s disease. Ann Surg Oncol. 4:287–292, 1997. 176. Anderson JM, Ariga R, Govil H, et al: Assessment of Her-2/Neu status by immunohistochemistry and fluorescence in situ hybridization in mammary Paget disease and underlying carcinoma. Appl Immunohistochem Mol Morphol. 11:120–124, 2003. 177. Haerslev T, Krag Jacobsen G: Expression of cytokeratin and erbB-2 oncoprotein in Paget’s disease of the nipple. An immunohistochemical study. APMIS. 100:1041–1047, 1992. 178. Lammie GA, Barnes DM, Millis RR, et al: An immunohistochemical study of the presence of c-erbB-2 protein in Paget’s disease of the nipple. Histopathology. 15:505–514, 1989. 179. Wolber RA, Dupuis BA, Wick MR: Expression of c-erbB-2 oncoprotein in mammary and extramammary Paget’s disease. Am J Clin Pathol. 96:243–247, 1991. 180. Meissner K, Riviere A, Haupt G, et al: Study of neu-protein expression in mammary Paget’s disease with and without
References underlying breast carcinoma and in extramammary Paget’s disease. Am J Pathol. 137:1305–1309, 1990. 181. Tani EM, Skoog L: Immunocytochemical detection of estrogen receptors in mammary Paget cells. Acta Cytol. 32:825–828, 1988. 182. Fu W, Lobocki CA, Silberberg BK, et al: Molecular markers in Paget disease of the breast. J Surg Oncol. 77:171–178, 2001. 183. Gillett CE, Bobrow LG, Millis RR: S100 protein in human mammary tissue–immunoreactivity in breast carcinoma, including Paget’s disease of the nipple, and value as a marker of myoepithelial cells. J Pathol. 160:19–24, 1990. 184. Lundquist K, Kohler S, Rouse RV: Intraepidermal cytokeratin 7 expression is not restricted to Paget cells but is also seen in Toker cells and Merkel cells. Am J Surg Pathol. 23:212–219, 1999. 185. Marucci G, Betts CM, Golouh R, et al: Toker cells are probably precursors of Paget cell carcinoma: a morphological and ultrastructural description. Virchows Arch. 441:117–123, 2002. 186. Zeng Z, Melamed J, Symmans PJ, et al: Benign proliferative nipple duct lesions frequently contain CAM 5.2 and anticytokeratin 7 immunoreactive cells in the overlying epidermis. Am J Surg Pathol. 23:1349–1355, 1999. 187. Yao DX, Hoda SA, Chiu A, et al: Intraepidermal cytokeratin 7 immunoreactive cells in the non-neoplastic nipple may represent interepithelial extension of lactiferous duct cells. Histopathology. 40:230–236, 2002. 188. Bader AA, Tio J, Petru E, et al: T1 breast cancer: identification of patients at low risk of axillary lymph node metastases. Breast Cancer Res Treat. 76:11–17, 2002. 189. Barth A, Craig PH, Silverstein MJ: Predictors of axillary lymph node metastases in patients with T1 breast carcinoma. Cancer. 79:1918–1922, 1997. 190. Chadha M, Chabon AB, Friedmann P, et al: Predictors of axillary lymph node metastases in patients with T1 breast cancer. A multivariate analysis. Cancer. 73:350–353, 1994. 191. Gajdos C, Tartter PI, Bleiweiss IJ: Lymphatic invasion, tumor size, and age are independent predictors of axillary lymph node metastases in women with T1 breast cancers. Ann Surg. 230:692– 696, 1999. 192. Van den Eynden GG, Van der Auwera I, Van Laere SJ, et al: Distinguishing blood and lymph vessel invasion in breast cancer: a prospective immunohistochemical study. Br J Cancer. 94:1643–1649, 2006. 193. Amparo RS, Angel CD, Ana LH, et al: Inflammatory breast carcinoma: pathological or clinical entity? Breast Cancer Res Treat. 64:269–273, 2000. 194. Bonnier P, Charpin C, Lejeune C, et al: Inflammatory carcinomas of the breast: a clinical, pathological, or a clinical and pathological definition? Int J Cancer. 62:382–385, 1995. 195. Le MG, Arriagada R, Contesso G, et al: Dermal lymphatic emboli in inflammatory and noninflammatory breast cancer: a French-Tunisian joint study in 337 patients. Clin Breast Cancer. 6:439–445, 2005. 196. Kahn HJ, Marks A: A new monoclonal antibody, D2-40, for detection of lymphatic invasion in primary tumors. Lab Invest. 82:1255–1257, 2002. 197. Kaiserling E: [Immunohistochemical identification of lymph vessels with D2-40 in diagnostic pathology]. Pathologe. 25:362– 374, 2004. 198. Kahn HJ, Bailey D, Marks A: Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi’s sarcoma and a subset of angiosarcomas. Mod Pathol. 15:434–440, 2002. 199. Cabanas RM: An approach for the treatment of penile carcinoma. Cancer. 39:456–466, 1977. 200. Mansi JL, Gogas H, Bliss JM, et al: Outcome of primary-breastcancer patients with micrometastases: a long-term follow-up study. Lancet. 354:197–202, 1999. 201. Steinhoff MM: Axillary Node Micrometastases: Detection and Biologic Significance. Breast J. 5:325–329, 1999. 202. Nasser IA, Lee AK, Bosari S, et al: Occult axillary lymph node metastases in “node-negative” breast carcinoma. Hum Pathol. 24:950–957, 1993. 203. Bass SS, Lyman GH, McCann CR, et al: Lymphatic Mapping and Sentinel Lymph Node Biopsy. Breast J. 5:288–295, 1999.
761.e5
204. Dabbs DJ, Johnson R: The optimal number of sentinel lymph nodes for focused pathologic examination. Breast J. 10:186–189, 2004. 205. Giuliano AE, McCall L, Beitsch P, et al: Locoregional recurrence after sentinel lymph node dissection with or without axillary dissection in patients with sentinel lymph node metastases: the American College of Surgeons Oncology Group Z0011 randomized trial. Ann Surg. 252:426–432; discussion 432–423, 2010. 206. Giuliano AE, Hawes D, Ballman KV, et al: Association of occult metastases in sentinel lymph nodes and bone marrow with survival among women with early-stage invasive breast cancer. JAMA. 306:385–393, 2011. 207. Weaver DL, Ashikaga T, Krag DN, et al: Effect of occult metastases on survival in node-negative breast cancer. N Engl J Med. 364:412–421, 2011. 208. Klevesath MB, Bobrow LG, Pinder SE, et al: The value of immunohistochemistry in sentinel lymph node histopathology in breast cancer. Br J Cancer. 92:2201–2205, 2005. 209. Dabbs DJ, Fung M, Landsittel D, et al: Sentinel lymph node micrometastasis as a predictor of axillary tumor burden. Breast J. 10:101–105, 2004. 210. den Bakker MA, van Weeszenberg A, de Kanter AY, et al: Nonsentinel lymph node involvement in patients with breast cancer and sentinel node micrometastasis; too early to abandon axillary clearance. J Clin Pathol. 55:932–935, 2002. 211. Kamath VJ, Giuliano R, Dauway EL, et al: Characteristics of the sentinel lymph node in breast cancer predict further involvement of higher-echelon nodes in the axilla: a study to evaluate the need for complete axillary lymph node dissection. Arch Surg. 136:688–692, 2001. 212. Mignotte H, Treilleux I, Faure C, et al: Axillary lymph-node dissection for positive sentinel nodes in breast cancer patients. Eur J Surg Oncol. 28:623–626, 2002. 213. Cserni G, Bianchi S, Vezzosi V, et al: The value of cytokeratin immunohistochemistry in the evaluation of axillary sentinel lymph nodes in patients with lobular breast carcinoma. J Clin Pathol. 59:518–522, 2006. 214. Czerniecki BJ, Scheff AM, Callans LS, et al: Immunohistochemistry with pancytokeratins improves the sensitivity of sentinel lymph node biopsy in patients with breast carcinoma. Cancer. 85:1098–1103, 1999. 215. Doglioni C, Dell’Orto P, Zanetti G, et al: Cytokeratinimmunoreactive cells of human lymph nodes and spleen in normal and pathological conditions. An immunocytochemical study. Virchows Arch A Pathol Anat Histopathol. 416:479–490, 1990. 216. Iuzzolino P, Bontempini L, Doglioni C, et al: Keratin immunoreactivity in extrafollicular reticular cells of the lymph node. Am J Clin Pathol. 91:239–240, 1989. 217. Carter BA, Jensen RA, Simpson JF, et al: Benign transport of breast epithelium into axillary lymph nodes after biopsy. Am J Clin Pathol. 113:259–265, 2000. 218. Diaz NM, Cox CE, Ebert M, et al: Benign mechanical transport of breast epithelial cells to sentinel lymph nodes. Am J Surg Pathol. 28:1641–1645, 2004. 219. Diaz NM, Mayes JR, Vrcel V: Breast epithelial cells in dermal angiolymphatic spaces: a manifestation of benign mechanical transport. Hum Pathol. 36:310–313, 2005. 220. Diaz NM, Vrcel V, Centeno BA, et al: Modes of benign mechanical transport of breast epithelial cells to axillary lymph nodes. Adv Anat Pathol. 12:7–9, 2005. 221. Hughes SJ, Xi L, Raja S, et al: A rapid, fully automated, molecular-based assay accurately analyzes sentinel lymph nodes for the presence of metastatic breast cancer. Ann Surg. 243:389– 398, 2006. 222. Viale G, Dell’Orto P, Biasi MO, et al: Comparative evaluation of an extensive histopathologic examination and a real-time reverse-transcription-polymerase chain reaction assay for mammaglobin and cytokeratin 19 on axillary sentinel lymph nodes of breast carcinoma patients. Ann Surg. 247:136–142, 2008. 223. Blumencranz P, Whitworth PW, Deck K, et al: Scientific Impact Recognition Award. Sentinel node staging for breast cancer: intraoperative molecular pathology overcomes conventional histologic sampling errors. Am J Surg. 194:426–432, 2007.
761.e6
Immunohistology of the Breast
224. Mansel RE, Goyal A, Douglas-Jones A, et al: Detection of breast cancer metastasis in sentinel lymph nodes using intra-operative real time GeneSearchtrade mark BLN Assay in the operating room: results of the Cardiff study. Breast Cancer Res Treat. 2008. 225. Martin Martinez MD, Veys I, Majjaj S, et al: Clinical validation of a molecular assay for intra-operative detection of metastases in breast sentinel lymph nodes. Eur J Surg Oncol. 2008. 226. Rubin BP, Skarin AT, Pisick E, et al: Use of cytokeratins 7 and 20 in determining the origin of metastatic carcinoma of unknown primary, with special emphasis on lung cancer. Eur J Cancer Prev. 10:77–82, 2001. 227. Tot T: Adenocarcinomas metastatic to the liver: the value of cytokeratins 20 and 7 in the search for unknown primary tumors. Cancer. 85:171–177, 1999. 228. Perry A, Parisi JE, Kurtin PJ: Metastatic adenocarcinoma to the brain: an immunohistochemical approach. Hum Pathol. 28:938– 943, 1997. 229. Wick MR, Lillemoe TJ, Copland GT, et al: Gross cystic disease fluid protein-15 as a marker for breast cancer: immunohistochemical analysis of 690 human neoplasms and comparison with alpha-lactalbumin. Hum Pathol. 20:281–287, 1989. 230. Pearlman WH, Gueriguian JL, Sawyer ME: A specific progesterone-binding component of human breast cyst fluid. J Biol Chem. 248:5736–5741, 1973. 231. Haagensen DE, Jr, Mazoujian G, Holder WD, Jr, et al: Evaluation of a breast cyst fluid protein detectable in the plasma of breast carcinoma patients. Ann Surg. 185:279–285, 1977. 232. Murphy LC, Lee-Wing M, Goldenberg GJ, et al: Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo: correlation with steroid receptor status. Cancer Res. 47:4160–4164, 1987. 233. Mazoujian G, Bodian C, Haagensen DE, Jr, et al: Expression of GCDFP-15 in breast carcinomas. Relationship to pathologic and clinical factors. Cancer. 63:2156–2161, 1989. 234. Mazoujian G, Margolis R: Immunohistochemistry of gross cystic disease fluid protein (GCDFP-15) in 65 benign sweat gland tumors of the skin. Am J Dermatopathol. 10:28–35, 1988. 235. Mazoujian G, Pinkus GS, Davis S, et al: Immunohistochemistry of a gross cystic disease fluid protein (GCDFP-15) of the breast. A marker of apocrine epithelium and breast carcinomas with apocrine features. Am J Pathol. 110:105–112, 1983. 236. Swanson PE, Pettinato G, Lillemoe TJ, et al: Gross cystic disease fluid protein-15 in salivary gland tumors. Arch Pathol Lab Med. 115:158–163, 1991. 237. Viacava P, Naccarato AG, Bevilacqua G: Spectrum of GCDFP15 expression in human fetal and adult normal tissues. Virchows Arch. 432:255–260, 1998. 238. Ormsby AH, Snow JL, Su WP, et al: Diagnostic immunohistochemistry of cutaneous metastatic breast carcinoma: a statistical analysis of the utility of gross cystic disease fluid protein-15 and estrogen receptor protein. J Am Acad Dermatol. 32:711–716, 1995. 239. Wick MR, Ockner DM, Mills SE, et al: Homologous carcinomas of the breasts, skin, and salivary glands. A histologic and immunohistochemical comparison of ductal mammary carcinoma, ductal sweat gland carcinoma, and salivary duct carcinoma. Am J Clin Pathol. 109:75–84, 1998. 240. Fiel MI, Cernaianu G, Burstein DE, et al: Value of GCDFP-15 (BRST-2) as a specific immunocytochemical marker for breast carcinoma in cytologic specimens. Acta Cytol. 40:637–641, 1996. 241. Kaufmann O, Deidesheimer T, Muehlenberg M, et al: Immunohistochemical differentiation of metastatic breast carcinomas from metastatic adenocarcinomas of other common primary sites. Histopathology. 29:233–240, 1996. 242. Raab SS, Berg LC, Swanson PE, et al: Adenocarcinoma in the lung in patients with breast cancer. A prospective analysis of the discriminatory value of immunohistology. Am J Clin Pathol. 100:27–35, 1993. 243. Striebel JM, Dacic S, Yousem SA: Gross cystic disease fluid protein-(GCDFP-15): expression in primary lung adenocarcinoma. Am J Surg Pathol. 32:426–432, 2008. 244. Domfeh AB, Carley AL, Striebel JM, et al: WT1 immunoreactivity in breast carcinoma: selective expression in pure and mixed mucinous subtypes. Mod Pathol. 21:1217–1223, 2008.
245. Dabbs DJ, Landreneau RJ, Liu Y, et al: Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg. 73:403–405; discussion 406, 2002. 246. Nash JW, Morrison C, Frankel WL: The utility of estrogen receptor and progesterone receptor immunohistochemistry in the distinction of metastatic breast carcinoma from other tumors in the liver. Arch Pathol Lab Med. 127:1591–1595, 2003. 247. Wallace ML, Longacre TA, Smoller BR: Estrogen and progesterone receptors and anti-gross cystic disease fluid protein 15 (BRST-2) fail to distinguish metastatic breast carcinoma from eccrine neoplasms. Mod Pathol. 8:897–901, 1995. 248. Ciampa A, Fanger G, Khan A, et al: Mammaglobin and CRxA-01 in pleural effusion cytology: potential utility of distinguishing metastatic breast carcinomas from other cytokeratin 7-positive/ cytokeratin 20-negative carcinomas. Cancer. 102:368–372, 2004. 249. Han JH, Kang Y, Shin HC, et al: Mammaglobin expression in lymph nodes is an important marker of metastatic breast carcinoma. Arch Pathol Lab Med. 127:1330–1334, 2003. 250. Bhargava R, Dabbs D: Mammaglobin versus GCDFP-15: An immunohistologic validation survey of sensitivity and specificity. Am J Clin Pathol. 2006, In Press. 251. Jager D, Stockert E, Gure AO, et al: Identification of a tissuespecific putative transcription factor in breast tissue by serological screening of a breast cancer library. Cancer Res. 61:2055–2061, 2001. 252. Theurillat JP, Zurrer-Hardi U, Varga Z, et al: NY-BR-1 protein expression in breast carcinoma: a mammary gland differentiation antigen as target for cancer immunotherapy. Cancer Immunol Immunother. 56:1723–1731, 2007. 253. Varga Z, Theurillat JP, Filonenko V, et al: Preferential nuclear and cytoplasmic NY-BR-1 protein expression in primary breast cancer and lymph node metastases. Clin Cancer Res. 12:2745– 2751, 2006. 254. Woodard AH, Yu J, Dabbs DJ, et al: NY-BR-1 and PAX8 immunoreactivity in breast, gynecologic tract, and other CK7+ carcinomas: potential use for determining site of origin. Am J Clin Pathol. 136:428–435, 2011. 255. Ozcan A, Shen SS, Hamilton C, et al: PAX 8 expression in nonneoplastic tissues, primary tumors, and metastatic tumors: a comprehensive immunohistochemical study. Mod Pathol. 24: 751–764, 2011. 256. Long KB, Srivastava A, Hirsch MS, et al: PAX8 Expression in well-differentiated pancreatic endocrine tumors: correlation with clinicopathologic features and comparison with gastrointestinal and pulmonary carcinoid tumors. Am J Surg Pathol. 34:723–729, 2010. 257. Tong GX, Yu WM, Beaubier NT, et al: Expression of PAX8 in normal and neoplastic renal tissues: an immunohistochemical study. Mod Pathol. 22:1218–1227, 2009. 258. Weissferdt A, Moran CA: Pax8 expression in thymic epithelial neoplasms: an immunohistochemical analysis. Am J Surg Pathol. 35:1305–1310, 2011. 259. Noguchi S, Motomura K, Inaji H, et al: Clonal analysis of fibroadenoma and phyllodes tumor of the breast. Cancer Res. 53:4071–4074, 1993. 260. Aranda FI, Laforga JB, Lopez JI: Phyllodes tumor of the breast. An immunohistochemical study of 28 cases with special attention to the role of myofibroblasts. Pathol Res Pract. 190:474–481, 1994. 261. Auger M, Hanna W, Kahn HJ: Cystosarcoma phylloides of the breast and its mimics. An immunohistochemical and ultrastructural study. Arch Pathol Lab Med. 113:1231–1235, 1989. 262. Yeh IT, Francis DJ, Orenstein JM, et al: Ultrastructure of cystosarcoma phyllodes and fibroadenoma. A comparative study. Am J Clin Pathol. 84:131–136, 1985. 262a. Jacobs TW, Chen Y, Guinee DG, et al: Fibroepithelial lesions with cellular stroma on breast core needle biopsy: Are there predictors of outcome on surgical excision? Am J Clin Pathol. 124:342–354, 2005. 263. Kleer CG, Giordano TJ, Braun T, et al: Pathologic, immunohistochemical, and molecular features of benign and malignant phyllodes tumors of the breast. Mod Pathol. 14:185–190, 2001. 264. Kuenen-Boumeester V, Henzen-Logmans SC, Timmermans MM, et al: Altered expression of p53 and its regulated proteins in phyllodes tumours of the breast. J Pathol. 189:169–175, 1999.
References 265. Niezabitowski A, Lackowska B, Rys J, et al: Prognostic evaluation of proliferative activity and DNA content in the phyllodes tumor of the breast: immunohistochemical and flow cytometric study of 118 cases. Breast Cancer Res Treat. 65:77–85, 2001. 266. Shpitz B, Bomstein Y, Sternberg A, et al: Immunoreactivity of p53, Ki-67, and c-erbB-2 in phyllodes tumors of the breast in correlation with clinical and morphologic features. J Surg Oncol. 79:86–92, 2002. 267. Millar EK, Beretov J, Marr P, et al: Malignant phyllodes tumours of the breast display increased stromal p53 protein expression. Histopathology. 34:491–496, 1999. 268. Suo Z, Nesland JM: Phyllodes tumor of the breast: EGFR family expression and relation to clinicopathological features. Ultrastruct Pathol. 24:371–381, 2000. 269. Tse GM, Putti TC, Kung FY, et al: Increased p53 protein expression in malignant mammary phyllodes tumors. Mod Pathol. 15:734–740, 2002. 270. Chen CM, Chen CJ, Chang CL, et al: CD34, CD117, and actin expression in phyllodes tumor of the breast. J Surg Res. 94:84– 91, 2000. 271. Esposito NN, Mohan D, Brufsky A, et al: Phyllodes tumor: a clinicopathologic and immunohistochemical study of 30 cases. Arch Pathol Lab Med. 130:1516–1521, 2006. 272. Sawyer EJ, Poulsom R, Hunt FT, et al: Malignant phyllodes tumours show stromal overexpression of c-myc and c-kit. J Pathol. 200:59–64, 2003. 273. Tse GM, Putti TC, Lui PC, et al: Increased c-kit (CD117) expression in malignant mammary phyllodes tumors. Mod Pathol. 17:827–831, 2004. 274. Djordjevic B, Hanna WM: Expression of c-kit in fibroepithelial lesions of the breast is a mast cell phenomenon. Mod Pathol. 21:1238–1245, 2008. 275. Kersting C, Kuijper A, Schmidt H, et al: Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression. Lab Invest. 86:54–61, 2006. 276. Tse GM, Lui PC, Vong JS, et al: Increased epidermal growth factor receptor (EGFR) expression in malignant mammary phyllodes tumors. Breast Cancer Res Treat. 2008. 277. Inoshita S: Phyllodes tumor (cystosarcoma phyllodes) of the breast. A clinicopathologic study of 45 cases. Acta Pathol Jpn. 38:21–33, 1988. 278. Khan SA, Badve S: Phyllodes tumors of the breast. Curr Treat Options Oncol. 2:139–147, 2001. 279. Shabahang M, Franceschi D, Sundaram M, et al: Surgical management of primary breast sarcoma. Am Surg. 68:673–677; discussion 677, 2002. 280. Kuijper A, de Vos RA, Lagendijk JH, et al: Progressive deregulation of the cell cycle with higher tumor grade in the stroma of breast phyllodes tumors. Am J Clin Pathol. 123:690–698, 2005. 281. Feakins RM, Mulcahy HE, Nickols CD, et al: p53 expression in phyllodes tumours is associated with histological features of malignancy but does not predict outcome. Histopathology. 35:162–169, 1999. 282. Asoglu O, Ugurlu MM, Blanchard K, et al: Risk factors for recurrence and death after primary surgical treatment of malignant phyllodes tumors. Ann Surg Oncol. 11:1011–1017, 2004. 283. Ben Hassouna J, Damak T, Gamoudi A, et al: Phyllodes tumors of the breast: a case series of 106 patients. Am J Surg. 192:141– 147, 2006. 284. Chen WH, Cheng SP, Tzen CY, et al: Surgical treatment of phyllodes tumors of the breast: retrospective review of 172 cases. J Surg Oncol. 91:185–194, 2005. 285. Cheng SP, Chang YC, Liu TP, et al: Phyllodes tumor of the breast: the challenge persists. World J Surg. 30:1414–1421, 2006. 286. Hawkins RE, Schofield JB, Fisher C, et al: The clinical and histologic criteria that predict metastases from cystosarcoma phyllodes. Cancer. 69:141–147, 1992. 287. Kapiris I, Nasiri N, A’Hern R, et al: Outcome and predictive factors of local recurrence and distant metastases following primary surgical treatment of high-grade malignant phyllodes tumours of the breast. Eur J Surg Oncol. 27:723–730, 2001. 288. Tan PH, Jayabaskar T, Chuah KL, et al: Phyllodes tumors of the breast: the role of pathologic parameters. Am J Clin Pathol. 123:529–540, 2005.
761.e7
289. Burga AM, Tavassoli FA: Periductal stromal tumor: a rare lesion with low-grade sarcomatous behavior. Am J Surg Pathol. 27:343– 348, 2003. 290. Garfein CF, Aulicino MR, Leytin A, et al: Epithelioid cells in myoid hamartoma of the breast: a potential diagnostic pitfall for core biopsies. Arch Pathol Lab Med. 120:676–680, 1996. 291. Mathers ME, Shrimankar J: Lobular Neoplasia within a Myoid Hamartoma of the Breast. Breast J. 10:58–59, 2004. 292. Cheng L, Binder SW, Fu YS, et al: Demonstration of estrogen receptors by monoclonal antibody in formalin-fixed breast tumors. Lab Invest. 58:346–353, 1988. 293. Harvey JM, Clark GM, Osborne CK, et al: Estrogen receptor status by immunohistochemistry is superior to the ligandbinding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 17:1474–1481, 1999. 294. McCarty KS, Jr, Miller LS, Cox EB, et al: Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med. 109:716–721, 1985. 295. Allred DC, Harvey JM, Berardo M, et al: Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 11:155–168, 1998. 296. Diaz LK, Sneige N: Estrogen receptor analysis for breast cancer: current issues and keys to increasing testing accuracy. Adv Anat Pathol. 12:10–19, 2005. 297. Press MF, Sauter G, Bernstein L, et al: Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res. 11:6598–6607, 2005. 298. Goldstein NS, Hewitt SM, Taylor CR, et al: Recommendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol. 15:124–133, 2007. 299. Shi SR, Liu C, Taylor CR: Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Technique: From Experiments to Hypothesis. J Histochem Cytochem. 2006. 300. Taylor CR: Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology. Biotech Histochem. 81:3–12, 2006. 301. Jacobs TW, Siziopikou KP, Prioleau JE, et al: Do prognostic marker studies on core needle biopsy specimens of breast carcinoma accurately reflect the marker status of the tumor? Mod Pathol. 11:259–264, 1998. 302. Keshgegian AA, Inverso K, Kline TS: Determination of estrogen receptor by monoclonal antireceptor antibody in aspiration biopsy cytology from breast carcinoma. Am J Clin Pathol. 89:24– 29, 1988. 303. Marrazzo A, Taormina P, Leonardi P, et al: Immunocytochemical determination of estrogen and progesterone receptors on 219 fine-needle aspirates of breast cancer. A prospective study. Anticancer Res. 15:521–526, 1995. 304. Masood S: Estrogen and progesterone receptors in cytology: a comprehensive review. Diagn Cytopathol. 8:475–491, 1992. 305. Masood S, Dee S, Goldstein JD: Immunocytochemical analysis of progesterone receptors in breast cancer. Am J Clin Pathol. 96:59–63, 1991. 306. Zidan A, Christie Brown JS, Peston D, et al: Oestrogen and progesterone receptor assessment in core biopsy specimens of breast carcinoma. J Clin Pathol. 50:27–29, 1997. 307. Beatson GT: On The Treatment of Inoperable Cases of Carcinoma of the Mamma: Suggestions for a New Method of Treatment, with Illustrative Cases. CA Cancer J Clin. 33:108–121, 1983. 308. Jacquemier JD, Hassoun J, Torrente M, et al: Distribution of estrogen and progesterone receptors in healthy tissue adjacent to breast lesions at various stages–immunohistochemical study of 107 cases. Breast Cancer Res Treat. 15:109–117, 1990. 309. Brdar B, Graf D, Padovan R, et al: Estrogen and progesterone receptors as prognostic factors in breast cancer. Tumori. 74:45– 52, 1988. 310. Castagnetta L, Traina A, Carruba G, et al: The prognosis of breast cancer patients in relation to the oestrogen receptor status of both primary disease and involved nodes. Br J Cancer. 66:167– 170, 1992.
761.e8
Immunohistology of the Breast
311. Clark GM, McGuire WL: Steroid receptors and other prognostic factors in primary breast cancer. Semin Oncol. 15:20–25, 1988. 312. Clark GM, McGuire WL, Hubay CA, et al: Progesterone receptors as a prognostic factor in Stage II breast cancer. N Engl J Med. 309:1343–1347, 1983. 313. Crowe JP, Hubay CA, Pearson OH, et al: Estrogen receptor status as a prognostic indicator for stage I breast cancer patients. Breast Cancer Res Treat. 2:171–176, 1982. 314. Lesser ML, Rosen PP, Senie RT, et al: Estrogen and progesterone receptors in breast carcinoma: correlations with epidemiology and pathology. Cancer. 48:299–309, 1981. 315. Pichon MF, Pallud C, Hacene K, et al: Prognostic value of progesterone receptor after long-term follow-up in primary breast cancer. Eur J Cancer. 28A:1676–1680, 1992. 316. Reiner A, Reiner G, Spona J, et al: Histopathologic characterization of human breast cancer in correlation with estrogen receptor status. A comparison of immunocytochemical and biochemical analysis. Cancer. 61:1149–1154, 1988. 317. Neumeister VM, Anagnostou V, Siddiqui S, et al: Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues. J Natl Cancer Inst. 104:1815– 1824, 2012. 318. Li X, Deavers MT, Guo M, et al: The effect of prolonged cold ischemia time on estrogen receptor immunohistochemistry in breast cancer. Mod Pathol. 26:71–78, 2013. 319. Yildiz-Aktas IZ, Dabbs DJ, Bhargava R: The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol. 25:1098–1105, 2012. 320. Yildiz-Aktas IZ, Dabbs DJ, Cooper KL, et al: The effect of 96-hour formalin fixation on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Am J Clin Pathol. 137:691–698, 2012. 321. Goldstein NS, Ferkowicz M, Odish E, et al: Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol. 120:86–92, 2003. 322. Tong D, Schuster E, Seifert M, et al: Expression of estrogen receptor beta isoforms in human breast cancer tissues and cell lines. Breast Cancer Res Treat. 71:249–255, 2002. 323. Esslimani-Sahla M, Simony-Lafontaine J, Kramar A, et al: Estrogen receptor beta (ER beta) level but not its ER beta cx variant helps to predict tamoxifen resistance in breast cancer. Clin Cancer Res. 10:5769–5776, 2004. 324. Hopp TA, Weiss HL, Parra IS, et al: Low levels of estrogen receptor beta protein predict resistance to tamoxifen therapy in breast cancer. Clin Cancer Res. 10:7490–7499, 2004. 325. Speirs V, Parkes AT, Kerin MJ, et al: Coexpression of estrogen receptor alpha and beta: poor prognostic factors in human breast cancer? Cancer Res. 59:525–528, 1999. 326. Stefanou D, Batistatou A, Briasoulis E, et al: Estrogen receptor beta (ERbeta) expression in breast carcinomas is not correlated with estrogen receptor alpha (ERalpha) and prognosis: the Greek experience. Eur J Gynaecol Oncol. 25:457–461, 2004. 327. Matsuyama S, Ohkura Y, Eguchi H, et al: Estrogen receptor beta is expressed in human stomach adenocarcinoma. J Cancer Res Clin Oncol. 128:319–324, 2002. 328. Pais V, Leav I, Lau KM, et al: Estrogen receptor-beta expression in human testicular germ cell tumors. Clin Cancer Res. 9:4475– 4482, 2003. 329. National Institutes of Health Consensus Development Conference statement: adjuvant therapy for breast cancer, November 1-3, 2000. J Natl Cancer Inst Monogr. 5–15, 2001. 330. Cheang MC, Treaba DO, Speers CH, et al: Immunohistochemical detection using the new rabbit monoclonal antibody SP1 of estrogen receptor in breast cancer is superior to mouse monoclonal antibody 1D5 in predicting survival. J Clin Oncol. 24: 5637–5644, 2006. 331. Pertschuk LP, Feldman JG, Kim YD, et al: Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays. Cancer. 77:2514–2519, 1996. 332. Hammond ME, Hayes DF, Dowsett M, et al: American Society of Clinical Oncology/College Of American Pathologists
guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol. 28:2784–2795, 2010. 333. Charpin C, Andrac L, Habib MC, et al: Immunodetection in fine-needle aspirates and multiparametric (SAMBA) image analysis. Receptors (monoclonal antiestrogen and antiprogesterone) and growth fraction (monoclonal Ki67) evaluation in breast carcinomas. Cancer. 63:863–872, 1989. 334. Esteban JM, Kandalaft PL, Mehta P, et al: Improvement of the quantification of estrogen and progesterone receptors in paraffinembedded tumors by image analysis. Am J Clin Pathol. 99:32– 38, 1993. 335. Layfield LJ, Saria EA, Conlon DH, et al: Estrogen and progesterone receptor status determined by the Ventana ES 320 automated immunohistochemical stainer and the CAS 200 image analyzer in 236 early-stage breast carcinomas: prognostic significance. J Surg Oncol. 61:177–184, 1996. 336. McClelland RA, Finlay P, Walker KJ, et al: Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer. Cancer Res. 50:3545–3550, 1990. 337. Veronese SM, Barbareschi M, Morelli L, et al: Predictive value of ER1D5 antibody immunostaining in breast cancer: a paraffinbased retrospective study of 257 cases. Appl Immunohistochem. 3:85–90, 1993. 338. Barnes DM, Harris WH, Smith P, et al: Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. Br J Cancer. 74:1445–1451, 1996. 339. Goulding H, Pinder S, Cannon P, et al: A new immunohistochemical antibody for the assessment of estrogen receptor status on routine formalin-fixed tissue samples. Hum Pathol. 26:291– 294, 1995. 340. Baehner FL, Maddala T, Alexander C, et al: A Kaiser-Permanente population-based study of ER and PR expression by the standard method, immunohistochemistry (IHC), compared to a new method, quantitative reverse transcription polymerase chain reaction (RT-PCR), ASCO Breast Cancer Symposium 2007, Abstract # 88. 341. Cronin M, Pho M, Dutta D, et al: Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol. 164:35–42, 2004. 342. Collins LC, Botero ML, Schnitt SJ: Bimodal frequency distribution of estrogen receptor immunohistochemical staining results in breast cancer: an analysis of 825 cases. Am J Clin Pathol. 123:16–20, 2005. 343. Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et al: Immunohistochemistry of estrogen and progesterone receptors reconsidered: experience with 5,993 breast cancers. Am J Clin Pathol. 123:21–27, 2005. 344. Baehner FL, Watson D, Shak S, et al: Quantitative RT-PCR analysis of ER and PR by Oncotype DX indicates distinct and different associations with prognosis and prediction of tamoxifen benefit, 29th Annual San Antonio Breast Cancer Symposium 2006, Abstract # 45. 345. Mohsin SK, Weiss H, Havighurst T, et al: Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study. Mod Pathol. 17:1545–1554, 2004. 346. Bhargava R, Beriwal S, Dabbs DJ, et al: Immunohistochemical surrogate markers of breast cancer molecular classes predicts response to neoadjuvant chemotherapy: a single institutional experience with 359 cases. Cancer. 116:1431–1439, 2010. 347. Bhargava R, Dabbs DJ, Beriwal S, et al: Semiquantitative hormone receptor level influences response to trastuzumabcontaining neoadjuvant chemotherapy in HER2-positive breast cancer. Mod Pathol. 24:367–374, 2011. 348. Cuzick J, Dowsett M, Pineda S, et al: Prognostic value of a combined estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor 2 immunohistochemical score and comparison with the Genomic Health recurrence score in early breast cancer. J Clin Oncol. 29:4273–4278, 2011. 349. Cohen DA, Dabbs DJ, Cooper KL, et al: Interobserver agreement among pathologists for semiquantitative hormone receptor scoring in breast carcinoma. Am J Clin Pathol. 138:796–802, 2012.
References 350. Kraus JA, Dabbs DJ, Beriwal S, et al: Semi-quantitative immunohistochemical assay versus oncotype DX((R)) qRT-PCR assay for estrogen and progesterone receptors: an independent quality assurance study. Mod Pathol. 25:869–876, 2012. 351. Press M, Spaulding B, Groshen S, et al: Comparison of different antibodies for detection of progesterone receptor in breast cancer. Steroids. 67:799–813, 2002. 352. Liu S, Chia SK, Mehl E, et al: Progesterone receptor is a significant factor associated with clinical outcomes and effect of adjuvant tamoxifen therapy in breast cancer patients. Breast Cancer Res Treat. 119:53–61, 2010. 353. Clark BZ, Dabbs DJ, Cooper KL, et al: Impact of Progesterone Receptor Semiquantitative Immunohistochemical Result on Oncotype DX Recurrence Score: A Quality Assurance Study of 1074 Cases. Appl Immunohistochem Mol Morphol. 2012. 354. Prat A, Cheang MC, Martin M, et al: Prognostic Significance of Progesterone Receptor-Positive Tumor Cells Within Immunohistochemically Defined Luminal A Breast Cancer. J Clin Oncol. 2012. 355. Coussens L, Yang-Feng TL, Liao YC, et al: Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science. 230:1132–1139, 1985. 356. Semba K, Kamata N, Toyoshima K, et al: A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1/epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma. Proc Natl Acad Sci U S A. 82:6497–6501, 1985. 357. Di Fiore PP, Pierce JH, Kraus MH, et al: erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science. 237:178–182, 1987. 358. Akiyama T, Sudo C, Ogawara H, et al: The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science. 232:1644–1646, 1986. 359. Slamon DJ, Clark GM, Wong SG, et al: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 235:177–182, 1987. 360. Owens MA, Horten BC, Da Silva MM: HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 5:63–69, 2004. 361. Yaziji H, Goldstein LC, Barry TS, et al: HER-2 testing in breast cancer using parallel tissue-based methods. JAMA. 291:1972– 1977, 2004. 362. Hayes DF, Thor AD, Dressler LG, et al: HER2 and response to paclitaxel in node-positive breast cancer. N Engl J Med. 357:1496–1506, 2007. 363. Konecny GE, Thomssen C, Luck HJ, et al: Her-2/neu gene amplification and response to paclitaxel in patients with metastatic breast cancer. J Natl Cancer Inst. 96:1141–1151, 2004. 364. Menard S, Valagussa P, Pilotti S, et al: Response to cyclophosphamide, methotrexate, and fluorouracil in lymph node-positive breast cancer according to HER2 overexpression and other tumor biologic variables. J Clin Oncol. 19:329–335, 2001. 365. Pritchard KI, Shepherd LE, O’Malley FP, et al: HER2 and responsiveness of breast cancer to adjuvant chemotherapy. N Engl J Med. 354:2103–2111, 2006. 366. Thor AD, Berry DA, Budman DR, et al: erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst. 90:1346–1360, 1998. 367. Ellis MJ, Coop A, Singh B, et al: Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/ or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial. J Clin Oncol. 19:3808–3816, 2001. 368. Konecny G, Pauletti G, Pegram M, et al: Quantitative association between HER-2/neu and steroid hormone receptors in hormone receptor-positive primary breast cancer. J Natl Cancer Inst. 95:142–153, 2003. 369. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, et al: Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer. N Engl J Med. 354:809–820, 2006. 370. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al: Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 353:1659–1672, 2005.
761.e9
371. Romond EH, Perez EA, Bryant J, et al: Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 353:1673–1684, 2005. 372. Smith I, Procter M, Gelber RD, et al: 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial. Lancet. 369:29–36, 2007. 373. Wolff AC, Hammond ME, Schwartz JN, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 25:118–145, 2007. 374. Portier BP, Wang Z, Downs-Kelly E, et al: Delay to formalin fixation “cold ischemia time”: effect on ERBB2 detection by in-situ hybridization and immunohistochemistry. Mod Pathol. 26:1–9, 2013. 375. Slamon DJ, Leyland-Jones B, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 344:783–792, 2001. 376. Egervari K, Szollosi Z, Nemes Z: Immunohistochemical antibodies in breast cancer HER2 diagnostics. A comparative immunohistochemical and fluorescence in situ hybridization study. Tumour Biol. 29:18–27, 2008. 377. Gouvea AP, Milanezi F, Olson SJ, et al: Selecting antibodies to detect HER2 overexpression by immunohistochemistry in invasive mammary carcinomas. Appl Immunohistochem Mol Morphol. 14:103–108, 2006. 378. Powell WC, Hicks DG, Prescott N, et al: A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility. Appl Immunohistochem Mol Morphol. 15:94–102, 2007. 379. Acs G, Wang L, Raghunath PN, et al: Role of different immunostaining patterns in HercepTest interpretation and criteria for gene amplification as determined by fluorescence in situ hybridization. Appl Immunohistochem Mol Morphol. 11:222–229, 2003. 380. Bhargava R, Naeem R, Marconi S, et al: Tyrosine kinase activation in breast carcinoma with correlation to HER-2/neu gene amplification and receptor overexpression. Hum Pathol. 32: 1344–1350, 2001. 381. Perez EA, Roche PC, Jenkins RB, et al: HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clin Proc. 77:148–154, 2002. 382. Garcia-Caballero T, Menendez MD, Vazquez-Boquete A, et al: HER-2 status determination in breast carcinomas. A practical approach. Histol Histopathol. 21:227–236, 2006. 383. Tsuda H, Akiyama F, Terasaki H, et al: Detection of HER-2/neu (c-erb B-2) DNA amplification in primary breast carcinoma. Interobserver reproducibility and correlation with immunohistochemical HER-2 overexpression. Cancer. 92:2965–2974, 2001. 384. Tubbs RR, Pettay JD, Roche PC, et al: Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message. J Clin Oncol. 19:2714–2721, 2001. 385. Chorn N: Accurate identification of HER2-positive patients is essential for superior outcomes with trastuzumab therapy. Oncol Nurs Forum. 33:265–272, 2006. 386. Mass RD, Press MF, Anderson S, et al: Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clin Breast Cancer. 6:240–246, 2005. 387. Clinical laboratory assays for HER-2/neu amplification and overexpression: quality assurance, standardization, and proficiency testing. Arch Pathol Lab Med. 126:803–808, 2002. 388. Rhodes A, Borthwick D, Sykes R, et al: The use of cell line standards to reduce HER-2/neu assay variation in multiple European cancer centers and the potential of automated image analysis to provide for more accurate cut points for predicting clinical response to trastuzumab. Am J Clin Pathol. 122:51–60, 2004. 389. Zarbo RJ, Hammond ME: Conference summary, Strategic Science symposium. Her-2/neu testing of breast cancer patients in clinical practice. Arch Pathol Lab Med. 127:549–553, 2003.
761.e10
Immunohistology of the Breast
390. Persons DL, Tubbs RR, Cooley LD, et al: HER-2 fluorescence in situ hybridization: results from the survey program of the College of American Pathologists. Arch Pathol Lab Med. 130: 325–331, 2006. 391. Chivukula M, Bhargava R, Brufsky A, et al: Clinical importance of HER2 immunohistologic heterogeneous expression in coreneedle biopsies vs resection specimens for equivocal (immunohistochemical score 2+) cases. Mod Pathol. 21:363–368, 2008. 392. Lal P, Salazar PA, Hudis CA, et al: HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring. Am J Clin Pathol. 121:631–636, 2004. 393. Bose S, Mohammed M, Shintaku P, et al: Her-2/neu gene amplification in low to moderately expressing breast cancers: possible role of chromosome 17/Her-2/neu polysomy. Breast J. 7:337– 344, 2001. 394. Striebel JM, Bhargava R, Horbinski C, et al: The equivocally amplified HER2 FISH result on breast core biopsy: indications for further sampling do affect patient management. Am J Clin Pathol. 129:383–390, 2008. 395. Bhargava R, Lal P, Chen B: Chromogenic in situ hybridization for the detection of HER-2/neu gene amplification in breast cancer with an emphasis on tumors with borderline and lowlevel amplification: does it measure up to fluorescence in situ hybridization? Am J Clin Pathol. 123:237–243, 2005. 396. Gupta D, Middleton LP, Whitaker MJ, et al: Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer. Am J Clin Pathol. 119:381–387, 2003. 397. Isola J, Tanner M, Forsyth A, et al: Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization. Clin Cancer Res. 10:4793– 4798, 2004. 398. Tanner M, Gancberg D, Di Leo A, et al: Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. Am J Pathol. 157:1467–1472, 2000. 399. Bartlett JM, Campbell FM, Ibrahim M, et al: A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay. Am J Clin Pathol. 135:157–162, 2011. 400. Bartlett JM, Campbell FM, Ibrahim M, et al: Chromogenic in situ hybridization: a multicenter study comparing silver in situ hybridization with FISH. Am J Clin Pathol. 132:514–520, 2009. 401. Brugmann A, Lelkaitis G, Nielsen S, et al: Testing HER2 in breast cancer: a comparative study on BRISH, FISH, and IHC. Appl Immunohistochem Mol Morphol. 19:203–211, 2011. 402. Baehner FL, Achacoso N, Maddala T, et al: Human epidermal growth factor receptor 2 assessment in a case-control study: comparison of fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories. J Clin Oncol. 28:4300–4306, 2010. 403. Dabbs DJ, Klein ME, Mohsin SK, et al: High false-negative rate of HER2 quantitative reverse transcription polymerase chain reaction of the Oncotype DX test: an independent quality assurance study. J Clin Oncol. 29:4279–4285, 2011. 404. Dvorak L, Dolan M, Fink J, et al: Correlation Between HER2 Determined by Fluorescence In Situ Hybridization and Reverse Transcription-Polymerase Chain Reaction of the Oncotype DX Test. Appl Immunohistochem Mol Morphol. 2012. 405. Bartlett JM, Starczynski J: Quantitative reverse transcriptase polymerase chain reaction and the Oncotype DX test for assessment of human epidermal growth factor receptor 2 status: time to reflect again? J Clin Oncol. 29:4219–4221, 2011. 406. Bhargava R, Dabbs DJ: Oncotype DX test on unequivocally HER2-positive cases: potential for harm. J Clin Oncol. 30:570– 571, 2012. 407. Barton MK: Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Oncotype DX. CA: Cancer J Clin. 2012. 408. Ignatiadis M, Sotiriou C: Breast cancer: Should we assess HER2 status by Oncotype DX? Nat Rev Clin Oncol. 9:12–14, 2012.
409. Golub TR, Slonim DK, Tamayo P, et al: Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science. 286:531–537, 1999. 410. Sorlie T: Molecular classification of breast tumors: toward improved diagnostics and treatments. Methods Mol Biol. 360:91– 114, 2007. 411. Bhargava R, Striebel J, Beriwal S, et al: Prevalence, morphologic features and proliferation indices of breast carcinoma molecular classes using immunohistochemical surrogate markers. Int J Clin Exp Pathol. 2:444–455, 2009. 412. Cheang MC, Chia SK, Voduc D, et al: Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst. 101:736–750, 2009. 413. Nielsen TO, Parker JS, Leung S, et al: A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptorpositive breast cancer. Clin Cancer Res. 16:5222–5232, 2010. 414. Eisinger F, Jacquemier J, Charpin C, et al: Mutations at BRCA1: the medullary breast carcinoma revisited. Cancer Res. 58:1588– 1592, 1998. 415. Kovi J, Mohla S, Norris HJ, et al: Breast lesions in black women. Pathol Annu. 24(Pt 1):199–218, 1989. 416. Liu X, Holstege H, van der Gulden H, et al: Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc Natl Acad Sci U S A. 104:12111–12116, 2007. 417. Mittra NK, Rush BF, Jr, Verner E: A comparative study of breast cancer in the black and white populations of two inner-city hospitals. J Surg Oncol. 15:11–17, 1980. 418. Natarajan N, Nemoto T, Mettlin C, et al: Race-related differences in breast cancer patients. Results of the 1982 national survey of breast cancer by the American College of Surgeons. Cancer. 56:1704–1709, 1985. 419. Rosen PP, Lesser ML, Kinne DW: Breast carcinoma at the extremes of age: a comparison of patients younger than 35 years and older than 75 years. J Surg Oncol. 28:90–96, 1985. 420. Jumppanen M, Gruvberger-Saal S, Kauraniemi P, et al: Basal-like phenotype is not associated with patient survival in estrogenreceptor-negative breast cancers. Breast Cancer Res. 9:R16, 2007. 421. Cheang MC, Voduc D, Bajdik C, et al: Basal-like breast cancer defined by five biomarkers has superior prognostic value than triple-negative phenotype. Clin Cancer Res. 14:1368–1376, 2008. 422. Niemeier LA, Dabbs DJ, Beriwal S, et al: Androgen receptor in breast cancer: expression in estrogen receptor-positive tumors and in estrogen receptor-negative tumors with apocrine differentiation. Mod Pathol. 23:205–212, 2010. 423. Farmer P, Bonnefoi H, Becette V, et al: Identification of molecular apocrine breast tumours by microarray analysis. Oncogene. 24:4660–4671, 2005. 424. Parker JS, Mullins M, Cheang MC, et al: Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol. 27:1160–1167, 2009. 425. Weigelt B, Horlings H, Kreike B, et al: Refinement of breast cancer classification by molecular characterization of histological special types. J Pathol. 216:141–150, 2008. 426. Rouzier R, Perou CM, Symmans WF, et al: Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res. 11:5678–5685, 2005. 427. Houssami N, Macaskill P, von Minckwitz G, et al: Meta-analysis of the association of breast cancer subtype and pathologic complete response to neoadjuvant chemotherapy. Eur J Cancer. 48:3342–3354, 2012. 428. van de Vijver MJ, He YD, van’t Veer LJ, et al: A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med. 347:1999–2009, 2002. 429. van ‘t Veer LJ, Dai H, van de Vijver MJ, et al: Gene expression profiling predicts clinical outcome of breast cancer. Nature. 415:530–536, 2002. 430. Glas AM, Floore A, Delahaye LJ, et al: Converting a breast cancer microarray signature into a high-throughput diagnostic test. BMC Genomics. 7:278, 2006. 431. Harris L, Fritsche H, Mennel R, et al: American Society of Clinical Oncology 2007 update of recommendations for the use of
References tumor markers in breast cancer. J Clin Oncol. 25:5287–5312, 2007. 432. Chang HY, Nuyten DS, Sneddon JB, et al: Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci U S A. 102:3738–3743, 2005. 433. Wang Y, Klijn JG, Zhang Y, et al: Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 365:671–679, 2005. 434. Foekens JA, Atkins D, Zhang Y, et al: Multicenter validation of a gene expression-based prognostic signature in lymph nodenegative primary breast cancer. J Clin Oncol. 24:1665–1671, 2006. 435. Paik S, Shak S, Tang G, et al: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 351:2817–2826, 2004. 436. Paik S, Tang G, Shak S, et al: Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol. 24:3726–3734, 2006. 437. Flanagan MB, Dabbs DJ, Brufsky AM, et al: Histopathologic variables predict Oncotype DX™ recurrence score. Mod Pathol. 21:1255–1261, 2008. 438. Klein ME, Dabbs DJ, Shuai Y, et al: Prediction of the oncotype DX recurrence score: Use of pathology generated equations derived by linear regression analysis. Mod Pathol. 26:658–664, 2013. 439. Ma XJ, Wang Z, Ryan PD, et al: A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell. 5:607–616, 2004. 440. Ma XJ, Hilsenbeck SG, Wang W, et al: The HOXB13:IL17BR expression index is a prognostic factor in early-stage breast cancer. J Clin Oncol. 24:4611–4619, 2006. 441. Fan C, Oh DS, Wessels L, et al: Concordance among geneexpression-based predictors for breast cancer. N Engl J Med. 355:560–569, 2006. 442. Ma XJ, Salunga R, Dahiya S, et al: A five-gene molecular grade index and HOXB13:IL17BR are complementary prognostic factors in early stage breast cancer. Clin Cancer Res. 14:2601– 2608, 2008. 443. Ma XJ, Salunga R, Tuggle JT, et al: Gene expression profiles of human breast cancer progression. Proc Natl Acad Sci U S A. 100:5974–5979, 2003. 444. Sotiriou C, Wirapati P, Loi S, et al: Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst. 98:262–272, 2006. 445. Gao JP, Uchida T, Wang C, et al: Relationship between p53 gene mutation and protein expression: clinical significance in transitional cell carcinoma of the bladder. Int J Oncol. 16:469–475, 2000. 446. Salinas-Sanchez AS, Atienzar-Tobarra M, Lorenzo-Romero JG, et al: Sensitivity and specificity of p53 protein detection by immunohistochemistry in patients with urothelial bladder carcinoma. Urol Int. 79:321–327, 2007. 447. Olivier M, Langerod A, Carrieri P, et al: The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer. Clin Cancer Res. 12:1157–1167, 2006. 448. Bartlett JM, Bloom KJ, Piper T, et al: Mammostrat as an immunohistochemical multigene assay for prediction of early relapse risk in the tamoxifen versus exemestane adjuvant multicenter trial pathology study. J Clin Oncol. 30:4477–4484, 2012. 449. Bartlett JM, Thomas J, Ross DT, et al: Mammostrat as a tool to stratify breast cancer patients at risk of recurrence during endocrine therapy. Breast Cancer Res. 12:R47, 2010. 450. Ring BZ, Seitz RS, Beck RA, et al: A novel five-antibody immunohistochemical test for subclassification of lung carcinoma. Mod Pathol. 22:1032–1043, 2009. 451. Bhargava R, Brufsky AM, Davidson NE: Prognostic/Predictive immunohistochemistry assays for estrogen receptor-positive breast cancer: back to the future? J Clin Oncol. 30:4451–4453, 2012. 452. Caly M, Genin P, Ghuzlan AA, et al: Analysis of correlation between mitotic index, MIB1 score and S-phase fraction as proliferation markers in invasive breast carcinoma.
761.e11
Methodological aspects and prognostic value in a series of 257 cases. Anticancer Res. 24:3283–3288, 2004. 453. Gonzalez-Vela MC, Garijo MF, Fernandez F, et al: MIB1 proliferation index in breast infiltrating carcinoma: comparison with other proliferative markers and association with new biological prognostic factors. Histol Histopathol. 16:399–406, 2001. 454. Molino A, Micciolo R, Turazza M, et al: Ki-67 immunostaining in 322 primary breast cancers: associations with clinical and pathological variables and prognosis. Int J Cancer. 74:433–437, 1997. 455. Nakagomi H, Miyake T, Hada M, et al: Prognostic and Therapeutic Implications of the MIB-1 Labeling Index in Breast Cancer. Breast Cancer. 5:255–259, 1998. 456. Colozza M, Azambuja E, Cardoso F, et al: Proliferative markers as prognostic and predictive tools in early breast cancer: where are we now? Ann Oncol. 16:1723–1739, 2005. 457. Perou CM, Jeffrey SS, van de Rijn M, et al: Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A. 96:9212–9217, 1999. 458. Dowsett M, Nielsen TO, A’Hern R, et al: Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. J Natl Cancer Inst. 103:1656– 1664, 2011. 459. Bhargava R, Gerald WL, Li AR, et al: EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations. Mod Pathol. 18:1027–1033, 2005. 460. Reis-Filho JS, Pinheiro C, Lambros MB, et al: EGFR amplification and lack of activating mutations in metaplastic breast carcinomas. J Pathol. 209:445–453, 2006. 461. Gholam D, Chebib A, Hauteville D, et al: Combined paclitaxel and cetuximab achieved a major response on the skin metastases of a patient with epidermal growth factor receptor-positive, estrogen receptor-negative, progesterone receptor-negative and human epidermal growth factor receptor-2-negative (triplenegative) breast cancer. Anticancer Drugs. 18:835–837, 2007. 462. Modi S, D’Andrea G, Norton L, et al: A phase I study of cetuximab/paclitaxel in patients with advanced-stage breast cancer. Clin Breast Cancer. 7:270–277, 2006. 463. Duffy MJ: Urokinase plasminogen activator and its inhibitor, PAI-1, as prognostic markers in breast cancer: from pilot to level 1 evidence studies. Clin Chem. 48:1194–1197, 2002. 464. Foekens JA, Schmitt M, van Putten WL, et al: Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. J Clin Oncol. 12:1648–1658, 1994. 465. Look MP, van Putten WL, Duffy MJ, et al: Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst. 94:116–128, 2002. 466. Visscher DW, Sarkar F, LoRusso P, et al: Immunohistologic evaluation of invasion-associated proteases in breast carcinoma. Mod Pathol. 6:302–306, 1993. 467. Schmitt M, Sturmheit AS, Welk A, et al: Procedures for the quantitative protein determination of urokinase and its inhibitor, PAI-1, in human breast cancer tissue extracts by ELISA. Methods Mol Med. 120:245–265, 2006. 468. Grimberg A, Cohen P: Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis. J Cell Physiol. 183:1–9, 2000. 469. Belfiore A, Frasca F: IGF and insulin receptor signaling in breast cancer. J Mammary Gland Biol Neoplasia. 13:381–406, 2008. 470. Ouban A, Muraca P, Yeatman T, et al: Expression and distribution of insulin-like growth factor-1 receptor in human carcinomas. Hum Pathol. 34:803–808, 2003. 471. Papa V, Gliozzo B, Clark GM, et al: Insulin-like growth factor-I receptors are overexpressed and predict a low risk in human breast cancer. Cancer Res. 53:3736–3740, 1993. 472. Bhargava R, Beriwal S, McManus K, et al: Insulin-like growth factor receptor-1 (IGF-1R) expression in normal breast, proliferative breast lesions, and breast carcinoma. Appl Immunohistochem Mol Morphol. 19:218–225, 2011. 473. Castiglione F, Sarotto I, Fontana V, et al: Bcl2, p53 and clinical outcome in a series of 138 operable breast cancer patients. Anticancer Res. 19:4555–4563, 1999.
761.e12
Immunohistology of the Breast
474. Ioachim EE, Malamou-Mitsi V, Kamina SA, et al: Immunohistochemical expression of Bcl-2 protein in breast lesions: correlation with Bax, p53, Rb, C-erbB-2, EGFR and proliferation indices. Anticancer Res. 20:4221–4225, 2000. 475. Kroger N, Milde-Langosch K, Riethdorf S, et al: Prognostic and predictive effects of immunohistochemical factors in high-risk primary breast cancer patients. Clin Cancer Res. 12:159–168, 2006. 476. Leek RD, Kaklamanis L, Pezzella F, et al: bcl-2 in normal human breast and carcinoma, association with oestrogen receptorpositive, epidermal growth factor receptor-negative tumours and in situ cancer. Br J Cancer. 69:135–139, 1994. 477. Abdel-Fatah TM, Powe DG, Ball G, et al: Proposal for a modified grading system based on mitotic index and Bcl2 provides objective determination of clinical outcome for patients with breast cancer. J Pathol. 222:388–399, 2010. 478. Badve S, Turbin D, Thorat MA, et al: FOXA1 expression in breast cancer–correlation with luminal subtype A and survival. Clin Cancer Res. 13:4415–4421, 2007.
479. Laganiere J, Deblois G, Lefebvre C, et al: From the Cover: Location analysis of estrogen receptor alpha target promoters reveals that FOXA1 defines a domain of the estrogen response. Proc Natl Acad Sci U S A. 102:11651–11656, 2005. 480. Thorat MA, Marchio C, Morimiya A, et al: Forkhead box A1 expression in breast cancer is associated with luminal subtype and good prognosis. J Clin Pathol. 61:327–332, 2008. 481. Badve S, Nakshatri H: Oestrogen receptor-positive breast cancer: towards bridging histopathologic and molecular classifications. J Clin Pathol. 2008. 482. Mehra R, Varambally S, Ding L, et al: Identification of GATA3 as a breast cancer prognostic marker by global gene expression meta-analysis. Cancer Res. 65:11259–11264, 2005. 483. Voduc D, Cheang M, Nielsen T: GATA-3 expression in breast cancer has a strong association with estrogen receptor but lacks independent prognostic value. Cancer Epidemiol Biomarkers Prev. 17:365–373, 2008.
C H A P T E R 2 0
IMMUNOHISTOLOGY OF THE NERVOUS SYSTEM PAUL E. MCKEEVER
Overview 762 Clinical and Radiologic Perspective of Lesions 764 Nonneoplastic Brain Lesions 767 Tumors of the Nervous System 775 Cysts of the Nervous System 818 Dementias 819 Demyelination 821 Epilepsy 822 Pitfalls in Diagnosis 824 Summary 828
Overview This chapter focuses on the diagnostic immunohistochemistry (IHC) of nervous system diseases. Concise information about pathologic entities is provided in Tables 20-2 to 20-13 and in the algorithms in Figures 20-1 through 20-3. A suspected specific disease can be checked directly in the individual table in which its structural, IHC, and topographic features are listed; the algorithms can also be used. The text and figures elaborate on these features.1-3 Features of unknown diseases may be found in individual algorithms and tables to assist diagnosis. For example, for a mass, specific tables in the chapter summarize differential features of the mass, as follows: • Fibrillar cells: Table 20-7; Figures 20-11, 20-12, and 20-18 • Epithelioid cells: Table 20-2; Figures 20-4, 20-22, and 20-39 • More than one type of cell: Table 20-8; Figure 20-34 • Small anaplastic cells: Table 20-9; Figures 20-24 and 20-41 to 20-44 • Syncytial cells: Table 20-10; Figure 20-49 762
These features are evident on cytologic and histologic preparations.3,4 Figure 20-1 focuses on the differential diagnosis of clear cell lesions, Figure 20-2 displays the differential diagnosis IHC of epithelioid tumors, and Figure 20-3 displays the differential diagnosis of so-called blue tumors. IHC stains that are particularly useful for diagnosis of nervous system diseases are listed in alphabetic order in Table 20-1. IHC should always be controlled. I prefer selecting a specimen with all of the following items: 1) the lesion of interest; 2) tissue with regions that should react positively to the stain; and 3) tissue with regions that should react negatively to the stain. Regions 2 and 3 serve as internal standard tissue controls.1,5 For example, a specimen stained with glial fibrillary acidic protein (GFAP) could contain regions of gliosis (positive control) and vessels (negative control) in the same block. This approach is better than using a separate tissue control block (STCB) that probably is neither fixed nor treated exactly the same as the one in question. These differences remain when a section of STCB is placed on the same slide with the specimen, and they are aggravated if additional heat is needed to make both sections adhere to one slide. Although normal and reactive tissues retain their expected immunophenotype, individual neoplasms may not stain for a marker generally representative of their type.5,6 Because of this, a positive immunostaining result is more meaningful than a negative result. We will emphasize the positive features in this chapter. If a lesion is not identified immediately, a differential diagnosis may be constructed for which a group of appropriate IHC stains is described in the text, algorithms, and tables. The following example describes the application of this approach to an actual case. Figure 20-4 shows a cerebral tumor from the lumbosacral region of a middle-aged woman. The hematoxylin and eosin (H&E)–stained slide reveals a neoplasm with mainly epithelioid cells and a few clear cells and abundant round to oval nuclei with fine chromatin (Table 20-2; see Fig. 20-4, A). Its IHC is focally positive to epithelial membrane antigen (EMA; see Fig. 20-4, B). Carcinoma, chordoma, craniopharyngioma, pituitary adenoma, and meningioma are EMA positive (see
Overview
Immunohistochemical stain response
Margin
763
Entity
Diffuse
Oligodendroglioma PXA
Syn–
Clear cell ependymoma KP1–
GFAP+
DNT
Sharp
Syn+
EMA–
Central neurocytoma Demyelination KP1+
Infarct
Syn– GFAP–
EMA+
Clear cell meningioma KP1–
Sharp
Renal cell carcinoma
EMA–
Hemangioblastoma
Figure 20-1 Differential diagnoses of clear cell lesions. Empty boxes reflect neoplasms for which the feature or the immunohistochemical stain response is not decisive. DNT, Dysembryoplastic neuroepithelial tumor; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; KP1, CD68; PXA, pleomorphic xanthoastrocytoma; Syn, synaptophysin. Modified from Gokden M, Roth KA, Garroll SL, et al: Clear cell neoplasms and pesudoneoplastic lesions of the central nervous system. Semin Diagn Pathol 1997;14:253-269.
Figs. 20-1 and 20-2). This tumor is negative for cytokeratin CAM5.2 (see Fig. 20-4, C), so it does not fit the IHC profile of carcinoma, chordoma, or craniopharyngioma. It is negative for chromogranin A (see Fig. 20-4, D) and negative for hormones of pituitary adenomas. It was also found to be negative for GFAP, synaptophysin, H&E
and human melanoma black 15 (HMB-45). Table 20-2 summarizes the differential diagnosis for epithelioid cells, confirms the IHC profile for meningioma, and notes common features and locations. The tumor in the example was observed to have rare whorls and to involve the spinal meninges. In comparison with descriptions of
Oligodendroglioma, astrocytoma Ependymoma Electron microscopy
+
Medulloepithelioma Transthyretin
+ or –
+ Choroid plexus tumor
+ Chordoma, craniopharyngioma
GFAP
S-100
+
– Carcinoma
+ Pit adenoma +
Pit hrm – Paraganglioma
–
CAM 5.2
+ or –
CgA
–
EMA
+ Meningioma
+ Melanoma –
HMB-45 – Hemangioblastoma
Figure 20-2 Immunohistochemical diagnosis of epithelioid cell tumors. CgA, Chromogranin A; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; H&E, hematoxylin and eosin; HMB-45, human melanoma black 45; Pit, pituitary; Pit hrm, pituitary hormone.
764
Immunohistology of the Nervous System
Posterior fossa
+
Medulloblastoma
Pineoblastoma
Pineal gland
PNET
Anterior fossa
Synaptophysin
Figure 20-3 Immunohistochemical diagnosis of tumor crowded with malignant nuclei and little cytoplasm. CLA, Common leukocyte antigen; PNET, primitive neuroectodermal tumor.
+ Lymphoma −
CLA, CD20 −
CAM 5.2
+ Carcinoma
meningiomas in the text, the cells were epithelioid and only focally syncytial. The clear cells were not prominent and lacked the cytoplasmic glycogen found in the clear cell variant. The tumor was a meningothelial meningioma with prominent epithelioid appearance.1,3 Brain biopsies for nonneoplastic diseases often require IHC combined with additional studies such as microbiologic culture, polymerase chain reaction (PCR), Western blot, or electron microscopy (EM).7-9 Specialized centers are available to assist with interpretations of the results of these studies.9-13
Clinical and Radiologic Perspective of Lesions Major categories of lesions of the brain, spinal cord, and meninges—such as solitary and multiple masses, cysts, vascular malformations, and abscesses—are likely to be recognized clinically through the use of computed tomography (CT), magnetic resonance imaging (MRI), or angiography. New methods of using flow voids with CT or MRI have provided noninvasive evaluations of
A
B
C
D
Figure 20-4 This actual case illustrates how the algorithms (see Figs. 20-1 and 20-2), tables, and text assist interpretation from initial impression to final diagnosis. A, Large mass is from the lumbosacral region of a middle-aged woman (hematoxylin and eosin). It is focally positive for epithelial membrane antigen (B), negative for CAM5.2 (C), and negative for chromogranin A (D). It also reacted positively to vimentin and negatively to glial fibrillary acidic protein and synaptophysin (not shown). This tumor is a meningothelial meningioma with prominent epithelioid cells. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC, 2004: Armed Forces Institute of Pathology.
Clinical and Radiologic Perspective of Lesions
765
TABLE 20-1 Immunohistochemical Stains Used for Nervous Tissue Primary Antibody, Source and Dilution*
Principal Lesions and Tissue Components
Antigen Rescue*
A6 (CD45RO), Zymed 1 : 50 or CD3
T lymphocytes
Mw 15 min in citrate, pH 6.0
Chromogranin A,† 1 : 160
Pituitary adenoma, paraganglioma, neuroendocrine tumors
Mw 15 min in citrate, pH 6.0
Collagen type IV, Dako 1 : 8
Fibrosis, abscess, sarcoma, teratoma, fibrous cyst and vessel walls, dura, pituitary fibrovascular stroma
Ventana protease 1, 16 min
CAM5.2 cytokeratin, BD 1 : 10
Carcinoma, craniopharyngioma, chordoma, epithelia
Ventana protease 2, 16 min
EMA, Dako 1 : 50
Carcinoma, meningioma, craniopharyngioma, chordoma, epithelia, (few ependymomas)
Mw 15 min in citrate, pH 6.0
GFAP, Dako 1 : 6400
Gliosis, gliomas, CNS parenchyma
Ventana protease 2, 16 min
Herpesvirus antigen, Dako 1 : 1000
Herpes simplex encephalitis, CMV, herpes zoster
None
IDH1 mutant
Gliomas
JC virus/SV40 viral antigen, Lee Biomolecular 1 : 500
Progressive multifocal leukoencephalopathy
None
KP1 (CD68), Dako 1 : 1600
Macrophages
Mw 15 min in citrate, pH 6.0
Leu-7 (CD57)
Most gliomas, particularly oligodendrogliomas, hemangiopericytomas
L26 (CD20), Dako 1 : 500
B lymphocytes, B lymphoma
Mw 15 min in citrate, pH 6.0
MIB-1, Immunotech 1 : 25
Proliferating cells
Mw 15 min in citrate, pH 6.0
Neuron-specific enolase
Darkest in medulloblastoma/PNET and hemangioblastoma, most gliomas
NeuN
Neuronal tumors, normal neurons
CD163
Macrophages and activated macrophages
Neurofilament, Dako 1 : 50
Ganglion cell tumors, neurofibroma, PNET, Alzheimer disease, CNS parenchyma
Neurofilament and Luxol fast blue
Brain: demyelination, necrosis
p53
Astrocytic gliomas
Phosphohistone H3
Screening for mitoses
Prealbumin, Dako 1 : 500
Choroid plexus tumors
None
S-100 protein, Dako 1 : 500
Gliomas, PNET, melanoma, schwannoma, neurofibroma, neuronal and chondroid tumors, chordoma, CNS, PNS
None
Synaptophysin, BioGenex 1 : 600
Neuronal and pineal tumors, PNET, medulloblastoma
Mw 10 min in citrate, pH 6.0
Toxoplasma, BioGenex neat
Toxoplasmosis
None
Vimentin, Dako 1 : 800
Many cells, excessive in meningiomas
Mw 15 min in citrate, pH 6.0
Mw 15 min in citrate, pH 6.0
*Data from McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G (eds): Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004, with the expert advice and careful assistance of the immunohistology staff of the Immunoperoxidase Laboratory, Department of Pathology, University of Michigan Medical School. † Courtesy Dr. Riccardo Lloyd, University of Wisconsin–Madison. BD, Becton Dickinson; CMV, cytomegalovirus; CNS, central nervous system; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; PNET, primitive neuroectodermal tumor; PNS, peripheral nervous system; Mw, microwave starting in cold buffer for time noted.
766
Immunohistology of the Nervous System
TABLE 20-2 Differential Diagnosis of a Mass of Epithelioid Cells Differential Features Diagnosis*
Structures
Antibody†
Locations‡
Gitter cells/ xanthogranuloma
Crowded macrophages engorged with lipid vacuoles; eccentric nucleus; noncohesive cells
α-ACT (S), KP1 (+), muramidase (S)
CNS
Ependymoma/ malignant ependymoma
Structures of ependymoma or malignant ependymoma plus epithelioid cells
GFAP (S), cytokeratin (R), EMA (R)
Cerebellum, cerebrum, spinal cord, CNS
Myxopapillary ependymoma
Cuboidal/columnar epithelium on hyaline fibrovascular papillae, variable fibrillarity
GFAP (S)
Regions of the filum terminale
Oligodendroglioma
Round cells and nuclei with prominent perinuclear halos, nests of cells between delicate vessels
Leu-7 (+), S-100 (+), GFAP (R), IDH1 mutant
Cerebrum, CNS
Anaplastic oligodendroglioma
Features of oligodendroglioma with mitoses and pleomorphism
Leu-7 (S), S-100 (S), GFAP (R), IDH1 mutant
Cerebrum, CNS
Choroid plexus papilloma
Large mass with structure of choroid plexus
Laminin (+), cytokeratin (+), transthyretin (S), synaptophysin, IGF-2
Fourth ventricle, lateral ventricle, CP angle, choroid plexus
Choroid plexus carcinoma
Features of choroid plexus papilloma with anaplasia and mitoses, (necrosis§)
Cytokeratin (+), CD44, synaptophysin, transthyretin (R)
Same as for choroid plexus papilloma
Medulloepithelioma
Columnar epithelium with basement membrane on both surfaces, fibrovascular base for papillae and tubules
GFAP, nestin
Deep cerebrum, cauda equina, CNS
Meningioma
Whorls, psammoma bodies, interdigitating cell processes and desmosomes, (thick collagen)§
Vimentin (+), EMA (S), S-100 (R)
Falx, tentorium, meninges, choroid plexus, (extracranial)§
Chordoma
Masses or cords of physaliphorous cells
Cytokeratin (+), S-100 (+), EMA (+), vimentin (+)
Cauda equina, clivus, spinal canal
Craniopharyngioma
Squamous, adamantinomatous
Cytokeratin (+)
Suprasellar, sellar
Carcinoma
Distinct margin with CNS; anaplasia, mitoses, necrosis
Cytokeratin (+), EMA (S)
Cerebrum, cerebellum, meninges, CNS, frequent multiple masses, systemic
Melanoma
Anaplasia, mitoses, necrosis
HMB-45 (S), S-100 (+)
CNS, meninges
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *The order of tabulated lesions follows the order of discussion in text. † Key to staining results: +, almost always strong, diffuse positivity; S, sometimes or focally positive; R, rare cells may be positive. ‡ The most common or most specific location is listed first. § Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. α-ACT, Alpha-antichymotrypsin; CNS, central nervous system; CP; cerebellopontine; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; HMB-45, human melanoma black 45; IGF-2, insulin-like growth factor II.
vessels similar to magnetic resonance angiography (MRA). MRI can be targeted on specific central nervous system (CNS) chemicals, such as choline and N-acetylaspartate (NAA), to help identify tumor tissue. Diffusion-weighted MRIs can differentiate tissues by their water movement in three dimensions.14 Multiple lesions can be produced by degenerative, vascular, and infectious diseases or by neoplasms. Regarding neoplasms, the M rule for differential diagnosis of common multiple CNS neoplasms includes metastases, malignant lymphoma, melanoma, and medulloblastoma.13
Depending upon its age, the tomographic density of hemorrhage is often sufficiently unique to identify it as a major component of a lesion. Calcifications and relationships with the skull are resolved well on CT, whereas gray and white matter, edema, and melanin are better seen on MRI. Vascular abnormalities are frequently defined by MRA, or, if needed, they may be defined angiographically. Nonneoplastic lesions are often evaluated by a neurologist. Thus a major neurologic symptom (pain, weakness, or visual loss) or category of neurologic disease (e.g., dementia) may focus the differential diagnosis (Table 20-3).
Nonneoplastic Brain Lesions
767
TABLE 20-3 Biopsies Directed Toward a Neurologic Symptom or Specific Disease Symptom/Suspected Disease*
Confirmatory Features of Suspected Disease Structures
Antibody
Locations†
Herpes simplex encephalitis
Encephalitis (Table 20-4); Cowdry A amphophilic nuclear inclusions of 90 to 100 nm target capsids
Herpes simplex virus
Temporal or basilar frontal lobe(s), CNS; frequently bilateral
Toxoplasmosis
Necrosis containing 3 to 5 µm tachyzoites; (cysts); (inflammation)‡
Toxoplasma
CNS, frequent multiple lesions
Progressive multifocal leukoencephalopathy
Demyelination; bizarre glia; amphophilic nuclear inclusions of 15 to 25 nm or 30 to 40 nm diameter filaments or spheres
JC virus/SV40, myelin, neurofilament, KP1
Cerebral white matter, CNS
Dementia/CreutzfeldtJakob disease
Cytoplasmic vacuoles indenting nuclei; gliosis
PrP, GFAP
Bilateral cerebral cortex, gray matter
Small vessel disease
Vasculitis, arterial sclerosis, or congophilic angiopathy
A6, L26, CD31, amyloid, muscle actin, elastin
Cerebrum, CNS, frequent multiple lesions
Dementia/Alzheimer disease
Argyrophilic plaques, neurofibrillary tangles of bihelical filaments
Neurofilament, tau, ubiquitin, Alz-50
Bilateral cerebral cortex
Demyelination
Loss of myelin; gliosis, gitter cells with or without axonal preservation
Myelin, neurofilament, KP1
Cerebral white matter, CNS
Epilepsy
Low-grade glioma or ganglioglioma, gliosis, or vascular malformation
GFAP, neurofilament, elastin
Cerebral cortex
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *The order of tabulated lesions follows the order of discussion in text. † Most common or most specific location is listed first. ‡ Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. CNS, Central nervous system; GFAP, glial fibrillary acid protein; HSV, herpes simplex virus; JCV, JC virus; PrP, antiprion protein.
Nonneoplastic Brain Lesions Reactive Changes GLIOSIS
Gliosis is a reaction of the CNS to injury of the brain or spinal cord. Although subtle changes occur earlier, gliosis is usually appreciated by 2 to 3 weeks after an injury. Nearly any injury of the CNS can cause gliosis, so its presence is not diagnostic of a specific pathologic entity (Table 20-4).15 Anti-GFAP immunostain (Fig. 20-5) highlights the dark-brown intense immunoreactivity, relatively low nuclear to cytoplasmic ratio, and separation of astrocytes in gliosis. When it is critical to distinguish gliosis from normal brain, an age- and site-matched control slide of normal CNS can be stained concurrently. Both the number and the density of GFAP-positive cells and cellular processes should be greater in the specimen than in the normal control. The GFAP stain helps distinguish gliosis from glioma (see Table 20-4). GFAP-positive cells are uniformly spaced in gliosis (see Fig. 20-5). This spacing of individual reactive astrocytes is more uniform than that found in the margin of an infiltrative glioma (see the “Gliomas” section later in the chapter). The nuclear/ cytoplasmic ratio of gliosis is less than that of a glioma.3
MACROPHAGES
After three days, phagocytic macrophages may be seen in any destruction or irritation (Fig. 20-6, A; see Tables 20-3 and 20-4). Macrophages are rich in enzymes such as α-antichymotrypsin and muramidase, and they possess markers of mononuclear phagocytic cells and thus react with antibodies CD68 (KP1) and MAC387. All of these features can be stained by using IHC. Around hemorrhages or traumatic lesions, macrophages contain iron-positive hemosiderin. Encephalitis simply means brain inflammation. Many things cause it—from a virus to surgical implants.13 In cerebritis, meningitis, or encephalitis, the macrophages are pleomorphic cells. Some are thin, and others are loaded with debris (see Fig. 20-6); additionally, they may contain yeast and other organisms. Macrophages swollen plump by phagocytosis within the CNS are referred to as granular or gitter cells. They are large and round with a foamy cytoplasm filled with lipid droplets (Fig. 20-7, A; see Fig. 20-6, A). Macrophages that are small cells with scant cytoplasm participate in the chronic inflammatory infiltrate centered on blood vessels in encephalitides; in glial nodules; around dying neurons (neuronophagia); and in other inflammatory, demyelinating, and degenerative processes.3,16 CD68 stains them well (see Fig. 20-6, A).
768
Immunohistology of the Nervous System
TABLE 20-4 Differential Diagnosis of Cells Infiltrating Central Nervous System Parenchyma Differential Features Diagnosis
Structures
Antibody
Locations*
Gliosis†
Cells fibrillar, uncrowded; round/oval nuclei
GFAP in stellate glial processes
CNS
Macrophages
Cells and nuclei round to elongated; cell content reflects injury
KP1, α-ACT
CNS, meninges
Encephalitis/cerebritis
Perivascular mixture of inflammatory cells
LCA, L26, A6, κ and λ Ig, α-ACT, KP1, microorganism
CNS gray matter/CNS
Hemorrhage
RBCs or macrophages with hemosiderin
Fibrin, KP1
Deep cerebrum, cerebellum, CNS
Margin of gliomas‡
Cells fibrillar; angular nuclei indent each other; (mitoses)‡§
GFAP
CNS
Lymphoma
Perivascular noncohesive small round cells
LCA, L26, A6, κ and λ Ig
Deep cerebrum, CNS, meninges
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS, ed: Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *Most common or most specific location is listed first. † Nonspecific reaction to injury. ‡ Suspicion of margin of glioma on frozen section should be followed by a request for another, more central biopsy. Mitoses suggest margin of a high-grade glioma. § Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. α-ACT, Alpha-antichymotrypsin; CNS, central nervous system; GFAP, glial fibrillary acid protein; Ig, immunoglobulin; LCA, leukocyte common antigen; RBC, red blood cell.
PERIVASCULAR INFLAMMATION
Perivascular inflammation consists of small round cells with high nuclear/cytoplasmic ratios. These can be mistaken for lymphoma and also for neuroectodermal clusters, which are particularly common in the brains of children. Leukocyte common antigen (LCA; CD45/45R), CD3-ε, CD5, CD20, and CD79-α markers distinguish the inflammation by highlighting polyclonal reactive lymphocytes (see Fig. 20-6, B-C).
Irritation of the CNS elicits inflammation around blood vessels.17 CD68-positive macrophages ingest the irritant or injured cellular constituents and move them to the perivascular space.16 In the absence of classic lymph nodes in the brain, this perivascular region is where cells that respond to antigen intermingle. Depending on the severity and duration of the illness, the perivascular inflammation varies substantially.3 Old hemorrhage exemplifies a minimal response characterized mainly by perivascular macrophages laden with hemosiderin (see Table 20-4). Surgical wounds and implants cause substantial reactions. Viral or allergic encephalitis produces a maximal response with abundant perivascular macrophages and many CD3-ε– positive T lymphocytes.3 Some diseases affect mainly veins, such as perivenous encephalitis (PVE). Others affect small arteries, such as
KEY DIAGNOSTIC POINTS Reactive Changes
Figure 20-5 Glial fibrillary acidic protein (GFAP) stain of gliosis. This gliosis is in the cerebral cortex from a patient with CreutzfeldtJakob disease. The glia are reactive and stellate with abundant brown cytoplasmic GFAP, quite different from the remote gliosis illustrated in the case of long-standing seizures (see Fig. 20-31, B). The coalescing vacuoles are a feature of Creutzfeldt-Jakob disease but not of gliosis in general (anti-GFAP with hematoxylin and eosin).
• Gliosis is the usual slow reaction to brain injury. • GFAP, the single most important brain immunostain, highlights the low nuclear/cytoplasmic ratios, stellate processes, and evenly separated reactive astrocytes in gliosis. • Polymorphonuclear cells, macrophages, and lymphocytes react to brain injury in a manner similar to the reaction in systemic injury. Without lymph nodes in brain, their interactions are less obvious, and their clusters are perivascular. • Common systemically, fibrosis is rare in brain tissue.
Nonneoplastic Brain Lesions
A
769
B
Figure 20-6 Inflammatory cells in severe encephalitis include KP1-positive macrophages with various shapes that reflect their immediate surroundings, states of activation, and engorgement with products of endocytosis (A); perivascular and parenchymal A6-positive T lymphocytes (B); and L26-positive B lymphocytes (C). Gitter cells are large, round macrophages swollen with products of endocytosis (A).
C
cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Unlike PVE, CADASIL has little or no inflammation (see the “Small Vessel Disease” section later in the chapter). Affected vessels may be distinguished with anti–smooth muscle actin (anti-SMA) or myosin, because cerebral arteries have a thicker circumferential layer of spindle-shaped smooth muscle cells than cerebral veins.
A
FIBROSIS
Fibrosis is rare in brain tissue, but it occurs around abscesses (see Fig. 20-7, A), in granulomas, and in desmoplastic and sarcomatous tumors. Fibrosis is more common in meninges. Meningeal fibrosis develops after traumatic injuries, meningitis, vasculitis involving meningeal vessels, and radiation therapy and as a desmoplastic response to a tumor.
B
Figure 20-7 A, This specimen from a brain abscess shows an inflammatory lesion with a distinctive wall of collagen-stained cyan with Masson trichrome stain. Brain around this wall (orange side) contains highly reactive (gemistocytic) astrocytes. Toward the center of the abscess (gray side) are leukocytes and swollen macrophages. B, Exserohilum rostratum fungal contaminant of an injected steroid compound stained with Gomori methenamine silver shows septated and branched hyphae. Courtesy Dr. Nathan Shaller, University of Michigan Department of Pathology.
770
Immunohistology of the Nervous System
Various constituents of fibrosis can be detected with IHC stains; this includes collagen, fibronectin, and laminin.18 Type IV collagen works best for most brain and meningeal tissues.3 For routine identification of fibrosis, standard tinctorial stains rival IHC stains (see Fig. 20-7, A).
Infectious Diseases Infections may produce meningitis, cerebritis, abscess, encephalitis, or encephalopathy.19 Except for encephalopathy, inflammation is a prominent feature. It proceeds from acute to chronic phases much like a systemic infection, and infection must be distinguished from lymphoma. Infections cause polyclonal inflammation and often show a prominent T-cell component that stains with IHC markers, including CD45RO, CD3-ε, and CD5 (see Table 20-1). Mature, EMA-positive plasma cells signify inflammation when present. On the other hand, large neoplastic cells with malignant nuclei in primary brain lymphomas are usually B cells that stain with CD20 or CD79 alpha (see the “Hematopoietic and Lymphoid Neoplasms” section later in the chapter). We will describe each histopathologic type of infection here and follow with discussions of the organisms that cause each type. HISTOPATHOLOGY
Meningitis is an inflammation of the meninges that cover the brain and spinal cord. Leptomeningitis affects the thin meninges, the pia and arachnoid. Pachymeningitis affects the thick dura and is less common than leptomeningitis in nonsurgical cases. Organisms access the meninges by local extension from sinuses or from the bloodstream. The perivascular space in the CNS is an extension of the subarachnoid space. Persistent meningitis travels along this space, where it can cause cerebritis or an abscess. Cerebritis is focal inflammation of brain parenchyma (myelitis in the spinal cord). Cerebritis precedes abscess formation but requires an early biopsy to be seen (Table 20-5; see also Table 20-4). The inflammatory infiltrate is composed of neutrophils, macrophages, lymphocytes, and plasma cells, with or without parenchymal necrosis. Septic cerebritis is usually caused by bacterial agents, most often streptococci or staphylococci and less commonly by gram-negative organisms, such as Escherichia coli, Pseudomonas, and Haemophilus influenzae. Cerebritis also occurs around neoplasms, ruptured vascular malformations, infarcts, and traumatic lesions. Granulomatous forms of meningitis and cerebritis are seen in tuberculosis and atypical mycobacterial infections; fungal, parasitic, or spirochetal infections; idiopathic conditions, such as sarcoidosis, systemic lupus erythematosus, and Wegener and lymphomatoid granulomatoses; and histiocytosis X. Some diagnoses are made through biopsy and culture, and others are made through clinical correlation.13,20,21 An abscess combines features of inflammation and fibrosis in response to a suppurative microorganism, often bacterial or fungal. A mixture of
TABLE 20-5 World Health Organization Criteria for Grading of Meningioma* Anaplastic Meningioma (Grade III)
Meningioma (Grade I)
Atypical Meningioma (Grade II)
<4 mitotic figures per 10 hpf (0.16 mm2)
Increased mitotic activity: 4 to 19 per 10 hpf (0.16 mm2)
Increased mitotic activity: >19 per 10 hpf (0.16 mm2)
Fails to meet diagnostic criteria to right
Or >2 of the following: 1. Increased cellularity 2. Small cells with high nuclear/ cytoplasmic ratio 3. Prominent nucleoli 4. Sheetlike and/or patternless growth 5. Foci of spontaneous or geographic necrosis
Or malignant and/or anaplastic cytologic appearance (e.g., resembling sarcoma, carcinoma, melanoma)
Fits into a welldifferentiated subtype
(WHO grade II is assigned to clear cell and chordoid meningiomas)
(WHO grade III is assigned to rhabdoid and papillary meningiomas)
Modified from McKeever PE, Boyer PJ: The brain, spinal cord, and meninges. In Mills SE, Carter D, Greenson, JK, et al (eds): Sternberg’s diagnostic surgical pathology, ed 4. New York, 2004, Lippincott Williams & Wilkins; pp 399-506. *Brain invasion is not a criterion for increased grade. hpf, High-power field; Or, either mitotic or cytologic criteria needed for diagnosis; WHO, World Health Organization.
polymorphonuclear leukocytes, polyclonal T and B lymphocytes, macrophages, and plasma cells (with or without necrosis) confirms inflammation. Polymorphism of inflammatory components can be verified in difficult cases with CD45RO and CD20 IHC stains for polyclonal T and B lymphocytes, CD68 for macrophages, and EMA for plasma cells (see Tables 20-1 and 20-5). The wall of a brain abscess consists of a lining of CD31-positive and CD34-positive vascular tissue and collagen surrounded by highly GFAP-positive reactive gliosis. Adjacent brain is edematous. Because collagen is rare within the CNS, its presence is an important diagnostic feature of an abscess (see Table 20-5). Collagen may be difficult to distinguish from fibrillary gliosis on a slide stained with H&E. It can be confirmed histochemically with Masson trichrome stain or immunohistochemically with staining for collagen (see Fig. 20-7, A, and Table 20-1). Encephalitis is inflammation of brain tissue (see Fig. 20-6) often caused by viral or rickettsial organisms that produce a more diffuse inflammation than cerebritis.7 Most viral infections are self-limited and cause only meningitis or mild meningoencephalitis. The entities
Nonneoplastic Brain Lesions
emphasized here require surgical attention and are more serious. Encephalopathy (which translates as “brain suffering”) that is caused by infection may show little or no inflammation. This is especially true for the spongiform encephalopathies caused by prions, such as CreutzfeldtJakob disease (CJD).9 Brain cell death followed by gliosis is the common feature of the encephalopathies. A leukoencephalopathy (“white brain suffering”) targets white matter. A myelopathy (“cord-medulla suffering”) generally targets the spinal cord. ORGANISMS
Every brain biopsy specimen should be handled so that if inflammation is found at surgery, it will be possible to culture the tissue for bacteria, mycobacteria, and fungi and to use special stains, immunostaining techniques, and electron microscopy. My experience has been that for organisms that grow in vitro, microbiologic culture is preferable to histochemical stains, IHC, or PCR assay if sampling of the lesion is uniform. Prior antibiotic treatment or nonuniform sampling of focal infection affects individual cases. Fungal, bacterial, and parasitic infections are increasingly common in immunocompromised hosts. The common organisms are Cryptococcus neoformans, Listeria monocytogenes, Aspergillus fumigatus, and conventional bacteria such as Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus epidermidis, and Pseudomonas species. Cryptococcal meningitis is the most common form of fungal meningitis, but brain inflammation may be minimal in its presence. Chronic infections with these organisms produce granulomas. The organisms can be cultured or found with special stains, such as periodic acid-Schiff (PAS) and Gomori methenamine silver (GMS), but IHC analysis with specific reagents is an option for organisms refractory to culture.3,13,21 Species identification can be accomplished with immunostaining.21 A new threat to the nervous system came recently in bottles of preservative-free methylprednisolone acetate from a single compounding pharmacy.22 This produced meningitis, posterior stroke, or abscess after injection, and over 25 fatalities resulted.23 Most isolates were Exserohilum rostratum, a saprophyte that also infects eye and skin in immunocompromised patients (see Fig. 20-7, B). The fungus produces a dark pigment that qualifies it as a “black mold.” The Centers for Disease Control (CDC) Fungus Reference Laboratory can examine fungal isolates under the microscope and confirm their identification by DNA sequencing methods (visit www.cdc.gov/fungal/index.html or call 800-232-4636). Tuberculosis can involve any region of the CNS and its coverings. The disease usually causes granulomatous inflammation with or without caseating necrosis, meningitis, or arteritis. The extensive time required to grow mycobacteria invites preliminary testing with IHC, PCR assay, or acid-fast stains.20 Syphilis is rising in incidence, predominantly among immunocompromised patients, and it is contributing to
771
the differential diagnosis of granulomatous inflammation. The responsible organism, Treponema pallidum, is refractory to culture. Silver stains for it also stain brain, which produces background staining that confounds detection. IHC offers an alternative and more specific test.24 The most common parasitic infection of the CNS is neurocysticercosis, which prevails in developing countries. If a brain cyst contains a typical Cysticercus with a characteristic invaginated scolex, the disorder can be identified without IHC; analysis using cerebrospinal fluid (CSF) from proven cases as the source of primary antibody is available for mangled or degenerated organisms in cases for which the glycocalyx remains to be found.25 Schistosomiasis infects the brain and spinal cord. It can be highlighted with the readily available IHC stain for standard high-molecular-weight cytokeratin (HMWCK).26 Because there is little cytokeratin in brain, bits of organism stand out; this is one example of using a surrogate IHC marker when a more specific marker is not readily available. Whenever possible, the more specific marker is preferable. Whipple disease rarely causes a primary brain disease without gastrointestinal symptoms.13 The causative bacillus is Tropheryma whippleii. The diagnosis can be made on a brain biopsy specimen evaluated by light microscopy with immunoperoxidase staining for group B streptococci.27 Histologic features include PASpositive, diastase-resistant rods in macrophages; microgranulomas; perivascular CD3-ε and CD20-positive lymphocytes; and microglia reactive for CD68. Lyme disease is caused by a tick-borne spirochete, Borrelia burgdorferi. It involves the CNS and can be detected in CSF.28 The most common cause of nonepidemic encephalitis, and the one most often found on biopsy, is herpes simplex virus (HSV; see Table 20-3).29 The process is usually but not always localized to the temporal and frontal lobes. The earliest lesion is an area of vascular engorgement with ischemic changes in neurons that is positive for HSV when an immunoperoxidase technique is performed on routine paraffin-embedded tissue. Perivascular inflammation is characteristic, composed predominantly of CD45RO-positive and CD20-positive lymphocytes mixed with CD68-positive macrophages and accompanied by varying degrees of focal necrosis and hemorrhage. Intranuclear inclusion bodies are consistent with HSV but may be produced by many viruses, such as cytomegalovirus (CMV), varicella zoster, JC virus (JCV), and simian virus 40 (SV40).7 Cowdry type A bodies of HSV are not easy to demonstrate in small brain biopsy specimens. This argues for sensitive and specific methods of identification such as in situ hybridization (ISH), PCR, and IHC.3,30 Electron microscopy (EM) may demonstrate viral particles within the nuclei or cytoplasm but is less sensitive and less specific. Culture and sequential serologic CSF evaluations are slow but are still the most accurate methods of diagnosis for many viral CNS infections, including HSV. Enterovirus (Coxsackievirus, echovirus) and arbovirus infections (Eastern equine, St. Louis, West Nile) lack
772
Immunohistology of the Nervous System
characteristic inclusions. West Nile virus is an arbovirus with mosquito and bird vectors and hosts that has caused human infection.3 Its transmission by blood transfusion and organ transplantation has been documented, and some cases are fatal. The findings include patchy meningitis, encephalitis, and poliomyelitis with variable involvement of the cerebrum, thalamus, basal ganglia, brainstem, and cerebellum. CD3-ε and CD68 highlight perivascular inflammation with evidence of meningoencephalitis, microglial nodules, and neuronophagia. The anterior horn cells are targeted in some patients.3 Rabies produces round or oval eosinophilic 1- to 7-µm cytoplasmic inclusions.31 Immunostains and PCR assay are available for diagnosis.13 Subacute sclerosing panencephalitis and milder encephalitis are caused by measles virus. Focal lymphocytic inflammation in the leptomeninges and perivascular spaces involves the cerebral cortex, with many CD4-positive cells, patchy GFAP-positive fibrillary astrocytosis, and occasional microglial nodules. Diffuse mononuclear inflammation, gliosis, and loss of myelin occur in subcortical white matter. Inclusion bodies are Cowdry type A and may be seen on H&E-stained slides; their specific identification requires IHC.32
Acquired Immunodeficiency Syndrome CNS lesions in acquired immunodeficiency syndrome (AIDS) reflect the entire spectrum of neuropathologic disease, beginning with cerebritis, meningitis, encephalitis, and vascular disease and ending with degenerativemetabolic changes and neoplasia. The lesions have been summarized in several detailed reviews.8,33 Diseases either are directly caused by human immunodeficiency virus (HIV) or are secondary opportunistic diseases resulting from immunosuppression. CNS diseases were extremely common in the early era of AIDS, with neurologic symptoms at clinical presentation and CNS abnormalities noted in more than 50% of patients. Highly active antiretroviral therapy (HAART) has altered the disease course, such that fewer CNS complications occur.33
A
PRIMARY MANIFESTATIONS OF HUMAN IMMUNODEFICIENCY VIRUS
HIV encephalitis can be reliably diagnosed by histologic evaluation. The hallmark of HIV encephalitis is multinucleated giant cells both in parenchyma and around vessels. They are mixed with macrophages and microglia, and they form multiple foci of various sizes within the white matter, deep gray matter, and cortex. IHC detection of HIV p23 and p24 antigen and ISH is useful (Fig. 20-8).3 HIV leukoencephalopathy is characterized by diffuse damage to the white matter with loss of myelin, reactive astrogliosis, multinucleated cells, and macrophages. IHC and ISH help confirm the association of HIV with the process. Leukoencephalopathy occasionally manifests as marked vacuolar myelin swelling. This finding is more common in the spinal cord, however, where it forms multiple foci of vacuolar myelopathy that resemble combined systems degeneration without pernicious anemia. Still another manifestation of HIV infection, lymphocytic meningitis, is remarkable for heavy lymphocytic infiltrates within the leptomeninges and perivascular spaces. HIV cerebral vasculitis and granulomatous angiitis may occur with lymphocytic or lymphoplasmahistiocytic multinucleated giant cell infiltration of cerebral vessel walls, occasionally accompanied by necrosis.3 Since the onset of HAART, a new form of HIV encephalitis with severe leukoencephalopathy and intensive perivascular macrophage and lymphocyte infiltrates has been described. It may be a response of the revived immune system.33 INFECTIONS SECONDARY TO ACQUIRED IMMUNODEFICIENCY SYNDROME
Opportunistic infections are common in patients with AIDS but may also be found in other immunodeficient patients. Toxoplasmosis is the most common of these infections and manifests as a necrotizing encephalomyelitis characterized by discrete lesions that contain free trophozoites or cysts filled with parasites at the
B
Figure 20-8 Multinucleated giant cells formed from coalescence of macrophages have cytoplasm that is less fibrillar than surrounding brain and display tapered nuclei with light chromatin. They are elusive on hematoxylin and eosin stain (A) but obvious upon immunohistochemical staining for human immunodeficiency virus p24 antigen (B). Courtesy Dr. Clayton Wiley, University of Pittsburgh, Pittsburgh, PA.
Nonneoplastic Brain Lesions
773
periphery of the necrotic foci.34 Immunoperoxidase or immunofluorescence stains pinpoint the organism, which is not easily found on routine H&E-stained sections.35 KEY DIAGNOSTIC POINTS Infectious Diseases • Lesions found to be inflammatory at biopsy should be sent sterile from the operating room to the microbiology laboratory for cultures. Cultures are more sensitive than tissue stains for nearly all microorganisms that grow in vitro. • A variety of serologic and tissue-based assays that include IHC, ISH, special stains, EM, and PCR are available. These should be selected individually on the basis of clinical situation and suspected organisms.
CMV infection follows toxoplasmosis in frequency and varies from virtually no associated inflammation to severe necrotizing meningoencephalitis and ependymitis.36 IHC, ISH, and PCR assay are useful for detecting the virus in paraffin-embedded tissue if bizarre giant cells with nuclear inclusions are not evident.37 Severe encephalitis results from coinfection with HIV and JCV,38 and tuberculosis and neurosyphilis affect patients with AIDS.39,40 Microscopic examination reveals focal lymphoplasmacytic inflammatory infiltrates in a predominantly perivascular arrangement. Exotic CNS infections in patients with AIDS include amebic encephalitis, trypanosomiasis, and strongyloidiasis.13,41 PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY
Progressive multifocal leukoencephalopathy (PML) is a disease manifested as multiple discrete foci of destruction of myelin with relative preservation of axons, often with no other evidence of inflammation; radiographically, it may simulate multiple sclerosis or a mass. PML is caused by DNA papovavirus (predominantly JC papovavirus [JCP] and rarely SV40 virus) in immunodeficient patients (see Table 20-3). JCP has nothing in common with prions. Common underlying diseases are leukemia and AIDS. PML also occurs in patients with various types of carcinoma, tuberculosis, systemic lupus erythematosus, and sarcoidosis or after the immunosuppression associated with organ transplantation. Brain biopsy may show a destructive process within the white matter, with multiple lipid-laden macrophages, frequent large glial nuclei with a ground-glass appearance, and many large, unusual glia with pleomorphic and hyperchromatic nuclei. Perivascular infiltrates of mature lymphocytes are prominent in some cases. The pathology of JCV infection is similar in patients with and without AIDS. However, in patients with AIDS, bizarre astrocytes are less common, and perivascular inflammatory cells are more common.13 Glial nuclei are filled with virions in PML. PML should be differentiated from multiple sclerosis, other demyelinating disorders, and astrocytic neoplasia.
Figure 20-9 Progressive multifocal leukoencephalopathy. The patient, a young woman, had systemic lupus erythematosus, which was treated with high-dose antiinflammatory and cytotoxic agents. Immunohistochemical detection of JC viral antigen in swollen oligodendroglial nuclei stained brown. Negative, smaller, round oligodendroglial and elongated astrocytic nuclei counterstained purple with hematoxylin. Courtesy Dr. Riccardo Valdez, University of Michigan, Ann Arbor, MI.
Random distribution of rather uniformly distorted astrocytes among multiple lipid-laden macrophages is helpful in differentiating this lesion from an astrocytic neoplasm. Bizarre astrocytes and abnormal oligodendrocytes with large nuclei that contain inclusion bodies are characteristic. Diagnosis of PML is confirmed by EM, ISH, immunostaining (Fig. 20-9), or PCR assay for JCV, SV40, and BK virus.42,43
Spongiform Encephalopathies Spongiform encephalopathies are characterized by vacuoles (spongiform change) in the gray matter.3 Vacuoles vary in size up to 30 µm in diameter and larger (Fig. 20-10) and are in the neuropil and cellular perikaryon. Their neuroanatomic distribution varies among specific diseases and in individual cases. Lack of inflammation is usual. Specimens in which a spongiform encephalopathy is suspected should be processed as described later (see the “Dementias” section). The spongiform encephalopathies include CJD, the much-publicized mad cow disease, scrapie, kuru, Gerstmann-Sträussler-Scheinker (GSS) syndrome, and fatal familial insomnia.3,8,44-46 They are caused by infectious proteins known as prions, modified forms of normal counterpart proteins. Hereditary prion diseases such as familial fatal insomnia, GSS syndrome, and familial CJD have germline mutations that produce prions. Infectious prion diseases such as mad cow disease, scrapie, kuru, and spontaneous CJD are transmitted by intimate contact with prions. As with catalysts, these pathogenic prions propagate by inducing their ubiquitous normal counterparts to refold into the pathogenic conformation. As this cycle continues, a growing percentage of normal counterpart proteins are converted to the pathogenic configuration. Prions are very difficult to inactivate. Agents that completely denature protein, such as bleach and strong
774
Immunohistology of the Nervous System
Figure 20-10 Creutzfeldt-Jakob disease. Biopsy specimen from the cerebral frontal lobe of an elderly man who displayed progressive behavioral and memory changes for a few weeks. Patches of vacuoles and synaptic depletion can be seen in the cortical gray matter. Each tiny brown dot is a synapse in the neuropil stained with synaptophysin. Vacuoles in neuronal cytoplasm indent their nuclei (antisynaptophysin with hematoxylin counterstain).
alkali or acid (see the “Dementias” section later in the chapter) are effective, but ultraviolet light, routine formalin fixative, and standard disinfectants fail to eradicate prions. CJD was a common diagnosis in one evaluation of cerebral biopsy specimens for dementia.44 Vacuoles in the neuropil and perikaryon of neurons are regionally and temporally variable in CJD (see Figs. 20-5 and 20-9). If not prominent, vacuoles can be overlooked or mistaken for artifacts.9 Spongiform changes usually diminish in late-stage disease (see Table 20-3). In contrast, GFAP-positive gliosis gradually increases (see Fig. 20-5). Immunostaining with antiprion protein (PrP) antiserum is a useful tool in the identification of isoforms of this protein for the rapid diagnosis of CJD.47 Definitive diagnosis can be achieved by Western blot analysis for prion proteins resistant to digestion by proteinase K enzyme.9 In 1996, the European Union banned imports of British beef following the mysterious deaths of young “fast food” enthusiasts in 1995 from an atypical variant of CJD. These deaths and the deaths of cattlemen with bovine spongiform encephalopathy (BSE) in their herds were highly publicized.3,48 Thus emerged mad cow disease. Microscopic plaques that stain immunohistochemically for prion protein are the most striking and consistent neuropathologic features of this atypical variant of CJD.48 They are even more distinctive when surrounded by spongiform change.
Cerebrovascular Diseases Hemorrhage into brain tissue has many causes and often accompanies other lesions within the CNS. The major role of IHC is to identify certain causes of hemorrhage such as amyloid and neoplasm. Amyloid angiopathy is a common cause of spontaneous intracerebral hemorrhage in the elderly (see Table 20-3). IHC staining with an antibody to β/A4 protein is more sensitive
than Congo red stain in demonstrating the extent of vascular amyloid.49 Most neoplasms that cause brain hemorrhages are metastatic. Melanoma, renal cell carcinoma (RCC), choriocarcinoma (Chapters 7, 8, 16, and 18), leukemia, and glioblastoma tend to hemorrhage. Glioblastoma contains GFAP-positive cellular processes and vimentinpositive vascular proliferations, and the MIB-1 (Ki-67) proliferation index is high among gliomas (see the “Grading Malignant Potential” section later in the chapter). Carcinomas express cytokeratins. Hemorrhages in patients with hypertension often occur within the cerebral hemispheres, especially in the lateral areas of the basal ganglia.50 Coagulopathy is an important cause of intracerebral hemorrhage, including drug-induced coagulopathy. Saccular aneurysms occasionally rupture into the brain, but radiography reveals their nature. Embolism is an important cause of hemorrhagic cerebral infarcts.51 Sinus thrombosis followed by venous infarction may occur, usually as a complication of a preexisting infectious or inflammatory disease.3 Nontraumatic subarachnoid hemorrhages are usually due to rupture of a saccular aneurysm, most often located at a branch of a major artery or in the circle of Willis. Their source is radiographically apparent. A subdural hematoma may follow a traumatic event and is seen in elderly patients and in those with systemic cancer and brain tumors.52 Membranes are formed on both sides of the hematoma, and membrane formation requires several weeks to complete. The membrane on the dural side is usually 2 to 5 vimentin-positive fibroblasts thick in a 5-day-old subdural hematoma. It eventually becomes as thick as normal dura with new collagen that reacts with IHC stains for type IV collagen and fibronectin. SMALL VESSEL DISEASE
Brain biopsy specimens obtained in search of small vessel disease may require sectioning through the entire block of tissue to yield diagnostic material. Excessively involved vessels may not be recognizable, but CD31 shows them by highlighting their endothelial cells (see Table 20-3); CD31 is the endothelial marker of choice for its sensitivity and specificity.52 Involvement of small cerebral veins that have few spindle-shaped smooth muscle cells (SMCs), compared with arteries of the same diameter with more SMCs, can be assessed with anti-SMA or myosin.53 Causes are often cryptogenic in isolated angiitis of the CNS.54 Systemic vasculitides that may affect brain are associated with lupus erythematosus; illicit drugs, including cocaine, heroin, and amphetamines; infection, such as varicella zoster virus and meningovascular syphilis; toxins; granulomatous disease; Wegener disease; relapsing polychondritis; and Behçet disease.7,13,15,55,56 IHC can aid in the identification of microorganisms and classification of inflammatory cell types. CADASIL affects small arteries57 and is a rare disorder that results from NOTCH3 gene mutations (chromosome 19).13 Characteristic vascular changes can be identified in brain, skin, and muscle biopsy specimens.
Tumors of the Nervous System
775
By light microscopy, the affected vessels have a thickened appearance, and basophilic granular material is seen by H&E stain. This material is PAS positive and displaces the smooth muscle cells, which is best seen with an IHC stain for SMA. EM reveals the presence of dark, granular, osmophilic deposits. IHC for NOTCH3 protein deposition is available.57
Different neoplasms of brain predominate in adults and children. More pediatric neoplasms occur in the posterior fossa than in the anterior fossa, and the opposite is true of adult neoplasms.
VASCULAR MALFORMATION
Grading Malignant Potential
The following five types of vascular malformations are recognized:3,58 • Capillary telangiectasia • Cavernous angioma • Arteriovenous malformation (AVM) • Venous malformation • Sturge-Weber disease (cerebrofacial or cerebrotrigeminal angiomatosis) Although they may occur anywhere in the CNS, AVMs have a predilection for the cerebral hemispheres (Table 20-6). Elastic stains identify medium to large arteries and their abnormal counterparts. In AVMs, these stains often show abnormal vessels with focal loss or duplication of elastin. A monoclonal antielastin antibody is present, but special stains are typically used, such as Movat pentachrome stain.3 Abnormal smooth muscle layers can be highlighted with muscle actin. Cerebral veins are reported to have a thinner circumferential layer of smooth muscle cells than cerebral arteries, and these features exist in vascular malformations.59 IHC can be used to identify and localize vascular collagen, fibronectin, myofibroblasts (vimentin and muscle actin), and endothelial cells (CD31).
The World Health Organization (WHO) has established uniform terminology and grading of brain tumors according to histologic criteria.2 Starting with the most benign as grade I, numerical grades II, III, and IV represent increasing malignancy. The numerical grades assigned by the WHO classification are included in parentheses after the tumor names in headings in this section of the chapter. The most important aids to assessing grade of malignancy provided by IHC are as follows:3,60 1. Cell type identification with markers (Tables 20-7 through 20-11; see also Tables 20-2 and 20-5). 2. Identification of tumor infiltration. A rule of thumb for grading is that primary brain tumors without mitotic activity and with a distinct margin tend to be grade I, whereas infiltrating tumors tend to be grade II and higher. Neurofilament (NF) and synaptophysin stains aid assessment of tumor infiltration by staining preexisting axons, especially in white matter, and preexisting synapses (in gray matter). With a good hematoxylin nuclear counterstain, infiltrating neoplastic cells are evident with such stains.61 Eosin can be applied in addition to hematoxylin if needed. 3. Identification of vascular proliferations with vimentin; CD31, CD34, Ulex europaeus (Ulex), and factor VIII (FVIII) endothelial markers; and SMA. 4. Mitotic activity is key to grading many gliomas, including diffuse astrocytoma, grade II versus grade III. A new marker, phosphohistone H3 (PHH3), stains mitotic figures, but it often stains a fraction of interphase, pyknotic, and apoptotic nuclei. My counts of H&E-stained versus PHH3stained gliomas usually result in slightly higher numbers with PHH3, which raises a critical unresolved issue about grading any tumor that relies on counting mitoses. This problem notwithstanding, it is helpful when looking for true mitoses to use PHH3 on a section serial to H&E. The PHH3 assists in screening, but the better chromosomal detail in the H&E stain “keeps the PHH3 honest” and helps to distinguish true mitoses from mimics. 5. Proliferation markers such as molecular immunology Borstel 1 (MIB-1), a marker similar to Ki-67 that works on paraffin sections, supplement mitotic activity in assessing growth potential (Fig. 20-11). Proliferation markers show nuclear antigens that appear during one or more proliferative phases of the cell cycle. A labeling index (LI), also known as a proliferation index (PI), can be derived from them.62,63 The LI of any of the proliferation antigens is the number of
TABLE 20-6 Vascular Malformation Type
Location
Histology
Arteriovenous malformation
Cerebral hemispheres, brainstem, cerebellum
Veins and arteries with often poorly formed elastic membrane; gliotic brain tissue
Venous malformation
Central nervous system, spinal leptomeninges
Veins and gliotic or normal brain tissue; no arteries
Capillary telangiectasia
Pons, brainstem, central nervous system
Thin-walled dilated capillaries within brain parenchyma
Cavernous angioma
Central nervous system
Clusters of abnormal, often fibrotic or hyalinized vessels with elastic lamina and without intervening brain tissue
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492.
Tumors of the Nervous System
776
Immunohistology of the Nervous System
TABLE 20-7 Differential Diagnosis of a Mass of Fibrillar Cells Differential Features Diagnosis*
Structures
Antibody†
Locations‡
Fibrosis
Spindle cells of meningeal or perivascular origin
Type IV collagen (+), vimentin (+)
Meninges, CNS
Granuloma
Like fibrosis with “whorls” and inflammation
Microorganisms (see Table 20-1)
Basal meninges, CNS
Pilocytic astrocytoma
Hypercellularity, hairlike fibrillarity, Rosenthal fibers, microcysts
GFAP (+), S-100, alpha B– crystalline
Cerebellum, thalamus/ hypothalamus, optic nerve, CNS
Astrocytoma
Hypercellularity; angular nuclei cluster and indent one another; infiltrates CNS
GFAP (S), S-100, p53, IDH1 mutant
Cerebrum, brainstem, spinal cord, CNS
Anaplastic astrocytoma
Increase in above features; mitoses
GFAP (S), S-100, p53, IDH1 mutant
Cerebrum, brainstem, CNS
Gemistocytic astrocytoma
Hypercellularity; cells swollen with hyaline pink cytoplasm and eccentric pleomorphic nuclei; infiltrates CNS
GFAP (S), p53, IDH1 mutant
Cerebrum
Giant cell astrocytoma
Giant astrocytes with thick fibrils; large round/oval nuclei
GFAP (S)
Lateral ventricle, subependymal
Astroblastoma
Perivascular rosettes with expanded glial cell processes
Nonfibrillar GFAP (S)
Cerebrum, CNS
Pleomorphic xanthoastrocytoma
Pleomorphic cells often vacuolated
GFAP (S), type IV collagen (S)
Leptomeninges, cerebral cortex
Ependymoma
Hypercellularity, ependymal or perivascular rosettes or both, round/ oval nuclei, cilia, basal bodies
GFAP (+)
Cerebrum, cerebellum, spinal cord, CNS
Tanycytic ependymoma/ subependymoma
Combination of astrocytoma and ependymoma; round/oval nuclei cluster among fibrillar mats
GFAP (+)
Spinal cord, fourth ventricle, subependymal, CNS
Anaplastic ependymoma
Preceding features with mitoses; necrosis
GFAP (S), S-100
Cerebrum, cerebellum
Glioblastoma multiforme
Regions of coagulation necrosis; mitoses; pleomorphism; endothelial proliferation
GFAP (S), S-100, p53, IDH-1 mutant, EGFR
Cerebrum, CNS
Gliosarcoma
Glioblastoma plus fibrosarcoma intermixed
GFAP (S), fibronectin, type IV collagen (S), laminin, vimentin (S)
Cerebrum
Ganglion cell tumors
Binucleated and pleomorphic neurons; diagnosis depends on gliomatous and neuroblastic elements
GFAP (S), synaptophysin (S), PGP9.5, neurofilament (S), type IV collagen
Cerebrum, CNS
Central neurocytoma
Round cells and nuclei, thin fibrils near vessels
Synaptophysin (+), neurofilament (R)
Septum pellucidum, lateral ventricles
Pineocytoma
Normal pineal structures
Synaptophysin (+), neurofilament (R)
Pineal
Polar spongioblastoma
Rhythmic palisades of fibrillary cells
Fibroblastic meningioma
Spindle cells, interdigitating cell processes and desmosomes, (thick collagen), (whorls)§
Vimentin (+), EMA (S), S-100 (R)
Falx, tentorium, meninges, choroid plexus
Fibrosarcoma/ maligant fibrous histiocytoma
Hypercellular; pleomorphic spindle cells and nuclei, mitoses, necrosis
Vimentin, collagen
Meninges, CNS
Cerebrum, CNS
Tumors of the Nervous System
777
TABLE 20-7 Differential Diagnosis of a Mass of Fibrillar Cells—cont’d Differential Features Diagnosis*
Structures
Antibody†
Locations‡
Schwannoma
Verocay bodies, Antoni A and B; thin pericellular basement membrane
S-100 (+), Leu-7, type IV collagen, GFAP (R)
Eighth cranial nerve, spinal roots, PNS
Neurofibroma
Multiple cell types spread axons
Neurofilament (R), EMA, S-100 (+), Leu-7
Spinal root, PNS, cranial nerve
Histiocytosis
Sheetlike pattern of macrophages, fibroblasts, and leukocytes
α-ACT, S-100 (S)
Parasellar, CNS, systemic
Melanoma
Anaplasia, mitoses, necrosis
HMB-45 (S), S-100 (+)
CNS/meninges, frequent multiple metastases, systemic
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *The order of tabulated lesions follows the order of discussion in text. † Key to staining results: +, almost always strong, diffuse positivity; S, sometimes or focally positive; R, rare cells may be positive. ‡ Most common or most specific location is listed first. § Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. α-ACT, Alpha-antichymotrypsin; CNS, central nervous system; EMA, epithelial membrane antigen; GFAP, glial fibrillary acid protein; HMB-45, human melanoma black 45; PGP9.5, protein gene product 9.5; PNS, peripheral nervous system.
antigen-positive cells divided by the total number of cells in sampled microscopic areas of the tumor. Histologic grading of astrocytomas correlates with LI.63 MIB-1 is an antibody that detects proliferating cells in various phases of the cell cycle. Properly standardized, MIB-1 LI helps to predict patient outcome.64-66 Proper standardization with tissue processed in the same laboratory from a group of tumors is needed to glean the best prognostic information from the labeling index of an individual patient’s tumor. Laboratories vary in their range of staining and LI assessments, which limits the value of comparison with published data. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase,67 although MIB-1 is preferable to PCNA for distinct staining and reproducible LI.63
A
Apoptosis is the programmed death of cells. From cytologic and cytochemical assays the pathologist can determine an apoptotic index analogous to the LI for proliferation mentioned earlier in this chapter. The balance between cell proliferation and cell death affects tumor growth.68 Tumor progression in gliomas is associated with an increase in the grade of malignancy, which results in a poorer prognosis. Cyclin-dependent kinase 4 inhibitor (CDKN2A, formerly p16) is a cell-cycle regulatory protein that has been demonstrated to be inactivated by mutations, deletions, or transcriptional silencing during pathogenesis of a variety of human malignancies. CDKN2A immunocytochemistry may identify those low-grade gliomas likely to progress and to have poor
B
Figure 20-11 Diffuse fibrillary astrocytomas, grade II. MIB-1 antibody distinguishes long and short survivals in patients with grade II astrocytoma. A, Specimen with few brown, MIB-1–positive nuclei was taken from a patient who survived more than 8 years. B, Specimen with many MIB-1 positive nuclei was taken from a patient who survived less than 6 months. Hematoxylin counterstain colors MIB-1–negative nuclei purple. From McKeever PE, Strawderman MS, Yamini B, et al: MIB-1 proliferation index predicts survival among patients with grade II astrocytoma. J Neuropathol Exp Neurol 1998;57:931-936.
778
Immunohistology of the Nervous System
outcome and that thus would need more aggressive therapy.69 Various other genes and their immunoreactive proteins are altered during glioma tumor progression; they have been reviewed elsewhere.1,2
Gliomas Glioma is a term that describes astrocytoma, glioblastoma, ependymoma, oligodendroglioma, and their various subtypes and combinations. An important general rule is that gliomas tend to contain GFAP and to lack collagen, reticulin, and fibronectin in their parenchyma, which distinguishes them from nonglial neoplasms.70,71 Uncommon variants such as xanthoastrocytoma may have parenchymal reticulin (see Table 20-7). However, oligodendroglioma cells are more variable in their GFAP expression, and they uniformly express only glial proteins of low specificity, such as Leu-7 and S-100 protein.3 Gliomas lack widespread cytokeratin (CK) in their parenchyma but have been misinterpreted because of cross-reactivity between some anti-CK antibodies and GFAP.5 Clinicians often focus on infiltrating astrocytomas and oligodendrogliomas that are grades II to IV when they speak of gliomas. This confuses pathologists. Clinical needs are expanding the pathologist’s role in the interpretation of gliomas. For example, the effectiveness of procarbazine/chloroethylcyclohexylnitrosourea (CCNU)/vincristine (PCV) chemotherapy for gliomas with an oligodendroglial component, especially malignant gliomas with 1p and 19q chromosomal deletions,72 has increased the value of recognizing these tumors. PCV chemotherapy has now been largely replaced by Temodar (Merck, Whitehouse Station, NJ), which is easier to use. However, this may change. Two studies by van den Bent and associates and Cairncross and colleagues, presented at meetings but not yet published in final form, show major survival benefit of PCV for anaplastic gliomas with 1p/19q deletions. Each study is a late subgroup analysis of a randomized trial (van den Bent et al 2006, EORTC European trial; Cairncross et al 2006, RTOG American-Canadian trial), showing that adding PCV to radiation therapy (RT) for anaplastic gliomas provided significant benefit for progression-free survival. These results have led to redesign of an ongoing trial for anaplastic gliomas with 1p/19q deletion to include PCV plus RT versus Temodar plus RT.73 Recent discoveries of major importance are the frequency of mutations in isocitrate dehydrogenases (IDHs) and their effect on prognosis in gliomas. The IDH enzymes normally catalyze the oxidative carboxylation of isocitrate to α-ketoglutarate, which results in reduced nicotinamide adenine dinucleotide phosphate (NADPH) an important antioxidant. For some reason, IDH genes mutate frequently in diffuse gliomas, and there are several IDH enzymes. IDH1 mutations occur in gliomas approximately 10 times more often than IDH2 mutations. The R132H mutation is the most common mutation in IDH1, occurring about 80% of the time. An antibody was developed to this particular mutant because of its high frequency and potential diagnostic and prognostic utility.1 IDH1 mutation
distinguishes many gliomas from gliosis. The presence of an IDH1 mutation in a glioma confers better patient survival.74 The histologic term low grade, as applied to astrocytomas and other gliomas, does not necessarily imply a benign neoplasm or even a favorable prognosis. A benign designation, which implies that the glioma will not recur once removed, is frequently encountered only among WHO grade I astrocytomas, gangliogliomas, and ependymomas. Even these tumors need to be in favorable locations where they can be completely resected, thus giving the patient a good chance for cure.13 A general tendency that seems to be emerging from studies of the molecular biology of gliomas is that grade I gliomas and gliomas that do not infiltrate brain tend to not show IDH mutations, not overexpress TP53 gene product, and not overexpress epidermal growth factor receptors (EGFRs).75,76 In contrast, high-grade and infiltrative gliomas tend to overexpress at least one of these.2,76 Postoperative systemic thromboses are a major complication of brain tumor surgery. The pathologist can identify patients likely to encounter this difficulty by reporting the tumors, usually malignant gliomas, that contain thrombosed vessels.77 TUMOR AND TUMOR MARGIN
It is important to recognize two types of specimens of glioma (Fig. 20-12). The first type is the tumor itself (see Table 20-2 and Tables 20-7 and 20-8), which has cellular density exceeding that of surrounding brain (see Fig. 20-12, B). This tumor nidus is optimal for histopathologic classification.78,79 The second type of specimen is brain tissue infiltrated by the margin of the glioma, and it is a product of the infiltrative nature of many gliomas.3 IHC stains for brain neuroanatomic components are helpful in identifying this brain tissue. NF protein localizes axons in white matter, where axons are neuroanatomically oriented in parallel, and also in gray matter.61 The extent that glioma cells infiltrate this axonal meshwork in brain tissue is evident from the hematoxylin counterstain in IHC preparations (Fig. 20-13). Synaptophysin stains a finely pixelated “carpet” of synapses in gray matter; glioma cells disrupt this carpet. If only the margin is available for examination, it is often impossible to determine the histologic grade and KEY DIAGNOSTIC POINTS Glioma • Proper surgical sampling is needed for accurate classification and grading of diffuse or heterogeneous gliomas. Intraoperative consultation of pathologist and surgeon optimizes sampling. • Most grade II to IV gliomas infiltrate CNS tissue, which makes total resection difficult to impossible. Exceptions include certain ependymomas and xanthoastrocytomas. • The proliferation marker MIB-1 augments grading and prediction of patient outcome.
Tumors of the Nervous System
A
779
B
Figure 20-12 Stereotactic biopsy specimens of a left temporoparietal mass in an elderly woman. A, This specimen shows gliosis and rare neoplastic glia, classification and grade uncertain. B, This one shows glioblastoma with highly pleomorphic fibrillar cells, mitotic spindles, vascular proliferation, and necrosis. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
TABLE 20-8 Differential Diagnosis of a Mass of Conspicuously Different Cells Differential Features Diagnosis*
Structures
Antibody†
Locations‡
Oligoastrocytoma
Mixture of astrocytoma (Table 20-7) and oligodendroglioma (Table 20-2)
GFAP (S), Leu-7 (+), S-100 (+), IDH1 mutant
Cerebrum, CNS
Anaplastic oligoastrocytoma
Above features with mitoses and pleomorphism
GFAP (S), Leu-7 (S), S-100 (S), IDH1 mutant
Cerebrum, CNS
Glioblastoma/ gliosarcoma with epithelial metaplasia
Structures of glioblastoma/gliosarcoma (Table 20-7) plus epithelial regions
GFAP (S), S-100 (S), cytokeratin (S), EMA (S), p53, IDH1 mutant, EGFR
Cerebrum, CNS
Ganglion cell tumors
Binucleated and pleomorphic neurons plus glioma (Table 20-7) plus fibrosis plus inflammation
GFAP (S), synaptophysin (S), PGP9.5, neurofilament (R), type IV collagen (R)
Cerebrum, CNS
Desmoplastic medulloblastoma
Regions of medulloblastoma and fibrosis
Synaptophysin, S-100, type IV collagen, neurofilament (R), GFAP (R)
Lateral cerebellum, CNS, meninges, (extraaxial)§
Transitional meningioma
Regions of fibrous (Table 20-7) and syncytial (Table 20-10) meningioma
Vimentin (+), EMA (S), S-100 (R)
Falx, tentorium, meninges, choroid plexus
Hemangioblastoma
Multivacuolated stromal cells between many capillaries; hypervascularity; (fibrillarity is frozen section artifact)§
CD31 (S), factor VIII (S), NSE (S)
Cerebellum, spinal cord, CNS
Desmoplastic carcinoma
Regions of carcinoma (Table 20-2) and fibrosis (Table 20-7); occasional inflammation
Cytokeratin (S), EMA (S)
Cerebrum, cerebellum, meninges, CNS, frequent multiple masses, systemic
Melanoma
Regions of fibrillar and epithelioid melanoma (Tables 20-7, 20-2)
HMB-45 (S), S-100 (+)
Cerebrum, cerebellum, meninges, CNS, frequently multiple masses, systemic
Modified from McKeever PE, Blaivas M. The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *The order of tabulated lesions follows the order of discussion in text. † Key to staining results: +, almost always strong, diffuse positivity; S, sometimes or focally positive; or mixed cell populations; R, rare cells may be positive. ‡ Most common or most specific location is listed first. § Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. CNS, Central nervous system; EGFR, epidermal growth factor receptor; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; HMB-45, human melanoma black 45; NSE, neuron-specific enolase; PGP9.5, protein gene product 9.5.
780
Immunohistology of the Nervous System
A
B
Figure 20-13 A, The neurofilament immunohistochemical stain highlights axons in brain to facilitate evaluation of glioma infiltration into brain tissue. Crowded round and elongated nuclei of a grade II oligoastrocytoma (stained purple with hematoxylin) diffusely infiltrate between long, brown axonal constituents of the underlying brain tissue. B, In contrast, this pleomorphic xanthoastrocytoma does not infiltrate between individual brown axons, and its margin with cerebral cortex is distinct; its other features, pleomorphic cells and nuclei and lipid vacuoles, may be seen without immunohistochemistry (see Fig. 20-16). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
type of glioma giving rise to an infiltrative margin of neoplastic glia. Further from the glioma itself, neoplastic glia in CNS parenchyma are difficult to distinguish from gliosis (Fig. 20-14; see Fig. 20-12, A). Nonetheless, GFAP can help identify gliosis by showing excess cytoplasmic GFAP and regular spacing between cells in gliosis (see Fig. 20-5). ASTROCYTOMAS
Astrocytomas are among the most fibrillar of the CNS neoplasms, more fibrillar than other gliomas except tanycytic ependymomas and subependymomas (see Table 20-7). Astrocytomas nearly always contain GFAP (Fig. 20-15, A; see Fig. 20-12, A), although the amount is variable. GFAP is the single most important IHC marker and distinguishes astrocytomas from nearly all
A
nonglial neoplasms.3 Nerve sheath tumors occasionally show focal GFAP in substantially lesser amounts than the fibrillary astrocytomas that resemble them. Many astrocytomas express vimentin, and when they do, this feature distinguishes them from vimentin-negative oligodendrogliomas.13 Pilocytic Astrocytoma (WHO Grade I)
Pilocytic is defined as “composed of hair cells,” which is a major feature of the pilocytic astrocytoma. Parallel bundles of elongated, fibrillar, cytoplasmic processes resemble mats of hair (see Fig. 20-15, A).80 These hairlike processes contain large amounts of glial fibrils, which stain well with immunoperoxidase for GFAP (see Table 20-7). A diagnosis of pilocytic astrocytoma is about the only good news within the group of astrocytomas. This tumor
B
Figure 20-14 Gliosis and nonneoplastic perineuronal oligodendroglia (A) near a metastatic carcinoma (not shown) appear similar to the margin of a grade II fibrillary astrocytoma (B) distant from its nidus. Nuclei are more pleomorphic and hyperchromatic in B; compare with lighter nuclei in capillary endothelium at sides of panels (hematoxylin and eosin). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Tumors of the Nervous System
A
B
C
D
781
Figure 20-15 This tumor was in the cerebellar midline of a young man with headaches and vomiting. A, Pilocytic astrocytoma features numerous brown glial fibrillary acidic protein (GFAP)–positive cytoplasmic processes. Pale gray refractile globules, some with brown GFAPpositive rims, are Rosenthal fibers (RFs). The GFAP-negative RF resides in astrocytic processes. B, Pale gray RFs in this tumor mingle with pink, periodic acid–Schiff (PAS)-positive protein droplets (PAS with diastase). Both are less uniform in diameter than the light-brown intravascular erythrocytes. C, The marker alpha B-crystallin is specific for RFs but is not very sensitive. Although glial fibrils and purple nuclear chromatin are appropriately negative, many RFs show little or no brown staining. D, The sensitive marker ubiquitin stains all RFs in this nearby section from the same block as panel C. Although not evident here, ubiquitin sometimes stains nuclei. Structural features that include elongated beading of RFs within longitudinal sections of astrocytic processes and absence of chromatin distinguish the RFs. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
has a better prognosis than its diffuse counterparts, especially when it occurs in the cerebellum rather than its other common location near the third ventricle.81,82 It is critical to distinguish pilocytic astrocytoma from fibrillary grade II astrocytoma, which has a poorer prognosis. Even the better prognosis is tempered, because adequate surgical removal of a pilocytic astrocytoma depends on its location, and some develop as multicentric disease. The 10-year survival rate of patients with supratentorial tumors is 100% after gross total resection and 74% after subtotal resection or biopsy.83 Pilocytic astrocytomas rarely manifest malignant degeneration, indicated by hypercellularity, mitoses, and necrosis. Pilocytic astrocytomas have a well-demarcated MRI appearance; some have discrete margins, but many incorporate elements of brain at their margins. However, diffuse grade II astrocytomas (see discussion later in this section) infiltrate brain to a much greater extent than pilocytic astrocytomas.84 The extent of microscopic
infiltration can be evaluated by comparing GFAP staining in serial sections to identify the edge of the highly GFAP-positive tumor and NF protein staining to identify axons at the edge of the tumor. Pilocytic astrocytomas show few axons in their margins, but grade II astrocytomas show many. A nearly even mix of axons and neoplastic cells signals a grade II astrocytoma. Most, but not all, pilocytic astrocytomas occur in children and young adults. They are most abundant in the posterior fossa and around the third ventricle, thalamus, hypothalamus, neurohypophysis, and optic nerve. Cerebral hemispheric pilocytic astrocytomas are less common, but it is important to recognize them to ensure appropriate treatment.85 Rosenthal fibers (RFs) are highly eosinophilic hyaline structures that are round, oval, or beaded, and have slightly irregular margins.3 Their beaded appearance results from their formation within glial processes. In comparison with erythrocytes, they are pink, rather than
782
Immunohistology of the Nervous System
orange, and have greater variation in size and shape. Although RFs assist in distinguishing the pilocytic astrocytoma from other variants, they are of no value in differentiating astrocytomas from gliosis, because they occur in both abnormalities. IHC reveals that RFs contain alpha B-crystallin, stain with ubiquitin, and are centrally GFAP negative (see Fig. 20-15, A). Alpha B-crystallin is a lens protein in the small heat-shock protein family. When abundant, RFs are easily appreciated on H&E stain; IHC is most needed when they are scarce, and the ideal marker for scarce RFs would be both sensitive and specific. Ubiquitin is sensitive and not specific, and alpha B-crystallin is only slightly more specific and less sensitive than ubiquitin (see Fig. 20-15). My experience is that ubiquitin is best for screening for scarce RF, but these should be confirmed with either alpha B-crystallin or H&E stain. Structural features and color on H&E are often sufficient to identify RFs. Eosinophilic granular bodies (EGBs) are droplets of protein often found in pilocytic astrocytomas. These eosinophilic protein droplets are usually smaller and more aggregated than RFs and are usually intracellular, but occasionally are extracellular, and they are up to 40 µm in diameter. They are PAS positive (see Fig. 20-15, B). Both EGB and RFs are immunoreactive with alpha B-crystallin, which also is reported to stain cortical Lewy bodies, other astrocytomas, schwannomas, hemangioblastomas, and chordomas.13,86 Observations of subtypes of S-100 protein suggest that they distinguish pilocytic astrocytomas from WHO grade II to IV astrocytomas.87 Pilocytic astrocytomas do not overexpress p53 protein,75 and they tend to lack EGFR abnormalities.88 These features may have future diagnostic use. Cystic cerebellar pilocytic astrocytomas resemble hemangioblastomas, which may have focally GFAPpositive cells and a GFAP-positive cyst wall. Unlike hemangioblastoma, the mural nodule of a pilocytic astrocytoma contains highly fibrillar and abundantly GFAP-positive neoplastic astrocytes without clear vacuoles from lipids. CD31 and other endothelial cell markers show less abundant capillaries in the astrocytoma than in the hemangioblastoma. Diffuse Astrocytoma (Low-Grade Astrocytoma; WHO Grade II)
The fibrillary astrocytoma is more common than the protoplasmic astrocytoma.78 Fibrillary astrocytomas are a mixture of cellular processes (fibrils) and nuclei of greater angularity and density than normal or reactive astrocytes (Fig. 20-16; see Fig. 20-14, B). They contain more intracytoplasmic fibrils, and their cellular processes are longer than those in protoplasmic astrocytomas. Thus only the fibrillary astrocytoma stains well with phosphotungstic acid hematoxylin (PTAH), which stains fibrillar protein arrays, whereas both astrocytomas contain GFAP that can be stained immunohistochemically.13 The term diffuse appropriately describes an astrocytoma whose margin gradually diminishes in cellularity. Within the extensive margin, neoplastic cells
Figure 20-16 Pleomorphism of blue nuclei along with crowding, touching, and indentation occurs in this diffuse grade II astrocytoma. These nuclei infiltrate between preexisting axons stained brown with antineurofilament (anti-NF) protein stain. Diffuse infiltration of brain distinguishes most grade II and higher astrocytomas from grade I tumors (immunoperoxidase anti-NF protein with hematoxylin). From McKeever PE: Neurofilament [NF] and synaptophysin stains reveal diagnostic and prognostic patterns of interaction between normal and neoplastic tissues. Presented at the Annual Meeting of the Histochemical Society, Bethesda, MD, March 24, 2000.
intermingle with brain parenchyma. NF staining highlights axons of brain infiltrated by neoplastic astrocytes (Fig. 20-16). Diffuse invasion of brain may also be evident as formations of secondary structures of Scherer, which are described in the “Gliosis Versus Glioma” section later in the chapter. The diffuse nature of the growth and infiltration of low-grade astrocytomas demonstrates why they are so seldom cured despite their relatively benign histologic features. Postoperative survival is highly variable but usually is 3 to 10 years. The extreme variation in prognosis among grade II diffuse astrocytomas places a premium on better measures of outcome for individual patients. A low MIB-1 LI identifies patients with good prognosis (Fig. 20-17; see Fig. 20-12).65 A protein involved in the cell cycle, p53 is often mutated in diffuse astrocytomas. Direct detection of mutations in p53 require special procedures not generally used, but loss of normal p53 function usually results in increased expression of p53. This overexpression of p53 is so excessive that the p53 can be stained by IHC in many astrocytoma nuclei. Overexpression of p53 helps to identify many astrocytomas of grades II to IV. It is possible that p53 overexpression is associated with tumor progression to glioblastoma.89 The variety of chromosomal abnormalities in astrocytomas includes losses in chromosomes 13, 22, X, and Y and gains in chromosome 7.1,5 These changes can be detected with fluorescent and immunohistochemically enhanced ISH procedures.90 Gemistocytic Astrocytoma (WHO Grade II)
Gemistocytes are cells swollen with hyaline pink cytoplasm that is reactive for GFAP (Fig. 20-18, A; see Table 20-7). Their hyperchromatic and angulated nuclei are at the rim of the cells and produce a bizarre caricature
Tumors of the Nervous System
Gemistocytic astrocytomas are distinguished from oligodendrogliomas with microgemistocytes by their more angulated and pleomorphic nuclei and their longer GFAP-positive cellular processes; their lack of synaptophysin-positive neoplastic neurons distinguishes them from gangliogliomas, and their smaller and more angulated nuclei and greater tendency to infiltrate brain distinguish from subependymal giant cell tumors.
80
60 Survival (%)
783
40
Anaplastic Astrocytoma (WHO Grade III)
20
0 <2%
>2%
Labeling index (%) Figure 20-17 Probabilities of survival of patients with grade II astrocytomas in which mean MIB-1 labeling indices were ≤2% and >2%. Data from McKeever PE, Strawderman MY, Yamini B, et al: MIB-1 proliferation index predicts survival among patients with grade II astrocytoma. J Neuropathol Exp Neurol 1998;57:931-936.
of a reactive astrocyte. Astrocytomas with at least 20% gemistocytes may be considered gemistocytic astrocytomas, which are considered more aggressive than their nongemistocytic counterparts. Whereas gemistocytic astrocytomas are particularly likely to progress to a higher grade, those without high-grade features are considered grade II astrocytomas before progression.2
A Figure 20-18 A to C, Three sections of an anaplastic astrocytoma. A is stained for glial fibrillary acidic protein (GFAP) and shows some fibrillar cells. B is stained with a monoclonal antibody cocktail for cytokeratin that contains AE1/AE3, demonstrating the known cross-reactivity of AE1/AE3 with GFAP. C is stained with CAM5.2 monoclonal antibody to cytokeratin, showing that this glioma is actually negative for cytokeratin. This malignant astrocytoma demonstrates high-grade cytologic features, including mitotic activity (C), anaplastic nuclei, and pleomorphic cells that range from gemistocytes to nuclei nearly devoid of cytoplasm. Vascular features and the degree of anaplasia were insufficient to confirm grade IV (glioblastoma), and no coagulation necrosis was evident; compare with Figure 20-9 (hematoxylin counterstains). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
The anaplastic designation emphasizes the high grade of malignancy of the anaplastic astrocytoma. Features shared by high-grade gliomas are mitotic activity, as well as increases in cellular density, and nuclear pleomorphism (see Fig. 20-18) and hyperchromatism. Anaplastic astrocytomas retain GFAP-positive cellular processes and GFAP reactivity around their anaplastic nuclei (see Fig. 20-18, A); this important feature distinguishes them from reactive astrocytes trapped in other tumors. Their MIB-1 LIs are intermediate among gliomas.63 The combined lack of foci of coagulation necrosis and lack of conspicuous vascular proliferation in an astrocytic glioma distinguish anaplastic astrocytomas from glioblastomas (see Table 20-7).2 Average survival of patients with anaplastic astrocytoma is slightly more than 2 years. In pediatric patients, a low MIB-1 L1 identifies a group of patients who have a better prognosis.91
B
C
784
Immunohistology of the Nervous System
A
B
Figure 20-19 Giant cell astrocytoma. A, Huge cells with large nuclei and nucleoli and abundant, finely granular cytoplasm variably reactive for glial fibrillary acidic protein (GFAP) mingle with bundles of thick, dark-brown GFAP-positive cellular processes that are part of the tumor. Despite their size, most nuclei and nucleoli lack sharp edges. B, Nuclei have delicately stippled chromatin and rare mitoses. A few neurofilament (NF)-positive and many NF-negative cells are evident in this subependymal giant cell astrocytoma. One neoplastic cell has a mitosis and shows NF reactivity, a rare indication of the neoplastic nature of this cell type. This tumor grew from the septum pellucidum of a 20-year-old woman with tuberous sclerosis (immunoperoxidase anti-GFAP with hematoxylin). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Other Variants of Astrocytoma
Subependymal giant cell astrocytoma (GCA or SEGA) has a distinctive location, histology, and association with tuberous sclerosis (TS).92 The suppressor gene product associated with TS, tuberin, is predictably lost in GCA associated with TS.93 The tumor arises from the medial portion of the floor of the lateral ventricle in the region where the subependymal nodules of giant astrocytes in TS, known as candle guttering, are frequently found (see Table 20-7). Tumors are composed of giant astrocytes with large nuclei and prominent nucleoli (Fig. 20-19). Although these tumors are pleomorphic, most nuclei lack sharp angulations, and the giant cells are not crowded. These cells may contain glial filaments variably positive for GFAP (see Fig. 20-19, A). IHC has revealed partial neuronal differentiation in some GCAs (see Fig. 20-19, B), which complicates their classification as astrocytomas (versus gangliogliomas). These giant astrocytes and their characteristically thick cytoplasmic processes have a tendency to form disoriented fascicles. It is very important to recognize this histologic entity, because their pleomorphism is at variance with their relatively benign behavior and WHO grade I, and many GCAs are associated with TS.3 Astroblastoma is rare.2 Astroblastic rosettes resemble perivascular pseudorosettes of ependymomas, except that the astroblastic processes remain thick the entire distance from cell body to adventitia of the vessel. Foot processes may even thicken near the adventitia. IHC stains used in conjunction with routine neurohistochemical stains define this neoplasm. Although astroblastomas express focal GFAP, they do not stain with PTAH, a dichotomy that may be due to expression of a nonfibrillar form of the GFAP molecule, which is different from the fibrils of ependymoma and astrocytoma that stain for both. Pleomorphic xanthoastrocytoma (PXA) is a supratentorial astrocytoma that often involves both the
KEY DIAGNOSTIC POINTS Astrocytoma • GFAP with a good hematoxylin counterstain is the most important IHC stain to show neoplastic nuclei surrounded by GFAP and the fibrillar GFAP-positive processes of astrocytomas. • Neurofilaments stain both tests for a ganglionic tumor component (see the “Neuronal Tumors” section later in this chapter) and often show axons of brain tissue that existed before the tumor.58 The latter helps assess infiltration by neoplastic cells. • Noninfiltrating astrocytomas that lack both p53 and EGFR overexpression tend to be grade I tumors. • MIB-1 is important in assessing proliferation, particularly among astrocytomas with inconspicuous mitoses.
leptomeninges and cerebral cortex (see Table 20-7).94 It has a more distinct margin with brain than most astrocytomas (see Fig. 20-13, B). Its fibrillarity and pleomorphic, hyaline, lipid-laden, and multinucleated cells are clues to the diagnosis (Fig. 20-20). Intracellular lipid content and protein granular degeneration vary from abundant to absent in individual tumors. PXA may assume a clear cell appearance and thus may require identification by a panel of IHC reagents (see Fig. 20-1).95 Astrocytes are identified from their characteristic strongly GFAP-positive cells, often with coexpression of α-1–antitrypsin. Sparse lipid droplets are conspicuously negative for GFAP. These cells may be surrounded by reticulin fibers and basement membranes positive for type IV collagen, breaking a general rule that glioma cells lack reticulin. Neuronal elements occur in some tumors, suggesting that a PXA may be the glial portion of a ganglioglioma.96 The grade II PXA has been confused with the grade IV glioblastoma; both are very pleomorphic. The pathologist must look for
Tumors of the Nervous System
785
(LFS), the result of a germline mutation of the TP53 gene on the short arm of chromosome 17 (17p13). Data suggest that approximately 1% of gliomas occur in patients with LFS.101 More than 50% of these are astrocytic gliomas, including astrocytoma grades II to IV (diffuse low-grade astrocytoma through glioblastoma).2 Clinical situations that should arouse suspicion of LFS in a glioma patient include any second neoplasm, a family history of sarcoma or choroid plexus (CP) tumor, or a young (<45 years) close blood relative of the patient with a cancer, lymphoma, or brain tumor.102-105 EPENDYMOMAS Figure 20-20 Pleomorphic xanthoastrocytoma. In this specimen, round lipid vacuoles are visible in pleomorphic astrocytes with hematoxylin and eosin. Note that nuclear/cytoplasmic ratios are low, chromatin is finely granular, and nuclear membranes have gentle curves despite the large pleomorphic nuclei and nucleoli.
low MIB-1, virtual lack of mitoses, EGBs, little invasion (see the “Pilocytic Astrocytoma” section earlier in this chapter), and little or no overexpression of EGFR or p53 to distinguish PXA from diffuse astrocytomas and especially from glioblastomas (Fig. 20-21).97,98 Theranostic Applications
Among groups of patients with grade II astrocytomas, MIB-1 distinguishes tumors with good prognosis by their low LI (see Fig. 20-11, A).65 MIB-1 LI differentiates between grade II and grade III gliomas.66 Approximately 33% to 50% of diffuse astrocytomas have p53 abnormalities, which makes TP53 gene mutations and/or p53 overexpression an early change in astrocytomas that progress to higher grades.99 These p53 abnormalities are used to distinguish between astrocytomas and oligodendrogliomas. Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Approximately 5% of gliomas are familial.100 A fraction of these are associated with Li-Fraumeni syndrome
A
Ependymomas are an excellent example of how IHC highlights structural features important to their interpretation. The cellular conformations of ependymomas vary between fibrillar and epithelial, posing special problems of differentiation not only from other gliomas but also from carcinomas and meningiomas (see Tables 20-2 and 20-7). These latter differentiations are facilitated by the understanding that even epithelioid ependymomas frequently stain with anti-GFAP, have distinctive ultrastructure, and often contain at least a few cells with fibrillar processes (Fig. 20-22; see Figs. 20-1 and 20-2). The anti-GFAP stain highlights these fibrillar processes and greatly facilitates their identification (see Fig. 20-22, A-B). A good place to look for these fibrillar processes is around vessels. In contrast to nonglial neoplasms, aggregated ependymoma cells in tumor parenchyma lack a basement membrane. Immunostaining shows no collagen or fibronectin in these aggregates.70 Low-grade ependymomas have round and oval nuclei with finely dispersed chromatin. These nuclei distinguish them from virtually all brain tumors other than meningiomas.3 The pathologist should look for rosettes to confirm suspicion of an ependymoma (see Tables 20-2 and 207). Perivascular rosettes are most useful, because they occur in nearly all ependymomas. They have a fibrillar zone that is at least three erythrocyte diameters wide around central vessels. Anti-GFAP stains the fibrillar zone, making a subtle zone easier to find (see Fig. 20-22, B). The processes taper to become very thin as they
B
Figure 20-21 A, This pleomorphic xanthoastrocytoma does not overexpress epidermal growth factor receptors. B, Rare cells overexpress p53.
786
Immunohistology of the Nervous System
A
C
radiate from the cells to the vascular adventitia, distinguishing them from the thick processes of astroblastic formations. True ependymal rosettes are characteristic, but some samples of ependymoma lack them. The ependymal rosette consists of ependymal cells spaced around a lumen (Fig. 20-23). Cilia often protrude into the lumen from the ependymal lining. Some tumors show expanded ependymal rosettes, and others have long ependymal linings that do not close into rosettes.3 Many ependymomas have a relatively discrete margin with brain compared with other gliomas. This margin is revealed best in white matter with the NF IHC stain, which shows an abrupt border between the NF-positive axons abundant in white matter and the NF-negative ependymoma. Synaptophysin shows a corresponding distinct border between positively reacting neuropil and negatively reacting tumor.61 EM is better than IHC for difficult ependymomas (see Fig. 20-22, C). It shows cilia, basal bodies, cytoplasmic inclusions of microvilli, and elongated intercellular junctions. In addition, rare ependymomas have sparse CK or EMA immunoreactivity on their most differentiated epithelium. However, even these have much less CAM5.2 than CP papillomas and carcinomas, and CAM5.2 is recommended to distinguish them. The
B
Figure 20-22 A and B, Sections of a tumor. A, This tumor is composed of epithelioid cells (hematoxylin and eosin). B, The tumor is glial fibrillary acidic protein (GFAP) positive, and this stain highlights slight fibrillarity near the appropriately GFAPnegative vessels. C, Electron microscopy reveals cilia and basal bodies of ependymoma. Structural but not ultrastructural features of this clear cell ependymoma mimic oligodendroglioma. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Figure 20-23 Myxopapillary ependymoma. This tumor originated in the cauda equina of a middle-aged woman who had experienced knee pain for several years. It has epithelioid cells and blue-tinged mucin both in the center of vague ependymal rosettes and in the perivascular space (Alcian blue and nuclear fast red). From McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS, ed: Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492.
Tumors of the Nervous System
A
787
B
Figure 20-24 One medulloblastoma with well-differentiated Homer-Wright rosettes, from the posterior fossa of a girl of elementary school age, was positive for both neurofilament (A) and synaptophysin (not shown). Another, less well-differentiated medulloblastoma, from the posterior fossa of a boy of the same age, was synaptophysin positive (B) and neurofilament negative (not shown). Synaptophysin highlights the sparse fibrillar cellular processes. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
general features of ependymoma just described are useful in identifying the variants of ependymoma discussed in this section, except where specifically excluded. Low-Grade Ependymoma
The low-grade designation is often dropped from the name for this group of tumors, which are referred to simply as ependymomas. The features just described and the nuclear features of low-grade ependymomas distinguish them from other tumors. Nuclei of ependymomas are typically round or oval with prominent light and dark regions stained with hematoxylin (see Fig. 20-22, A, and Fig. 20-23). In the parenchyma away from rosettes, ependymoma nuclei tend to be more uniformly crowded (Fig. 20-22) than nuclei in low-grade astrocytomas (Figs. 20-11 and 20-14) and less crowded than in medulloblastomas (Fig. 20-24) and primitive neuroectodermal tumors (PNETs). Epithelioid ependymomas occasionally have remarkably distinct margins with brain that imitate margins of nonglial neoplasms (see Fig. 20-2). Anti-GFAP stain for glial filaments is extremely helpful in differentiating these ependymomas from carcinomas, pituitary adenomas, craniopharyngiomas, and meningiomas (see Fig. 20-22, B, and Table 20-2). The stain accentuates fibrillar cellular processes, which distinguish the ependymoma.3 Papillary ependymomas closely resemble CP papillomas. Solid regions of ependymoma parenchyma, where GFAP-positive neoplastic cells grow on one another, rather than on fibrovascular stroma, can be appreciated from their lack of collagen and fibronectin with IHC staining.3 Histologic grade is less predictive of survival in ependymoma than in astrocytoma.13 Radiographic evidence of residual disease after surgery predicts markedly reduced survival, putting a premium on correct intraoperative interpretation and total removal of the tumor. In infratentorial ependymomas, expression of large amounts of GFAP is associated with better prognosis.106
Clear Cell Ependymoma
Clear cell ependymoma (see Fig. 20-22, A) resembles oligodendroglioma and central neurocytoma (Fig. 20-25, A, and 20-26, A). It is an epithelioid ependymoma that also has clear perinuclear halos. IHC staining for GFAP may reveal ependymal features such as perivascular fibrils (see Fig. 20-22, B). The clear cell appearance of these ependymomas requires a panel of IHC reagents or, often, EM to differentiate them from other clear cell tumors (see Fig. 20-1 and 20-22, C).95 Tanycytic Ependymoma
Tanycytic ependymomas are found within the brain and particularly in the spinal cord. Their round to oval nuclei with distinctly light and dark regions of chromatin resemble those in ependymoma, and abundant GFAP-positive cellular processes resemble those in astrocytomas. They form structures replete with nuclei next to zones of fibrillar cellular processes. These structures are distinguished from Verocay bodies by their diffuse, extensive GFAP positivity and their lack of type IV collagen. They are not limited to surrounding GFAP-negative vessels such as the perivascular rosettes of other ependymomas. The margins of tanycytic ependymomas with surrounding parenchyma tend to be discrete, to exclude NF-positive axons of spinal cord tracts, and to be potentially resectable. Diffuse astrocytoma cells infiltrate between NF-positive axons.3,61 Subependymoma (WHO Grade I)
The subependymoma protrudes from the wall of a ventricle into the ventricular space.3 Its histologic and IHC features closely resemble those of tanycytic ependymoma (see Table 20-7). It is usually benign. Myxopapillary Ependymoma (WHO Grade I; Rarely Grade II)
The myxopapillary ependymoma (MXPE) appears the least glial in H&E-stained slides. It is nearly always
788
Immunohistology of the Nervous System
A
C
found in the region of the filum terminale, cauda equina, sacrum, and adjacent extravertebral soft tissues (see Fig. 20-23 and Table 20-2). This ependymoma differs from others in its amount of mucin production. Its hallmark is parenchymal and perivascular mucin produced by ependymal cells (see Fig. 20-23). MXPE is often papillary but may be solid. Although the differential features described in the general discussion of ependymomas can be useful, the
A
B
Figure 20-25 Both of these tumors are considered oligodendrogliomas according to current criteria, primarily on the basis of the roundness of their nuclei and also because of their perinuclear halos. A, The first tumor has glial fibrillary acidic protein (GFAP)–negative neoplastic cells with interlaced GFAP-positive processes from intermingling of reactive astrocytes with smaller nuclei. B, The second tumor contains numerous GFAP-positive microgemistocytes with brown balls of cytoplasmic GFAP and relatively short processes. C, The hematoxylin and eosin preparation of the second tumor shows perinuclear halos in many cells better than GFAP does. From McKeever PE: Insights about brain tumors gained through immunohistochemistry and in situ hybridization of nuclear and phenotypic markers. J Histochem Cytochem 1998;46:585-594.
peculiar morphology and growth of MXPEs pose unique problems. Individual tumors vary dramatically between epithelial and fibrillar cells. The most difficult variants of MXPE to recognize are those that are nearly all myxoid or all epithelial. The highly myxoid variety may produce cords of cells in a mucoid matrix that resembles chordoma, a neoplasm found in the same location. Presence of GFAP is the key IHC feature distinguishing MXPE from chordoma.
B
Figure 20-26 Central neurocytoma. A, The fibrillar perivascular zone and round cells with halos around round nuclei imitate features of ependymoma and oligodendroglioma (hematoxylin and eosin). B, Neoplastic cells ubiquitously express synaptophysin. From McKeever PE: Insights about brain tumors gained through immunohistochemistry and in situ hybridization of nuclear and phenotypic markers. J Histochem Cytochem 1998;46:585-594.
Tumors of the Nervous System
A
789
B
Figure 20-27 Schwannoma of a peripheral nerve in the brachial plexus of a young man. A, Type IV collagen–positive basement membranes surround each tumor cell in compact Antoni A tissue. The darker brown vessel in the corner has more collagen. B, Nuclei and cytoplasm are abundantly immunoreactive for S-100 protein, but the vessel in the corner is not.
Fibrillary MXPE may be confused with fibrous meningioma and schwannoma. The epithelial and papillary variants may resemble carcinoma or meningioma,3 although a positive GFAP stain response differentiates MXPE from GFAP-negative carcinoma and meningioma. MXPE lacks type IV collagen and fibronectinpositive basement membranes around each cell, a feature of schwannoma (Fig. 20-27, A). In contrast to metastatic carcinoma, MXPEs lack malignant cytology, have a lower MIB-1 LI, and are focally fibrillar.7 Paragangliomas may mimic MXPEs, but MXPEs lack chromogranin A and express GFAP. Anaplastic Ependymoma (Malignant Ependymoma; WHO Grade III)
Anaplastic ependymomas are those with malignant features, including conspicuous mitotic activity, nuclear and cellular pleomorphism, multinucleated and giant cells, high cellular density, necrosis, and vascular proliferation (see Tables 20-2 and 20-7).3 Histopathologic features of malignancy do not accurately predict poor survival.13 This problem may eventually be solved by IHC: the combination of increased vimentin expression and decreased GFAP expression may predict poor survival in infratentorial anaplastic ependymomas,106 and anaplastic ependymomas are more likely to overexpress p53 or EGFR protein than are low-grade ependymomas.76 OLIGODENDROGLIOMA
Oligodendroglioma and its anaplastic counterpart in particular have been found to respond to PCV and Temodar chemotherapy.107,108 This feature has put a premium on the recognition of both tumors.2 The pure oligodendroglioma differs from other gliomas, except for a few ependymomas, in having an epithelioid, rather than a fibrillar, appearance (see Table 20-2). This appearance is most evident within the central portion of the neoplasm, which is most crowded with neoplastic cells.3 Perinuclear halos are an important feature of formalin-fixed paraffin sections of oligodendroglioma
(see Fig. 20-25). Well-differentiated oligodendrogliomas have remarkably round and regular nuclei centrally placed within cells, which resemble fried eggs. Their vessels are usually numerous, fine, CD31-positive capillaries that sometimes segregate the parenchyma into small lobules.13 Microgemistocytes have been considered oligodendroglial and are distinguished from gemistocytic astrocytoma cells by their round nuclei and short processes. Microgemistocytes have a ball of cytoplasmic GFAP immunoreactivity near their nucleus and have shorter cellular processes than gemistocytic astrocytes and astrocytomas (see Fig. 20-25, B). The epithelioid appearance of pure oligodendrogliomas (see Fig. 20-25, A) imitates that of true epithelial neoplasms (Table 20-2).60 Suprasellar oligodendrogliomas may be mistaken for pituitary adenomas (see Fig 20-2). Oligodendrogliomas may be confused with meningotheliomatous or clear cell meningiomas or with lipoid metaplasia in meningiomas. Anaplastic oligodendrogliomas simulate metastatic carcinoma, particularly renal cell carcinoma. GFAP-positive tumor cells (see Fig. 20-25, B) distinguish the oligodendroglioma from these other tumors, but not all oligodendrogliomas have GFAP-positive neoplastic cells. In their absence, the tumor margin with brain is key (see Fig. 20-1). All types of oligodendroglioma, including oligoastrocytoma, have diffuse margins (see Fig. 20-13, A). It is also helpful to find reactive astrocytes widely dispersed within an oligodendroglioma (see Fig. 20-25, A), a pattern not seen in meningioma. Even macroscopically discrete oligodendrogliomas show a more diffuse margin with brain than adenomas, carcinomas, and meningiomas. Either NF or synaptophysin used as a brain tissue marker61 or the presence of GFAP-positive gliosis delineates a sharp margin with brain in these other tumors, even carcinomas that engulf chunks of brain (Figs. 20-28 and 20-29, B). Among these tumors, secondary structures are seen only with the oligodendroglioma (see the sections on astrocytomas and gliomas). Precise localization of the biopsy specimens is helpful, because oligodendrogliomas do not
790
Immunohistology of the Nervous System
expression of synaptophysin by as many as 18% of oligodendrogliomas109 should be noted so that they are not confused with central neurocytomas and dysembryoplastic neuroepithelial tumors (DNTs); both have distinct margins with brain, in contrast to the diffuse margin of oligodendroglioma. Oligoastrocytoma (Mixed OligodendrogliomaAstrocytoma; WHO Grade II)
Figure 20-28 This metastatic carcinoma in an elderly man was clinically judged to be a glioma because of its solitary nature and unknown primary. The clear cells and crowded anaplastic nuclei resemble those of an anaplastic oligodendroglioma. Here at the margin, however, it engulfs brain, excluding, rather than intermingling with, the resulting islands of brown, glial fibrillary acidic protein (GFAP)–positive gliosis. Subsequent clinical evaluation revealed the primary cytokeratin-positive and GFAP-negative carcinoma in the left lung base. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
The oligoastrocytoma is a mixed glioma composed of both astrocytoma and oligodendroglioma, as described in the respective sections on these tumors (see Fig. 20-13, A). Much less common than oligoastrocytoma, mixed gliomas may have a component of ependymoma.13 Difficulties are encountered in the attempt to determine whether individual tumors contain a mixture of both oligodendroglial and astrocytic elements and whether both elements are neoplastic. Each element must be conspicuous for this diagnosis. Oligodendroglioma cells must be distinguished from infiltrating macrophages with lipid; the former have neoplastic nuclei and lack IHC macrophage markers such as KP1 (see Figs. 20-6, A, and 20-25, A).
KEY DIAGNOSTIC POINTS
originate from the adenohypophysis or dura and rarely invade them. A panel of immunostains for chromogranin and pituitary hormones can identify adenomas, but no specific marker for oligodendroglia withstands paraffin embedding. The discovery of such a marker would be a major contribution to neuropathology. The broad specificity of Leu-7 and S-100 protein limits their use for IHC analysis of oligodendroglioma. However, an oligodendroglioma invading the meninges can be differentiated from a syncytial meningioma by its positivity with the Leu-7 monoclonal antibody, because meningiomas are typically Leu-7 negative. The
A
Oligodendroglioma • Prepare to use molecular markers to test for 1p and 19q deletions that signal better prognosis and PCV sensitivity. • Criteria for anaplastic oligodendroglioma are different than those for anaplastic astrocytoma. An occasional mitosis and limited vascular proliferation may be found in a grade II oligodendroglioma. • A few otherwise typical and diffusely invasive oligodendrogliomas may show some cells with positive neuronal markers, particularly synaptophysin.
B
Figure 20-29 Glioblastoma vs. trapped gliosis. The importance of examining the perikaryon (cytoplasm around the nucleus) for the marker is shown in these two tumors stained for glial fibrillary acidic protein (GFAP). A, GFAP surrounds and touches neoplastic nuclei and a mitosis in the glioblastoma. B, GFAP intimately contacts only reactive astrocytic nuclei in this gliosis trapped by metastatic carcinoma. The carcinoma was cytokeratin positive (not shown; see Fig. 20-28). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Tumors of the Nervous System
The astrocytic component can be assessed for neoplastic cells with hematoxylin on H&E and/or GFAP stains. The purpose of GFAP is to reveal neoplastic nuclei in cells with long cellular processes. If necessary, GFAP staining can be reduced to avoid hiding the purple nuclei in brown. For automatic stainers, the primary anti-GFAP antibody can be titrated to a dilution that shows a discernible light brown. In manual staining, either this titrating can be done, or the diaminobenzidine (DAB) substrate can be used for less than half the usual time. Regular-strength hematoxylin counterstain then reveals nuclear details without interference from a dark-brown IHC reaction product, which somehow obscures nuclei, even though glial fibrils are cytoplasmic. A good nuclear counterstain should always be used with IHC analysis of brain lesions. Indistinct cell borders and infiltrations in brain require the counterstain for orientation. The counterstain provides important information about cell type and reactive versus neoplastic cells. Anaplastic Oligodendroglioma (Malignant Oligodendroglioma; WHO Grade III)
The features of anaplastic transformation in oligodendroglioma are similar to those in anaplastic ependymoma (Fig. 20-30). However, limited amounts of vascular proliferation are frequently present in oligodendroglioma, and in isolation, they cannot be considered evidence of malignant transformation (see Table 20-2). Vascular proliferation is highlighted by vimentin and CD31 stains. Anaplastic and low-grade oligodendrogliomas may express neuronal markers and vimentin (Fig. 20-31). Synaptophysin-positive cells are scattered sparsely when present. Theranostic Applications. MIB-1 is useful in predicting good outcome among patients with oligodendrogliomas. An MIB-1 LI less than or equal to 5% has been found to correlate with better survival.110 Vimentin expression and molecular alterations in CDKN2A, PTEN, and EGFR genes correlate with poor prognosis.72,109 The presence of IDH1 mutations carries a strong favorable prognostic significance for overall survival in anaplastic oligodendroglial tumors.111 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications. Molecular characterization of tumors with oligodendroglioma components is being used as a guide to therapy. Chromosomal deletions in 1p and 19q are associated with favorable prognosis and good response to combined PCV chemotherapy— procarbazine, CCNU, and vincristine—for patients with oligodendroglioma and particularly its anaplastic counterpart.107,108,112 Neurooncologists at my institution have dropped vincristine from PCV, preferring PC chemotherapy, and many centers have done the same. Temozolomide is now the first-line chemotherapy for most grade II to IV gliomas.113 This has occurred without a study proving that temozolomide is superior to PCV or PC, although it is likely that it is at least equal. Molecular characterization of chromosomal deletions in 1p and 19q is used by some neuropathologists
791
in diagnosis. A new IHC marker of oligodendroglioma has appeared every year or two, but none have lasted. Arguably the best positive marker seems to be deletions on the short arm of chromosome 1 (1p) and on the long arm of chromosome 19 (19q).114,115 Should the absence of at least one sensitive and specific IHC marker for oligodendroglioma cells continue, the combined 1p/19q chromosomal marker will eventually be incorporated into diagnostic criteria for oligodendrogliomas. Chromosomal deletions in 1p and 19q can be detected either by PCR, ISH enhanced by immunostaining, or fluorescence in situ hybridization (FISH; see Fig. 20-30, D). Anaplastic Oligoastrocytoma (Malignant Mixed Oligodendroglioma-Astrocytoma; WHO Grade III)
Oligoastrocytomas can be anaplastic. Histologic features of cytologic anaplasia, high cell density, conspicuous mitoses, and microvascular proliferation distinguish these anaplastic oligoastrocytomas from grade II oligoastrocytomas. A frequent feature of anaplastic oligoastrocytoma is a prominence of fibrillar, GFAPpositive malignant astrocytes. These may overgrow the oligodendroglial component and may ultimately result in a glioblastoma. Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications. The utility of chemotherapy when deletions are present in 1p and 19q is thought to be higher than when deletions are not present. It is not clear whether oligodendroglioma without deletions is any different from mixed oligoastrocytoma in this regard. Studies of the potential utility of chemotherapy in anaplastic oligoastrocytomas and its relation to 1p/19q status are available.112,113 GLIOBLASTOMA MULTIFORME (GLIOBLASTOMA; WHO GRADE IV)
In large part as a result of revelations about its frequent expression of GFAP-positive fibrillar cellular processes, rather than being classified as an embryonal neuroglial malignancy, glioblastoma is now considered the most malignant of astrocytomas. Glioblastoma often contains focal astrocytoma, less often oligodendroglioma, and, rarely, ependymoma. The diagnostic criteria for glioblastoma were relaxed in the 1990s. Formerly, the cytologic criteria of anaplastic astrocytoma—mitotic activity, hypercellularity, pleomorphism, and nuclear hyperchromasia—plus both vascular proliferation and spontaneous necrosis were required (see Fig. 20-12, B). Now, either vascular proliferation or spontaneous coagulation necrosis is a sufficient addition to anaplasia for the diagnosis of glioblastoma (see the “Anaplastic Astrocytoma” section for a comparison). If necrosis is absent, vascular proliferation, increased density of cells in vascular walls, should be unequivocal; it can be highlighted with vascular endothelial growth factor (VEGF),116 vimentin, collagen type IV, or fibronectin stain. Other malignant features of glioblastomas are bizarre nuclei, multinucleated cells, and extreme pleomorphism (Fig. 20-32; see Figs. 20-12, B, and 20-29, A). Unfortunately, the heterogeneity of glioblastomas
792
Immunohistology of the Nervous System
A
D
B
C for these histologic features compromises diagnoses obtained on small specimens, such as those obtained with stereotactic needle biopsy, and this jeopardizes accurate grading (see Fig. 20-12).117 Most confusion arises in distinguishing glioblastoma from malignant meningioma and carcinoma. Unlike carcinoma and meningioma, glioblastomas contain fibrillar neoplastic cells that express GFAP in their cellular processes (see Figs. 20-4; 20-12, B; 20-28; 20-29; and 20-32 and Tables 20-2, 20-8, and 20-9). The cytologic features of the GFAP-positive cells should be checked for anaplasia to confirm glioblastoma, because both carcinoma
Figure 20-30 A, This grade III anaplastic oligodendroglioma is crowded with more pleomorphic nuclei than the grade II oligodendrogliomas shown in Figure 20-25. Despite their pleomorphism, nuclei tend to be round, and mitotic figures are numerous (hematoxylin and eosin). B, Crowded hyperchromatic nuclei do not overexpress p53. C, Diffuse margin of this anaplastic oligodendroglioma in gliotic brain highlights glial fibrillary acidic protein– negative branching microvascular proliferations. D, One of the deletions often found in oligodendroglioma is on the short arm of chromosome 1 (1p). In this fluorescent in situ hybridization (FISH) preparation, the test probe for 1p32 is red, and the reference probe for 1q42 is green. Each nucleus is counterstained blue. The single red dot in each of the three whole nuclei demonstrates a deletion on 1p. The reference probe shows two green dots, which reflects a pair of chromosomes giving this signal, as expected in these diploid interphase nuclei. FISH preparation courtesy Dr. Arie Perry, Division of Neuropathology, Department of Pathology, Washington University School of Medicine, St Louis, MO. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
and malignant melanoma can trap islands of CNS parenchyma and stimulate gliosis (see Figs. 20-28 and 20-29). Sarcoma is easily confused with glioblastoma on H&E staining, but GFAP reveals the glioblastoma (see Fig. 20-32). Although the rapid growth of a glioblastoma may produce a pseudocapsule, neoplastic glia are evident beyond this margin within brain tissue (Fig. 20-33). Gliosarcoma (WHO Grade IV)
A gliosarcoma is a mixture of glioblastoma and sarcoma (Fig. 20-34). Regions of collagen-positive and
Tumors of the Nervous System
793
70 60
Positive (%)
50 40 30 20 10 0 Vimentin
GFAP
NSE Synaptophysin
Immunohistochemical markers Figure 20-31 Immunohistochemical staining responses among more than 80 cases of oligodendroglioma (grades II and III). Bars represent percentages of cases positive for these four markers. GFAP, Glial fibrillary acidic protein; NSE, neuron-specific enolase. Data from Dehghani F, Schachenmayr W, Laun A, et al: Prognostic implication of histopathological, immunohistochemical and clinical features of oligodendrogliomas: a study of 89 cases. Acta Neuropathol 1998;95:493-504.
Figure 20-32 Extreme pleomorphism of cells and nuclei in a glioblastoma includes a huge, brown, glial fibrillary acidic protein (GFAP)–positive cell with multiple nuclei. Giant cell glioblastomas were called monstrocellular sarcomas before recognition of their GFAP reactivity. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
TABLE 20-9 Differential Diagnosis of a Mass of Small, Crowded Anaplastic Cells Differential Features Diagnosis
Structures
Antibody*
Locations†
Ependymoblastoma
Like PNET; ribbons/cords of cells, true ependymal rosettes
Vimentin (S), GFAP (R)
Cerebrum, cerebellum
Medulloblastoma/ pineoblastoma/ neuroblastoma/PNET
Slight fibrillarity, (HomerWright rosettes), (palisades), “carrot” nuclei, (neural or glial foci)‡
Synaptophysin (+), PGP9.5, S-100, neurofilament (R), GFAP (R)
Cerebellum, brainstem, pineal gland, CNS, (extra-axial)‡
Rhabdomyosarcoma/ medullomyoblastoma
Like PNET; muscle striations
Desmin (S), muscle-specific actin
Pineal, cerebellum, CNS
Atypical teratoidrhabdoid tumor
Like PNET; more cytoplasm
Vimentin (+), GFAP (S), cytokeratin (S), EMA (S), synaptophysin (R), chromogranin (R)
Cerebellum, brain
Hemangiopericytoma
Hypercellularity, thick pericellular matrix, mitoses
Vimentin (+)
Falx, tentorium meninges, (extracranial)‡
Lymphoma
Noncohesive round cells, vascular wall invasion
L26 (+), LCA (S), monoclonal κ and λ Ig
Deep cerebrum, CNS, meninges; may be multiple
Small cell carcinoma
Cohesive cells, (epithelioid), (desmosomes)‡
Cytokeratin (+), EMA, synaptophysin (S)
CNS, meninges; frequent multiple masses; systemic
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *Key to staining results: +, almost always strong, diffuse positivity; S, sometimes or focally positive; R, rare cells may be positive. † Most common or most specific location is listed first. ‡ Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. CNS, Central nervous system; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; Ig, immunoglobulin; LCA, leukocyte common antigen; PGP9.5, protein gene product 9.5; PNET, primitive neuroectodermal tumor.
794
Immunohistology of the Nervous System
GFAP-negative sarcoma cells bridge the glioblastoma in a marbled configuration (see Table 20-7). Differences in collagen messenger RNA (mRNA) and cellular DNA content indicate the extent of variation between these regions.118,119 Despite the fact that gliosarcomas often seem more circumscribed than glioblastoma, gliosarcoma can metastasize.79 Tumor progression from gliosarcoma to pure sarcoma lacking GFAP-positive cells can occur.120 The glial and mesenchymal elements have similar genetic alterations.121,122 KEY DIAGNOSTIC POINTS Glioblastoma • All glioblastomas have some cells that meet cytologic criteria of astrocytoma: long cellular processes, GFAP, and elongated nuclei. They often will have additional cells that resemble other types of glioma. • All meet the cytologic criteria of anaplasia: mitotic activity, hypercellularity, pleomorphism, and nuclear hyperchromasia. • In addition to the above, a glioblastoma must also have either necrosis or microvascular proliferation and may have both.
Figure 20-33 This neurofilament-negative giant cell glioblastoma has a relatively abrupt margin with brown neurofilament positive cerebral white matter. However, near the appropriately neurofilament-negative vessel and large abnormal mitotic spindle, the tumor infiltrates between brown long axons.
A
B
C
D
Figure 20-34 A, This gliosarcoma, shown with hematoxylin and eosin staining, had regions and bands of collagen-positive, glial fibrillary acidic protein (GFAP)–negative sarcoma cells and other regions of GFAP-positive, collagen-negative malignant astrocytes. B, Shown under high magnification here, sarcoma cells produce cyan collagen as they wander away from a vessel into tumor parenchyma (Masson trichrome stain). C, This nearby section shows most collagen-associated cells to be GFAP negative, whereas malignant astrocytes further from the vessel are GFAP positive. D, Epithelial metaplasia of brown CAM5.2-positive cells was present in small foci in other regions of this gliosarcoma. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Tumors of the Nervous System
Glioblastoma and Gliosarcoma with Epithelial Metaplasia (WHO Grade IV)
Rarely, gliosarcomas and glioblastomas produce adenoid formations or epithelial foci with squamous differentiation and keratin pearls.3,13 These regions stain immunohistochemically for CK and EMA (see Table 20-8 and Fig. 20-34, D). To avoid confusion of this tumor with carcinoma, it is necessary to obtain adequate sampling and to be aware that these regions are focal and that other regions will show the familiar fibrillar, GFAPpositive neoplastic cells of a glioblastoma. It is important to remember that carcinoma cells are GFAP negative. Glioblastomas manifest other peculiar features. Rare glioblastomas occur with granular cell tumors. Some epithelioid glioblastomas contain diffuse cytoplasmic lipids.13 Theranostic Applications
Two structurally similar varieties of glioblastoma are called primary and secondary glioblastoma. Primary glioblastomas are those that arise de novo in older patients, who have higher MIB-1 proliferation indices and shorter survival than their younger counterparts.64 Primary glioblastomas are often associated with CDKN2A deletions, PTEN alterations, and EGFR gene amplification plus EGFR overexpression. Secondary glioblastomas are those that arise by progression from lower-grade gliomas. Secondary glioblastomas are associated with TP53 gene mutations and/or with p53 protein overexpression.2,13 TP53 gene mutations and p53 overexpression often, but not always, coincide. EGFR and p53 overexpression can be defined immunohistochemically (Fig. 20-35). Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Loss of genetic material in chromosome 10 is the most frequent genetic abnormality in glioblastomas and occurs in approximately 66%. Loss of genetic material in the short arm of chromosome 10 (10p) is nearly
A
795
always associated with primary glioblastomas.2 Loss in the long arm (10q) is found in both primary and secondary glioblastomas. These losses can be observed with comparative genomic hybridization (CGH),123 FISH,124 ISH enhanced with immunostaining,121 or PCR assay. Amplifications of chromosome 7 DNA are also demonstrated by modifications of these techniques.125 Molecular oncology studies show variable responses of glioblastomas to chemotherapy. Some of this is due to O6 methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme that removes methyl groups from O6 methylguanine, an abnormal guanine that is harmful to DNA. This is a praiseworthy enzymatic pursuit when protecting normal cells. Unfortunately, MGMT also removes methyl groups from O6 methylguanine produced in glioblastoma cells by alkylating chemotherapy agents, such as temozolomide given to kill the tumor. In this way MGMT hinders the action of these important drugs. In an odd twist of fate, however, the MGMT gene promoter is methylated in nearly half of glioblastoma tumors, stopping their production of MGMT and rendering these tumors sensitive to alkylating agents. Methylation of the MGMT gene promoter can be detected by PCR.126 An assay of MGMT is offered by LabCorp (Research Triangle Park, NC) and by Mayo Medical Laboratories. Some neurooncology centers are using MGMT gene methylation to predict which patients have glioblastomas that are sensitive to temozolomide.
Neuronal Tumors Neuronal tumors contain an abnormal proliferation of neurons. They range from the most benign gangliocytoma to the anaplastic ganglioglioma and PNET (formerly ganglioneuroblastoma and CNS neuroblastoma; see Tables 20-7 through 20-9). Most low-grade ganglion cell neoplasms have a better prognosis than gliomas found in the same location, and their proper identification is important. The identification and evaluation of ganglion cell neoplasms has four important stages:
B
Figure 20-35 Molecular marker expression of glioblastomas. This glioblastoma arose de novo in a 70-year-old woman with a 2-week history of headaches. A, Tumor overexpresses epidermal growth factor receptors (EGFRs) but does not overexpress p53 (not shown). B, This glioblastoma progressed from a grade II astrocytoma in a 52-year-old man. It overexpresses p53, as shown here, but not EGFR (not shown).
796
Immunohistology of the Nervous System
A
B
Figure 20-36 Ganglioglioma from the parietal lobe of a middle-aged man with progressive unilateral loss of coordination exhibits binucleated ganglion cells with large nucleoli and Nissl substance on hematoxylin and eosin (A) and neurofilament (NF) staining (B). This specimen was prepared with immunoperoxidase anti-NF protein with hematoxylin.
1. Recognition of neurons (Fig. 20-36) 2. Confirmation that neurons are neoplastic 3. Determiation of whether glia are present 4. Evaluation of any glia for neoplasia Many neoplastic cells—particularly those of glioblastoma, melanoma, and astrocytoma—resemble neurons because of their large size or prominent nucleoli.127,128 These cells lack NF and synaptophysin markers of neurons (see Fig. 20-33 and 20-44, D). The most important step in using NF or synaptophysin to identify a neuron is to trace the marker back to the cell body (perikaryon). Synaptophysin is present in the neuropil, which makes evaluation of cellular surface staining difficult. Commercial anti-NF and anti-synaptophysin immunoperoxidase markers of neurons must be chosen carefully, and their use must be controlled by staining of normal brain specimens in the same batch of slides and preferably in the same slide as the unknown tumor (see Fig. 20-13). Whereas neuron-specific enolase (NSE) has been described as a marker of neuronal tumors in reviews and texts, it has better uses than this. The shortcoming of neuronal markers is their propensity to also stain glial tumors, which commonly need to be distinguished from neuronal tumors.2,3 Experimental IHC stains such as NeuN identify neurons by showing their on-target nuclear features rather than their cytoplasmic or confusing surface features.129,130 In cases refractory to IHC stains, EM positively identifies Nissl substance, neurofilaments (NFs), neurosecretory granules, and synapses in neoplastic cells. A common error in determining whether identified neurons are neoplastic is to interpret a field of normal neurons infiltrated by glioma cells as a ganglioglioma. As previously described, the synaptophysin or NF should be traced back to a cell body, and hematoxylinstained nuclear features should be used to determine whether the cell is neoplastic according to standard criteria. Evidence of neuronal neoplasia includes hypercellularity and disarray of neurons, binucleated neurons, nuclear atypia, and pleomorphism in cells that respond positively to staining for synaptophysin or NF (see
Fig. 20-36, B). Degenerative changes in such neoplastic neurons may occur.127 Ganglion cell tumors may show heavy bands of collagen and fibronectin-positive fibrous tissue, or they may show perivascular round cells, but neither of these is invariably present. GANGLIOCYTOMA (WHO GRADE I), GANGLIOGLIOMA (WHO GRADE I OR II), AND ANAPLASTIC GANGLIOGLIOMA (WHO GRADE III)
Gangliocytoma, ganglioglioma, and anaplastic ganglioglioma may arise anywhere but are most common in the cerebrum, particularly the temporal lobe. Once a ganglion cell neoplasm has been identified, the glial element must be evaluated (see Table 20-7). A section lightly stained for GFAP with immunoperoxidase (either with less than half the usual time in DAB substrate for manual staining, or with use of a lower primary antibody titer in an automatic stainer) and fully counterstained with hematoxylin facilitates this determination by providing a better view of glial nuclei. If the light-brown cells appear reactive, cluster near the margin of the neoplasm, and do not meet the criteria for neoplasia (described in the “Astrocytoma” section earlier in this chapter), the tumor is a central neurocytoma or gangliocytoma. Gangliocytomas tend to be benign. They often contain immunocytochemical positivity for at least one neurotransmitter peptide or amine, including somatostatin, corticotropin-releasing hormone, β-endorphin, galanin, vasoactive intestinal peptide, calcitonin, serotonin, catecholamines, and met-enkephalin.131 If the GFAP-positive cells are neoplastic but not anaplastic, the neoplasm is a ganglioglioma (Fig. 20-37, B). If these glial elements are anaplastic (see the sections on astrocytomas and glioblastoma earlier in this chapter), the neoplasm is an anaplastic ganglioglioma. The proliferative capacity of the GFAP-positive glial component of ganglion cell tumors is critical to their histologic grade and can be assessed immunohistochemically with MIB-1 or PCNA. Usually, the astrocytic component has immunoreactivity for such proliferation markers, and it is routinely low.13
Tumors of the Nervous System
A
B
C
D
E
797
Figure 20-37 A-B, Desmoplastic infantile ganglioglioma. A, This massive tumor of the left cerebral hemisphere of an infant boy contains red-stained abnormal binucleated neurons and glia mixed within a complex network of blue desmoplasia (trichrome). B, Glial fibrillary acidic protein (GFAP) highlights islands of brown astrocytes. C to E, Rosette-forming glioneuronal tumor. C, Predominantly round nuclei form perivascular pseudorosettes around vessels (hematoxylin and eosin). Tumor cells stain with variable intensities with synaptophysin (D) and GFAP (E) stains.
DYSPLASTIC GANGLIOCYTOMA OF THE CEREBELLUM (WHO GRADE I)
DYSEMBRYOPLASTIC NEUROEPITHELIAL TUMOR (WHO GRADE I)
An unusual variant of a gangliocytoma, dysplastic gangliocytoma of the cerebellum (DGC) is also known as Lhermitte-Duclos disease.92 Hyperplastic and disordered synaptophysin-positive granular cell neurons enlarge part of the cerebellum into bizarre “megafolia.” This rare tumor looks dysplastic but has recurred after surgery. The growth potential of individual tumors can be monitored by staining for MIB-1. Some DGCs are familial, and others are associated with Cowden syndrome or multiple hamartoma-neoplasia syndrome.132
Dysembryoplastic neuroepithelial tumor (DNT) may be the hamartomatous counterpart of a ganglioglioma. It is multinodular within the cerebral cortex, most often in the temporal lobe cortex. Some DNTs are cystic (Fig. 20-38, A). Some are incidental findings, but many others are associated with long-standing partial complex seizure disorders in children and young adults.133 These tumors have prominent cells that resemble oligodendroglia and NF-positive neurons, synaptophysinpositive neurons, or both, that often appear to float
798
Immunohistology of the Nervous System
A
Figure 20-38 Dysembryoplastic neuroepithelial tumor. A, This cystic tumor has multiple cortical nodules surrounded by brown glial fibrillary acidic protein–positive astrocytes near a collapsed cyst. B, Neurons float in cystic spaces. C, Brown, large, neurofilament-positive “kissing neurons” compress and mold into one another. This specimen is from a 40-year-old man who had long-standing, medically refractory epilepsy and a rightfrontal multinodular cortical tumor. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
within Alcian blue–positive acid mucopolysaccharide. These are known as floating neurons (see Fig. 20-38, B). Other large neurons lack their normal spacing (see Fig. 20-38, C). GFAP-positive astrocytes are variably present within the tumor and often surround it. This tumor’s low MIB-1 LI, mature histotopographic appearance, and association with cortical dysplasia suggest a maldevelopmental origin.13 Oligodendrogliomas can produce mucopolysaccharide and can even float an occasional normal neuron. Discrete cortical location, lack of mass effect, low MIB-1 LI, and lack of brain infiltration typify DNTs and distinguish them from oligodendrogliomas.2 Evaluating the tumor margin with serial sections analyzed immunohistochemically for NF, MIB-1, and synaptophysin shows these features.61 Neuronal nuclear antigen, if present, may also identify a DNT.134 GLIONEURONAL TUMORS
These tumors feature separate GFAP-positive and synaptophysin-positive elements. Currently, glioneuronal tumors include a diverse group of tumors with patterned and almost histiotypic features that include elements of glia and neurons (see Fig. 20-37, C-E). The cerebral papillary glioneuronal tumor (PGT) shows layers of glia with compact nuclei next to layers of oligodendroglia-like cells and neurons. Some layers cover vessels, and some look papillary.135,136 In the posterior fossa, the rosette-forming glioneuronal tumor resembles a PGT with the addition of perivascular
B
C pseudorosettes (see Fig. 20-37, C) or neurocytic rosettes with fibrillar cores and a more astrocytic glial component.137 The neurocytic rosettes have fibrillar cores, as do Homer-Wright rosettes. Most glioneuronal tumors are grade I, except for the glioneuronal tumor with neuropil-like islands reported to occur within grade II and III diffuse astrocytomas.2 KEY DIAGNOSTIC POINTS Neuronal Tumors • Neuronal brain tumors tend to have better patient prognoses than their glial counterparts (ganglioglioma vs. fibrillary astrocytoma). This tendency has been enhanced by recent reclassifications of malignant neuronal brain tumors as PNETs. • Key markers of neuronal tumors are synaptophysin and neurofilaments. High-quality hematoxylin counterstain is needed to reveal neoplastic nuclei in positive cells. • Many neuronal markers, including NSE, are not reliably specific for neuronal tumors. Test new markers on glioma before using them to identify neuronal tumors. • Low-grade neuronal neoplasms resemble hamartomas and dysplasias. Use MIB-1 and interval growth on serial radiographs to distinguish the true neoplasms.
Glioneuronal tumors are a heterogeneous group of tumors in need of further definition. When their definition is complete, the glioneuronal tumors may encompass other focally synaptophysin-positive tumors
Tumors of the Nervous System
also interpreted as odd variants of gliomas, such as oligodendrogliomas and ependymomas with neuronal differentiation.108,138,139 DESMOPLASTIC INFANTILE GANGLIOGLIOMA (WHO GRADE I)
Desmoplastic infantile gangliogliomas (DIGs) often attain considerable size and resemble very fibrous gangliogliomas (see Fig. 20-37, A). These neoplasms are found in patients younger than 3 years of age, are frequently cystic, and often involve the meninges.3 Their substantial differentiation produces a mixture of GFAPpositive glial cells (see Fig. 20-37, B), NF-positive and synaptophysin-positive neurons, and vimentin-positive fibrovascular cells. DIGs have a low MIB-1 and do not overexpress p53.140 CENTRAL NEUROCYTOMA (WHO GRADE II)
Recognized recently, central neurocytoma (CN) has stimulated much interest because of its structural beauty, hidden identity, generally benign prognosis, and fluctuating interpretations (see Fig. 20-26, A).3,141 Its previous WHO classification was grade I, more representative of the benign appearance and low proliferation of nearly all CN. It is the most common neoplasm involving the septum pellucidum in young adults, and although it often has slightly more fibrillarity, CN resembles oligodendroglioma (see Tables 20-2 and 20-7 and Fig. 20-1). Careful application of IHC markers facilitates proper identification of the tumor, which for years had been mistaken for a glioma (see Fig. 20-26). CNs express much synaptophysin (see Fig. 20-26, B).60 They are usually GFAP negative, but many contain reactive astrocytes. CNs do not amplify EGFR.142 If the synaptophysin stain is equivocal, EM is recommended to distinguish CN from oligodendroglioma and ependymoma. Although radiotherapy has been used to treat CN, a good prognosis usually follows total surgical excision.141 CN rarely shows anaplastic features. CEREBELLAR LIPONEUROCYTOMA
Except for lipids in some of its cells, the rare cerebellar liponeurocytoma resembles a CN.2 Microtubules, 100to 200-nm dense-core vesicles, and clear vesicles identify its true neuronal lineage. GANGLIONEUROBLASTOMA AND NEUROBLASTOMA
Ganglioneuroblastoma and neuroblastoma are discussed in the “Primitive Neuroectodermal Tumor” section later in the chapter.
Choroid Plexus Epithelial Tumors Most choroid plexus (CP) neoplasms appear in childhood. They can occur in any portion of the CP (see Table 20-2) but are more common in the lateral
799
ventricles of children and in the fourth ventricle of adults.3 CHOROID PLEXUS PAPILLOMA (WHO GRADE I)
In contrast to papillary ependymoma, the CP papilloma contains a layer of columnar to cuboidal epithelial cells over a basement membrane and fibrovascular stroma. The type IV collagen and laminin in this stroma contrast with ependymomas, which have solid parenchyma without collagen or laminin (see Tables 20-2 and 20-7; see the “Ependymomas” section earlier in this chapter). Focal GFAP reactivity in certain CP papillomas suggests focal ependymal differentiation. This overlap in immunostaining responses includes other markers, although CP papillomas express substantially more CAM5.2 cytokeratin than ependymomas.13 The CAM5.2 response is often particularly robust (Fig. 20-39, A). Transthyretin is a potential marker of this papilloma (see Fig. 20-39, B), but its spectrum of reactivity is broad.143 Newer potential markers of CP papillomas include insulin-like growth factor II (IGF-2) and synaptophysin. IGF-2 is found in papillomas but not in normal CP.144 Synaptophysin is present in some normal CP, CP papilloma, and CP carcinoma but not in metastatic papillary carcinoma.145 Both markers may assist in the differential diagnoses of some tumors, but I find them most useful combined with traditional markers. CD44 is preferentially expressed on atypical papilloma and CP carcinoma and may be a marker of aggressive CP tumors. Aggressive tumors have higher mean MIB-1 LIs of 6%.146 These various results should be verified with larger series of cases. Meningioma and carcinoma enter the differential diagnosis. An epithelial lining of CAM5.2-positive cells in the CP papilloma, lack of whorls, and lack of syncytial foci distinguish it from papillary meningioma. Secretory meningioma has a focal CK response, but not in an epithelial lining with a fibrovascular core. CP papilloma lacks the necrosis and anaplasia seen in metastatic papillary carcinoma. KEY DIAGNOSTIC POINTS Choroid Plexus Tumors • A variety of IHC markers are available for CP tumors, none of which are ideal. Individual markers lack either sensitivity or specificity, but used as a panel, they confirm difficult cases. • Location and structural features are sufficient to confirm CP papillomas in many cases. • CP carcinomas are difficult to distinguish from metastatic carcinomas.
CHOROID PLEXUS CARCINOMA (WHO GRADE III OR IV)
CP carcinoma, or anaplastic CP papilloma, is a rare neoplasm that is most difficult to distinguish from metastatic carcinoma (see Table 20-2). Each of these tumors produces cytokeratin, and each may produce
800
Immunohistology of the Nervous System
A
B
Figure 20-39 This choroid plexus papilloma from the lateral ventricle of a child is composed of well-differentiated columnar epithelium resting upon a fibrovascular stroma. It expresses CAM5.2 low-molecular-weight cytokeratin (A) and transthyretin (B). The fibrovascular stroma is easily identified from its negativity for cytokeratin (immunoperoxidase anti-CAM5.2 and antiprealbumin). From McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS: Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492.
transthyretin. A transitional zone between papilloma and carcinoma of the CP confirms CP carcinoma. Primary carcinoma of the CP so closely resembles metastatic carcinoma that the latter must be carefully excluded before the diagnosis of primary CP carcinoma can be made. Occult pulmonary or gastrointestinal primary tumors are common sources, and the paucity of such primary systemic carcinomas in children facilitates diagnosis of CP carcinoma in a patient in this age group. Some CP carcinomas express CD44 cell-adhesion molecule not seen in the most benign papillomas. The mean MIB-1 LI of CP carcinomas is 14%, which is higher than that of papillomas, but the LI varies among laboratories.146 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Earlier in this chapter we discussed LFS, the result of a germline mutation of the TP53 gene on the short arm of chromosome 17. The percentage of CP tumors associated with LFS is higher than the percentage of astrocytomas associated with LFS.102-104,147 Consider the possibility of LFS in a patient with a CP tumor, particularly one with a CP carcinoma.103
nuclei are divided by fibrovascular stroma into lobules. Other cells surround fibrillary centers.92 The cells are fibrillar and often radiate toward the vessels.148 IHC stains for NFs may reveal expansions at the tips of these processes that resemble clubs, and EM shows their similarity to neurons.3 These neural features distinguish pineocytoma from glioma. The major source of confusion with this histologic picture is with normal pineal gland. An MIB-1 LI higher than normal pineal gland, a specimen larger than the 0.5-cm diameter of the normal pineal gland, or invasion beyond the pineal gland confirms the diagnosis of pineocytoma. PINEAL CELL TUMOR OF INTERMEDIATE DIFFERENTIATION (LIKELY GRADE II OR III)
Primary pineal cell tumors are less differentiated than pineocytomas and more differentiated than pineoblastomas. They show mitotic activity and moderate crowding of their synaptophysin-positive cells and may contain
Pineal Cell Tumors Tumors described in this section arise from pineal cells or their precursors. Because tumors that arise from pineal cells are composed of neurons, synaptophysin immunoreactivity is common. Some tumors also react for retinal-S antigen (Fig. 20-40).148 Many other tumors occur in the pineal region, including gliomas, meningiomas, and germ cell tumors. These are described in their respective sections in this chapter. PINEOCYTOMA (WHO GRADE I)
Pineocytoma simulates the normal pineal gland (see Table 20-7). Synaptophysin-positive cells with round
Figure 20-40 Pineocytoma. Neoplastic cells with round nuclei include some that express retinal-S antigen. Courtesy Dr. Hernando Mena, Armed Forces Institute of Pathology, Washington, DC.
Tumors of the Nervous System
regions that resemble pineocytoma near regions similar to pineoblastoma. Their proliferation is regionally variable, with hot spots of proliferation higher than that of pineocytoma. PAPILLARY TUMOR OF THE PINEAL REGION (LIKELY GRADE II OR III)
The papillary tumor of the pineal region (PTPR) is a rare tumor found in patients of all ages. It has a prealbumin- and CAM5.2-positive cuboidal epithelium wrapped around vessels, and closely resembles a CP tumor. Some PTPRs may be distinguished by their robust microtubule-associated protein 2 (MAP-2) immunoreactivity, lack of membranous Kir7.1, and lack of cytoplasmic stanniocalcin-1 staining.149 Papillary tumors of the pineal region have an uncertain prognosis and a high risk of local recurrence. They have been treated with surgery and radiotherapy. Their only known prognostic factors are more than 4 mitoses per 10 highpower fields (hpf) and extent of surgical resection.2,150 PINEOBLASTOMA (GRADE IV)
Pineoblastoma resembles medulloblastoma (Fig. 20-41; see Fig. 20-24) except for its origin in the pineal gland (see Table 20-9). Fibrillary rosettes may be evident and are more common than Flexner-Wintersteiner rosettes. Synaptophysin is most useful in identifying neuronal differentiation in these tumors (see Fig. 20-41, A), because NF immunoreactivity is often negative (see Fig. 20-3). Some tumors express retinal-S antigen.148 It would be interesting to know whether this site-specific marker is more common in pineoblastomas than in other medulloblastomas.
Embryonal Tumors (WHO Grade IV) MEDULLOBLASTOMA
The medulloblastoma is a PNET that arises in the cerebellum or in the roof of the fourth ventricle (see Figs. 20-3 and 20-24 and Table 20-9). It is most common in
A
801
children but also occurs among young adults151 and, rarely, in patients older than 35 years.78 Because medulloblastomas commonly spread along CSF pathways, treatment should be directed at the entire neuraxis. Approximately 5% of medulloblastomas metastasize to a systemic location, particularly to bone marrow, where synaptophysin staining aids their recognition.13 Nuclear crowding and high nuclear/cytoplasmic ratio impart a distinctly blue macroscopic appearance to the medulloblastoma on H&E staining. These malignant cells have higher nuclear/cytoplasmic ratios than malignant gliomas, and they rarely amplify EGFR.152,153 Rosettes in the CNS that have cores filled with fibrils (Homer-Wright rosettes) are characteristic of medulloblastoma (see Fig. 20-24, A); however, in many biopsy samples of medulloblastoma, rosettes are only vague to nonexistent. In the absence of desmoplasia or large malignant cells, this type is called classic medulloblastoma. Virtually all medulloblastomas express synaptophysin, its most reliable marker of neuronal differentiation, which is not seen in lymphoma and is uncommon in carcinoma (see Fig. 20-24, B).3 Synaptophysin can be focal or diffuse. Special care is needed to trace it back to malignant cells, particularly in gray matter where synaptophysin abounds; the pathologist must look for regions of solid tumor and regions where tumor involves white matter. Compare regions of high versus low tumor cell density, and see whether synaptophysin increases with tumor cell density. Inspect individual tumor cells with high magnification and a good nuclear counterstain to determine whether malignant cells have synaptophysin. Cytoplasmic synaptophysin is often present and is easier to evaluate than surface staining. Protein gene product 9.5 (PGP9.5) is described in texts as another marker of neuronal differentiation of these tumors. S-100 protein may be found in medulloblastomas. In this author’s experience, findings of PGP9.5, NSE, and S-100 protein must be carefully interpreted because of the presence of these markers in other neoplasms in the differential diagnosis, including most gliomas and some carcinomas.3 These markers
B
Figure 20-41 Crowded malignant cells with pleomorphic and hyperchromatic nuclei in this pineal tumor express synaptophysin (A) but not glial fibrillary acidic protein (B) in serial sections of this pineoblastoma.
802
Immunohistology of the Nervous System
seem to work better for peripheral neuroblastoma than for medulloblastoma. Staining for synaptophysin and the presence of fibrillar cellular processes works best (see Fig. 20-24, B). Difficult cases can be examined for their ultrastructure. This collection of small malignant cells must be distinguished from small cell undifferentiated carcinoma and lymphoma (see Table 20-9). The examiner should look for cellular processes. To indicate medulloblastoma, the fibrillar cellular processes must come directly from the neoplastic cells. Synaptophysin, S-100 protein, and, less often, NF protein stains highlight these processes. An uncommon variant of medulloblastoma, large cell medulloblastoma has large cells with prominent nucleoli, nuclear molding, and high rates of mitosis and apoptosis. This variant is aggressive and has a poor prognosis.2 Although confirmation by larger studies would be appropriate, evidence suggests that GFAP reactivity in medulloblastoma is associated with longer survival than nonreactivity.154 Desmoplastic Medulloblastoma
Regions of a medulloblastoma may contain proliferating cells that can be demonstrated by reticulin staining (Fig. 20-42; see Table 20-8). Pale reticulin-free islands of cells
with perinuclear halos are often neuroblastic and react positively to staining for synaptophysin (see Fig. 20-42, B).155 Desmoplastic medulloblastoma has been recently redefined to include only nodular medulloblastoma with neuronal and sometimes glial differentiation in the nodules only and nodules surrounded by highly cellular “desmoplasia” composed of very proliferative cells that make reticulin.2 With standard treatments used now, this group of tumors has a better prognosis than classic medulloblastoma. Medullomyoblastoma
Medullomyoblastoma occurs in the midline posterior fossa of children and contains smooth or striated muscle fibers.78 These tumors are mixtures of stainable muscle cells and small neuroectodermal cells that resemble medulloblastoma, in contrast to pure primary intracranial rhabdomyosarcomas, which do not contain cells derived from neuroectoderm.80 Synaptophysin and retinal-S antigen markers facilitate detection of these neuroectodermal cells. Immunoperoxidase stains for desmin and muscle-specific actin (MSA) markers confirm muscular differentiation (see Table 20-9). Mesodermal elements other than muscle are occasionally found in medulloblastomas and medullomyoblastomas.13 The medullomyoblastoma has lately been considered a subtype of medulloblastoma.2
A
B
C
D
Figure 20-42 This desmoplastic medulloblastoma shows pale islands on hematoxylin and eosin (A) that contain synaptophysin-positive cells (B). Adjacent sections of the same region of tumor show that densely crowded cells form reticulin-positive bands (C) around the pale islands and that these bands contain most of the MIB-1–positive cells (D).
Tumors of the Nervous System
PRIMITIVE NEUROECTODERMAL TUMOR
803
KEY DIAGNOSTIC POINTS
The features and differential diagnosis of central nervous PNET (cPNET) can be found in the “Medulloblastoma” section earlier in this chapter. Although cerebral PNET, cerebellar medulloblastoma, and pineoblastoma are all PNETs by histology alone, the recent tendency has been to recognize the cerebral tumor as a PNET, the posterior fossa tumor as a medulloblastoma, and the pineal tumor as a pineoblastoma (see Fig. 20-3). It is this author’s opinion that these tumors are all different and that their differences will eventually be revealed with better markers. The cerebral ganglioneuroblastoma and neuroblastoma are referred to as PNET with advanced neuronal differentiation.2 Markers of neuronal differentiation, such as synaptophysin, usually show PNETs with neuronal differentiation (see Fig. 20-3). Expression of a variety of other ectodermal and neuroectodermal antigens and association with neural tube defects reflect the embryonal nature of these neoplasms.156 Peripheral PNETs (pPNETs) are intensely immunoreactive with MIC2 (Fig. 20-43) and NSE. Sites of occurrence of pPNET include neural crest derivatives, gonads, chest wall, and bone, including vertebral column, cranial vault, and cauda equina.157 Peripheral PNETs are highly aggressive, recur locally, and metastasize to specific organs.13
In contrast to the cPNET, the peripheral PNET (pPNET) is defined by its t(11;22q24;q12) chromosomal translocation. This 11;22 translocation can be seen with FISH. PCR is an elegant assay for precise definition of the specific chimeric gene of EWSR1-FLI1.157 Earlier in this chapter, I introduced LFS, the result of a germline mutation of the TP53 gene on the short arm of chromosome 17. Medulloblastoma and PNET account for approximately 10% of all brain tumors associated with a germline mutation of this gene.2,99
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
ATYPICAL TERATOID/RHABDOID TUMOR
The medulloblastoma is a CNS primitive neuroectodermal tumor (cPNET) in the cerebellum. The most frequent genetic abnormality associated with medulloblastoma is loss of chromosome arm 17p. This is often seen as an isochromosome 17q (one chromosome composed of two long arms of chromosome 17), which can be detected by ISH enhanced by immunostaining.153 In a study of 8 cerebral cPNETs and 35 medulloblastomas, no cerebral cPNETs showed this abnormality, whereas 13 medulloblastomas showed it.158 Cerebral cPNETs are uncommon, and numbers are small, but this suggests that cerebral cPNETs are different from medulloblastomas.
Embryonal Tumors • These multipotential tumors may express almost any neuroectodermal marker, but they express synaptophysin in particular. Some show additional markers, such as vimentin. • Many embryonal tumors are seen in childhood. • High cellular density is characteristic. • High proliferation is reflected in a high MIB-1 LI, often greater than 20%, and frequent mitotic activity. • Despite their categorical grade of IV, specific diagnosis is important, because some entities are very sensitive to radiotherapy or chemotherapy.
Atypical teratoid/rhabdoid tumor (AT/RT) was defined relatively recently (see Table 20-9). Infants and young children suffer from this highly malignant tumor,159 and its high frequency in patients younger than 3 years may account for 10% of CNS tumors in infants.160 AT/ RT occurs throughout the CNS, especially in the posterior fossa, and it metastasizes early through the CSF. Malignant cells have a pinker cytoplasm and are more epithelial than those in medulloblastoma. The plethora of IHC markers that this tumor expresses contributes significantly to its definition (Fig. 20-44). AT/RT contains multiple intermediate filament (IF) types: it nearly always stains for vimentin and EMA and often expresses focal SMA, GFAP, cytokeratin, or other IF types. Synaptophysin and chromogranin may be present. AT/RTs have abnormalities of chromosome 22, particularly involving loss or mutation at the INI1 locus at 22q11.2. The INI1 nuclear protein is lost in most AT/RTs, thus a loss of INI1 immunostaining can help to distinguish AT/ RTs from PNETs and medulloblastomas that retain INI1 expression.161 RARE EMBRYONAL TUMORS
Figure 20-43 This peripheral primitive neuroectodermal tumor is diffusely and intensely positive for MIC2.
The medulloepithelioma looks like carcinoma but occurs in childhood, an unlikely age for carcinoma. The pseudostratified columnar epithelium of medulloepithelioma is crowded with cells that resemble those that line the embryonic neural tube. It rests on a type IV collagen basement membrane and fibrous stroma. The basal layer of the epithelium expresses nestin,
804
Immunohistology of the Nervous System
A
B
C
D
E
vimentin, and MAP type 5 immunoreactivity. Focal differentiation and expression of either GFAP, S-100 protein, NSE (Fig. 20-45), NF protein, cytokeratin, or EMA immunoreactivity frequently occur.162 Ependymoblastoma usually occurs in the cerebrum of children (see Table 20-9).163 It resembles a PNET decorated with well-formed rosettes lined by mitotically active epithelioid cells. Unlike medulloepithelioma, the rosettes in ependymoblastoma merge into densely cellular malignant cells without a collagenous stroma. Ependymoblastomas contain vimentin, and their GFAP-negative rosettes stain differently from GFAP-positive rosettes in ependymomas.2,3,80 Ependymoblastoma is more cellular than anaplastic ependymoma and has less vascular proliferation.
Figure 20-44 Immunohistochemistry distinguishes the highly malignant atypical teratoid-rhabdoid tumor from medulloblastoma. This posterior fossa tumor in an infant (A, hematoxylin and eosin) contains vimentin (B), shows focal immunoreactivity to epithelial membrane antigen (C) and glial fibrillary acidic protein (D), and displays a focally weak response to synaptophysin (E). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Meningeal and Related Tumors MENINGIOMA
Meningeal location is a major discriminator of meningioma from other primary intracranial neoplasms (Table 20-10; also see Tables 20-7 and 20-8). Meningiomas are attached to dura or falx, which facilitates their recognition, and they arise less commonly from the CP and rarely arise within the CNS parenchyma.164 The classic genetic abnormality in meningioma is partial or complete loss of chromosome 22.1,5 This loss can be detected by chromosomal ISH visualized by immunostaining. In addition, certain features provide evidence of meningioma. A syncytial appearance is a distinctive feature of meningothelial meningiomas and
Tumors of the Nervous System
805
100 90 80 Positive %
70 60 50 40 30 20 10 0 Vimentin
EMA
CEA
Cytokeratin
Immunohistochemical markers Figure 20-45 Medulloepithelioma. Anaplastic cells of this papillary neoplasm are weakly positive for gamma-enolase (neuron-specific enolase). They form a pseudostratified epithelium with dense material on its surface. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Figure 20-46 Immunohistochemical staining results in 29 cases of meningioma stained with broad-spectrum antibodies to epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), and cytokeratin AE1/AE3. Percentages positive for all antibodies except for vimentin. From Ng HK, Tse CC, Lo ST: Meningiomas and arachnoid cells: an immunohistochemical study of epithelial markers. Pathology 1987;19:253-257.
a focal feature of other subtypes of meningioma (see Fig. 20-49; see Table 20-10). The structural bases of this syncytial appearance are numerous, tightly interdigitating cellular processes held together by desmosomes rather than a true syncytium. Psammoma bodies, concentrically laminated calcifications, and meningothelial whorls typify meningiomas, and the presence of these features on H&E-stained slides diminishes the need for IHC analysis.92 Meningiomas contain EMA,3 although it is often expressed focally and can be missed in small specimens. EMA positivity plus lack of GFAP distinguishes meningiomas from gliomas (Fig. 20-46; see Fig. 20-4, B).165 Positivity for EMA is the most decisive IHC marker of meningioma, and expression of progesterone receptors is variable. The intensity of vimentin expression by meningiomas has stimulated its use in these tumors, although vimentin markers should be used only in combination with other markers, because many other tumors
express vimentin. Meningiomas are variable and focal with these three markers, but they often have more reticulin, fibronectin, and collagen in their parenchyma than low-grade gliomas. Fibrous Meningioma (Fibroblastic Meningioma; WHO Grade I)
Fibrous meningiomas are firm tumors composed of spindle cells (see Table 20-7). They resemble schwannomas, solitary fibrous tumors (SFTs), fibrillary astrocytomas, and pilocytic astrocytomas.1,166 Fibrous meningiomas contain parenchymal collagen—often in large, pink bundles on H&E staining—that distinguishes them from astrocytomas.3 The similarity of fibrous meningiomas to schwannomas poses a particular diagnostic problem with tumors in the cerebellopontine angle and around spinal nerve roots. Structures that identify meningiomas in this context include whorls, psammoma bodies, and very
TABLE 20-10 Differential Diagnosis of a Mass that Includes Syncytial Cells Differential Features Diagnosis
Structures
Antibody*
Locations†
Meningiomas
Whorls, psammoma bodies, interdigitating cell processes and desmosomes, (thick collagen)‡
Vimentin (+), EMA (S), S-100 (R)
Falx, tentorium, meninges, choroid plexus, (extracranial)‡
Anaplastic meningioma
Decrease in preceding features, mitoses, necrosis, CNS invasion
Vimentin (+), EMA (R), S-100 (R)
Same as above
Hemangiopericytoma
Hypercellularity, thick pericellular matrix, mitoses
Vimentin (+)
Same as above
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *Key to staining results: +, almost always strong, diffuse positivity; S, sometimes positive; R, rare cells may be positive. † The most common or most specific location is listed first. ‡ Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when found. CNS, Central nervous system; EMA, epithelial membrane antigen.
806
Immunohistology of the Nervous System
80 70
Positive (%)
60 50 40 30 20 10 0 CD34
p53
EMA
S-100
Immunohistochemical markers Figure 20-47 Immunostaining characteristics of 20 fibrous meningiomas. EMA, epithelial membrane antigen. Data from Perry A, Scheithauer BW, Nascimento AG: The immunophenotypic spectrum of meningeal hemangiopericytoma: a comparison with fibrous meningioma and solitary fibrous tumor of meninges. Am J Surg Pathol 1997;21:1354-1360.
Figure 20-49 Meningotheliomatous meningioma. When the patient contemplated pregnancy, the tumor’s hormonal receptors were assessed. This meningioma has a syncytial appearance and rounded nuclei and is positive for progesterone receptors (dark brown nuclei). It could not be totally resected because of its location.
thick bundles of collagen. Fibrous meningiomas express EMA, which schwannomas lack, and the former usually show more vimentin and less S-100 protein than schwannomas.3 Fibrous meningiomas can be distinguished from SFTs by their IHC profiles.166,167 Fibrous meningiomas express more EMA, S-100 protein, and glycogen and less CD34 than SFTs (Figs. 20-47 and 20-48).167
bodies are often sparse (see Table 20-10), and some meningothelial meningiomas are divided into lobules by their fibrovascular stroma.168 Meningothelial meningioma occasionally appears fibrillar or epithelioid (see Fig. 20-4, A) and simulates ependymoma, but it lacks GFAP and usually contains enough stromal type IV collagen, reticulin, or EMA (see Fig. 20-4, B) to be distinctive. Confusion with myxopapillary ependymoma can be a problem, although this tumor usually shows at least focal GFAP. Intraparenchymal meningiomas can be confused with oligodendrogliomas (see Fig. 20-2), although EMA reactivity identifies most meningiomas. The margin of meningiomas with CNS parenchyma—evaluated with GFAP, synaptophysin, and neurofilament CNS markers—is more discrete than that of gliomas. Some meningothelial meningiomas lack classic whorls and psammoma bodies, whereas others show distinct epithelioid cellular margins (see Fig. 20-4, A). These tumors may be confused with carcinoma and adenoma. In general, meningiomas express little or no CAM5.2 cytokeratin and no pituitary peptides or chromogranin A.
Meningotheliomatous Meningioma (Syncytial Meningioma; WHO Grade I)
Meningotheliomatous meningioma is the classic syncytial meningioma (Fig. 20-49) that resembles the small clusters of meningeal cells found normally in the meninges and choroid plexus (CP). Whorls and psammoma
100 90 80
Transitional Meningioma (Mixed Meningioma; WHO Grade I)
Positive (%)
70 60 50 40 30 20 10 0 CD34
p53
Immunohistochemical markers Figure 20-48 Eight solitary fibrous tumors. Data from Perry A, Scheithauer BW, Nascimento AG: The immunophenotypic spectrum of meningeal hemangiopericytoma: a comparison with fibrous meningioma and solitary fibrous tumor of meninges. Am J Surg Pathol 1997;21:1354-1360.
Transitional meningiomas are composed of syncytial and fibroblastic components, as described previously. The presence of cells intermediate in type between syncytial and fibroblastic justifies their designation.168 Prominent whorls, psammoma bodies, and clusters of syncytial cells make these very common meningiomas among the easiest to identify, therefore IHC is usually not necessary (see Table 20-8). Psammomatous Meningioma (WHO Grade I)
Psammomatous meningiomas are crowded with psammoma bodies168 and are often spinal in location. This benign variant is recognized as meningioma from the finding of syncytial vimentin and EMA-positive cells
Tumors of the Nervous System
between the conspicuous, concentrically laminated psammoma bodies. Angiomatous Meningiomas (WHO Grade I)
Angiomatous meningiomas are highly vascular meningiomas.3,168 Their vessels can be highlighted with CD31, CD34, FVIII, and Ulex endothelial markers along with vimentin and muscle actin. For these meningiomas, CD34 causes less background staining than the other markers. Other Grade I Meningiomas
Recognizing the other meningioma variants is critical3 and helps to avoid mistaking them for similar entities, which includes tumors that require different treatments. Meningioma variants include lipoblastic,169 arachnoid trabecular,170 microcystic,171 lymphoplasmacyte-rich,2 osteogenic,168 cartilaginous,168 and secretory (Fig. 20-50) types. Robust vimentin and focal EMA help to identify these tumors.172,173 Keys to distinguishing these variants of meningioma from carcinoma and sarcoma are finely granular chromatin, few mitoses, and meningeal features (see Table 20-10). Meningiomas generally express more vimentin and much less CAM5.2 cytokeratin than carcinomas.3 Secretory meningioma stains spectacularly (see Fig. 20-50). It displays focal CAM5.2 reactivity in cells immediately surrounding its secretory granules, which are positive for carcinoembryonic antigen (CEA),
A
Figure 20-50 Secretory meningioma. Strikingly cytokeratinpositive structures that resemble acini suggest carcinoma, but the cytologic features are meningothelial. The secretory meningioma contains pink globules (A, hematoxylin and eosin) that are oddly reactive for carcinoembryonic antigen (B) and are surrounded by cytokeratin-positive cells (C). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
807
carbohydrate antigen 19-9, and PAS.174 Properly stained, this tumor can be nothing other than secretory meningioma. AGGRESSIVE OR MALIGNANT MENINGIOMA Brain Invasion
The invasion of brain tissue or of small blood vessels by tumor cells is considered to connote a higher risk of recurrence of a meningioma. At this time, however, brain invasion does not constitute a criterion for increased grade.2 Brain invasion is considered more ominous than dural invasion, a common finding in meningiomas. Chordoid Meningioma (WHO Grade II)
The chordoid meningioma resembles a chordoma, and it is found more often than most meningiomas in childhood,173 distinguished by its meningothelial features and the lack of cytokeratin.3 Its diagnosis carries a WHO grade of II because of its tendency to recur. Clear Cell Meningioma (WHO Grade II as an Intracranial Tumor)
One of the many reasons to subclassify meningiomas is to identify more aggressive variants. The subtype clear cell meningioma is exemplary (Fig. 20-51). Although they look benign, many clear cell meningiomas are
B
C
808
Immunohistology of the Nervous System
A
B
C
D
E
F
Figure 20-51 This clear cell meningioma has recurred several times (A, hematoxylin and eosin) and contains red periodic acid–Schiffpositive glycogen (B), eliminated by enzymes that digest glycogen (C). This tumor contained scattered S-100 protein–positive cells (D) and no cytokeratin-positive cells (E). Clear cell meningioma tends to be aggressive and difficult to manage surgically. As with most meningiomas, it is abundantly vimentin positive (F). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
biologically aggressive.2 Their clear cells resemble those in oligodendroglioma and clear cell ependymoma (see Fig. 20-1),93 and a mixture of clear cells and meningothelial features is a key to its diagnosis.3 Cytoplasmic glycogen helps to confirm the diagnosis (see Fig. 20-51, B-C).93 Diffusely positive vimentin and focally positive EMA can aid in the identification of its meningothelial origin (see Fig. 20-51, F, and Table 20-10). This variant is particularly common in the lumbar and cerebellopontine angle regions.175
Atypical Meningioma (WHO Grade II)
Specific criteria for the distinction among benign meningioma, atypical meningioma, and anaplastic meningioma have been adopted by the WHO (see Table 20-5).2 Atypical meningioma has 4 to 19 mitotic figures in 10 hpf. Alternatively, this diagnosis can be made if three or more of the following histologic features are noted: increased cellularity; small cells with a high nuclear/ cytoplasmic ratio; large prominent nucleoli; patternless,
Tumors of the Nervous System
sheetlike growth; and foci of spontaneous or geographic necrosis. Atypical meningioma tends to be more aggressive than grade 1 meningioma and is likely to recur locally. Chromosomal abnormalities, in addition to standard loss of 22 and increased MIB-1 LI, may forecast greater aggressiveness.2,176 This tumor is recognized as a distinct diagnostic entity with histopathologic features between those of benign and malignant meningiomas. Atypical meningiomas usually retain vimentin and at least slight focal EMA immunoreactivity. EMA may show in more differentiated foci that often look meningotheliomatous. Rhabdoid Meningioma (WHO Grade III)
A highly aggressive tumor, rhabdoid meningioma contains barely cohesive cells filled with abundant whorls of filaments that show immunocytochemical reactivity for vimentin.6,177 These filaments push the meningothelial nuclei to the side of the cell. Entirely rhabdoid tumors are difficult to distinguish from gemistocytic gliomas. In such cases, vimentin predominance, EMA reactivity, and lack of GFAP reveal their true identity. Papillary Meningioma (WHO Grade III)
Papillary configurations in meningiomas are associated with high rates of local recurrence and metastases. The papillae have a CD31-positive vascular core. Meningioma cells produce rosettes around these vessels, and, in addition to expected vimentin and S-100 protein, they may express cytokeratin.178 The recognition of a papillary meningioma in other than the dural locations characteristic of meningioma is difficult. Papillary meningiomas resemble papillary ependymomas, CP papillomas, and carcinomas.168 The pathologist should look for a high ratio of vimentin to cytokeratin and an absence of GFAP to identify the meningioma. Anaplastic (Malignant) Meningioma (WHO Grade III)
Anaplastic meningiomas are those with 20 or more mitoses per 10 hpf or regions with malignant cytologic features.2,3,179 Nuclear staining for p53 tumor suppressor gene product is evident in 10% of malignant meningiomas. The mean MIB-1 LI among 20 tumors in one series
KEY DIAGNOSTIC POINTS Meningioma • Most meningiomas are benign, grade I neoplasms. Location affects their chance for total removal and thus affects individual patient prognosis. • Grade II and III aggressive meningiomas occur as both specific subtypes (e.g., chordoid, rhabdoid) and as the standard subtypes of meningioma with specific ominous features, such as mitotic indices of atypical and malignant meningioma. • EMA is the most specific positive marker of meningioma. • Virtually all meningiomas express vimentin, but so do many other tumors.
809
was 11.7%; a wide range, between 1% and 24%, was noted in individual tumors (MIB-1 PI).180 Theranostic Applications
For decades surgeons knew that meningiomas in some women grew during pregnancy. Now we know why: many meningiomas have progesterone receptors (PRs).181 Prudence demands assessment of the PR in meningiomas for patient management, particularly in meningiomas that have not been totally removed surgically. PR status may affect decisions about pregnancy (see Fig. 20-49)1 or about taking progesterone supplements. In the future, PR-positive meningiomas may respond to a therapy that blocks the hormone in question.182 It is worth exploring to determine which highergrade meningiomas may have decreased or absent PR receptors.2 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
The neurofibromatosis 2 (NF2) tumor suppressor gene is located on the long arm of chromosome 22 at 22q12. It encodes for the merlin (schwannomin) protein, which is responsible for the inherited disease neurofibromatosis 2. NF2 gene mutations predominantly occur in transitional and fibroblastic meningiomas, whereas the meningothelial and secretory meningioma variants are less affected.183,184 ISH is an efficient and reliable method for routinely assessing NF2 gene deletion in formalinfixed paraffin-embedded (FFPE) tissues.185 An IHC assay for merlin is also available.183 HEMANGIOPERICYTOMA (WHO GRADE II OR III)
Hemangiopericytoma (HPC) is a highly cellular and mitotically active neoplasm rich in pericellular reticulin and stainable with anti–type IV collagen. Eighty percent of these tumors recur, and 23% metastasize.13,186 HPCs are distinguished from benign meningiomas by their hypercellularity, higher mitotic index, and microscopic tendency to bulge into vascular lumina without bursting through the endothelium (see Table 20-9). Although exceptions are found, these tumors tend to lack markers other than the relatively ubiquitous factor XIIIa, mesenchymal markers, and Leu-7.172,187 The spectrum of IHC markers for HPC overlaps with that for fibrous meningioma, but lack of EMA has become symbolic of HPC (Fig. 20-52).166 HPC is distinguished from malignant glioma and metastatic carcinoma by foci of extensive reticulin around individual neoplastic cells. HPC also lacks the GFAP found in glioma, and its nuclei are less pleomorphic and less spindled than those of fibrosarcoma. SOLITARY FIBROUS TUMOR
The solitary fibrous tumor (SFT) is a soft tissue tumor (see Chapter 4) that resembles fibrous meningioma but has a different IHC profile (see Figs. 20-47 and 2048).3,166,188 Its parenchymal cells express abundant CD34 and collagen but lack EMA. IHC is critical in distinguishing SFT from meningioma. SFT and HPC
810
Immunohistology of the Nervous System
90 80
Positive (%)
70 60 50 40 30 20 10 0 CD34
p53 Vimentin Factor Xllla
Leu 7 Desmin Cytokeratin
Immunohistochemical markers Figure 20-52 Immunochemical staining results of 27 meningeal hemangiopericytomas. Data from Perry A, Scheithauer BW, Nascimento AG: The immunophenotypic spectrum of meningeal hemangiopericytoma: a comparison with fibrous meningioma and solitary fibrous tumor of meninges. Am J Surg Pathol 1997;21:1354-1360.
have been said to be the same tumor, a matter that deserves further consideration. SFT is more common in pleura than dura, and malignant transformation of the SFT may be associated with trisomy 8.189
Chordoma and Sarcoma CHORDOMA
Approximately 40% of chordomas arise in the clivus; in the vertebrae, 10% arise along the cervical spine 2% along thoracic, and 2% along lumbar vertebrae; and more than 45% arise in the sacral portions of the spinal canal.3 Physaliphorous cells of chordomas contain characteristic large, intracytoplasmic vacuoles (Fig. 20-53; see Table 20-2). Because the cells frequently grow in cords, these vacuoles occasionally line up like beads on a string, distinguishing chordoma from chondroid neoplasms, which have individual cells embedded in cartilage.190
A
Chordomas contain cytokeratin (see Fig. 20-53, A), EMA, 5′-nucleotidase, and desmosomes, whereas chondrosarcomas lack these features.190 Presence of cytokeratin (CK) is the standard discriminator of chordoma from CK-negative chondrosarcoma.3 Chordoma cells are exuberantly bifilamentous (see Fig. 20-53) and contain vimentin and CK in the same cell. The vacuoles of physaliphorous cells contain mucin and glycogen. Their structure is distinct from that of watery perinuclear oligodendroglial halos and the multiplicity of smaller lipid vacuoles of hemangioblastomas (see Figs. 20-25, 20-53, and 20-59, B). Malignant histologic transformation of chordoma is uncommon. Nevertheless, relentless local invasion of clinically sensitive regions results in a poor long-term prognosis. Chondroid chordomas contain regions of classic chordoma that are positive for EMA, CK, and S-100 as well as chondroid regions that are S-100-positive and lack EMA and CK.190 The existence of chondroid chordoma has been challenged by some pathologists, who prefer to interpret such tumors as either chordomas or low-grade chondrosarcomas. SARCOMAS
Sarcomas are rare among brain tumors.81 Reported incidences of primary intracranial sarcomas vary from 0.08% to 4.3%, and the lower percentage is more contemporary.191 GFAP IHC analysis has demonstrated that tumors formerly considered sarcomas are actually primary brain tumors, particularly glioblastomas, medulloblastomas, and primary lymphomas.60 Causes of some sarcomas are known, although intracranial radiation is a surprisingly common cause of sarcoma.192,193 Intracranial Kaposi sarcoma is rare, and most cases are associated with immunodeficiency.194 The mesenchymal chondrosarcoma is rare and originates in the intracranial and spinal meninges and cauda equina in childhood and young adulthood.195 Mesenchymal chondrosarcoma resembles the HPC except for islands of S-100 protein–positive cartilage.
B
Figure 20-53 Chordoma. A, Cords of physaliphorous cells are positive for cytokeratin, a most important immunohistochemical feature in distinguishing chordoma from cytokeratin-negative chondrosarcoma. B, The tumor was also positive for vimentin. This specimen demonstrates the propensity of chordomas to express more than one intermediate filament.
Tumors of the Nervous System
A
Figure 20-54 Decisive and supplemental markers of a melanoma with rare pigmented cells. A, All sections are of the edge of this melanoma in brain (hematoxylin and eosin). B, The melanoma only is positive for human melanoma black 45, a decisive marker of melanocytic cells. C, Both the melanoma and brain are positive for S-100 protein. S-100 is best for screening but does not distinguish melanoma from primary brain tumors, such as glioma. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Poorly differentiated chondrosarcomas are rare and usually involve the meninges. A key feature is evidence of cartilage production, which is often sparse. Presence of S-100 protein as evidence of chondroid differentiation must be interpreted cautiously in meningeal and CNS neoplasms, because most gliomas, chordomas, melanomas, nerve sheath tumors, and an occasional meningioma contain this protein (Fig. 20-54, C; see Figs. 20-27 and 20-51, D).3,165 Primary cerebral rhabdomyosarcomas are rare.196 Synaptophysin-negative staining differentiates them from synaptophysin-positive medullomyoblastomas. Lack of GFAP-positive neoplastic glia distinguishes fibrosarcoma from glioblastoma that invades the meninges and from gliosarcoma.3 For specific features that differentiate individual sarcomas, see Chapter 4.
Nerve Sheath Tumors Benign nerve sheath tumors (WHO grade I) can be subclassified as either schwannoma or neurofibroma, as described in the discussions of these tumors (see Table 20-7). Malignant nerve sheath tumors (WHO grade III or IV) are much more difficult to subclassify when they lose the characteristics of their benign counterparts. Leu-7 and S-100 protein markers differentiate nerve sheath tumors from other tumors known to lack these markers.13,197
811
B
C
SCHWANNOMA, NEURILEMMOMA, AND NEURINOMA (WHO GRADE I)
The presence of a noninvasive tumor next to a peripheral nerve suggests the diagnosis of schwannoma. Verocay bodies are more distinctive of schwannomas than the Antoni A and Antoni B patterns but are not seen in all schwannomas. Bilateral eighth-nerve schwannomas indicate neurofibromatosis type 2 (NF-2), and unusual locations and associations with meningeal proliferation are also seen with NF-2.198 Both NF-2 and schwannomas are associated with abnormalities in chromosome 22.1,5 The histologic appearance of schwannoma is similar to that of fibrous meningioma, tanycytic ependymoma, subependymoma, and astrocytoma. Schwannoma is distinguished from these tumors by its very robust and abundant parenchymal reticulin, which is positive for type IV collagen (see Fig. 20-27, A). Schwannomas have continuous basement membranes all along the exterior surfaces of their cells (see Table 20-7). Focal reactivity of some schwannomas with anti-GFAP requires care in the use of these antisera to distinguish these tumors from astrocytomas.3 However, a negative GFAP response supports the diagnosis of schwannoma. When they lack the characteristic features of meningioma, such as meningeal whorls and psammoma bodies, fibrous meningiomas are more difficult than gliomas to distinguish from schwannomas. Antoni A and Antoni B
812
Immunohistology of the Nervous System
growth patterns in schwannomas resemble those seen in fibrous meningiomas. Schwannomas contain Leu-7 and S-100 protein13 and lack EMA. Reactivity of meningiomas for EMA is therefore a useful discriminator. Both tumors contain S-100 protein, but S-100 is more ubiquitous and abundant in schwannomas (see Fig. 20-27, B).3 Evidence suggests that sole expression of the beta subunit of S-100 may distinguish eighth-nerve schwannomas from some meningiomas.199 If present, GFAP-positive foci can distinguish a schwannoma from a meningioma. Meningioma cells are GFAP negative. Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
The NF2 gene is a tumor suppressor on chromosome 22. Loss of expression of the NF2 protein product, merlin (schwannomin), is associated with both sporadic and NF2-related schwannomas.200 Merlin is a cytoskeleton-associated tumor suppressor protein regulated by phosphorylation at serine 518 (S518). Unphosphorylated merlin restricts cell proliferation by inhibiting Rac and p21-activated kinase (Pak).201 In merlin-deficient schwannoma cells, Rac causes nonfunctional intercellular adhesion in aggregation assays that could cause increased proliferation rates because of loss of contact inhibition.202 NEUROFIBROMA (WHO GRADE I)
The key to recognition of neurofibromas is their involvement within peripheral nerve rather than next to it (see Table 20-7). Neurofibroma differs from schwannoma in that it has stainable NF-positive axons running through the tumor itself rather than confined to the periphery.3 This is because neurofibroma is a swelling of the nerve itself, with a mixture of Schwann cells, fibroblasts, collagen, and mucoid material enclosed in a weakly EMApositive perineurium. In contrast, schwannomas grow next to and compress the nerve, so that NFs are not evident within the central tumor nidus. The larger the neurofibroma, the more the axons are “diluted” with neoplastic cells. Fortunately, today’s anti-NF antibodies can detect individual axons in a “haystack” of tumor tissue (Fig. 20-55). Neurofibroma may occur as a sporadic tumor or as part of the dominantly inherited tumor syndrome known as von Recklinghausen disease, or neurofibromatosis 1 (NF-1).1 Plexiform neurofibromas are multiple swollen fascicles associated with NF-1. Other nervous system signs of NF-1 are more than one neurofibroma, optic nerve glioma (more appropriately called optic nerve pilocytic astrocytoma), and malignant peripheral nerve sheath tumor (MPNST) with glandular or rhabdomyoblastic regions.2 Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
NF-1 is caused by constitutional mutations in the NF1 gene, located in chromosome band 17q11. The NF1 gene codes for a protein known as neurofibromin, a guanosine triphosphatase–activating protein (GAP) and tumor suppressor protein.203 Although NF1 gene
Figure 20-55 Neurofibroma. Occasional long brown axons within the tumor are dispersed among proliferating cells (antineurofilament stain with hematoxylin counterstain). Courtesy Drs. Andrew Flint and Victor Elner, University of Michigan, Ann Arbor, MI.
involvement in the development of neurofibroma in von Recklinghausen patients has been characterized, it has been harder to prove the significance of inactivation of this gene in sporadic neurofibromas. However, wellstudied sporadic neurofibromas have shown inactivation of both NF1 gene alleles.204 MALIGNANT PERIPHERAL NERVE SHEATH TUMOR
Malignant peripheral nerve sheath tumors are discussed in Chapter 4, “Immunohistology of Neoplasms of Soft Tissue and Bone.”
Neuroendocrine Tumors Neuroendocrine tumors are discussed in Chapter 10, “Immunohistology of Endocrine Tumors.”
Germ Cell Tumors Within the cranial vault are found 95% of primary germ cell tumors (GCTs), especially along the midline in the pineal region but also in suprasellar regions. Approximately 10% involve both regions, and 25% arise in the suprasellar cistern. The mixed GCT and lymphoma (gerlymphoma) has only been seen in the sella turcica.205 GCTs rarely involve spinal cord or peripheral nerve.3 Germinomas are the most common intracranial germ cell neoplasm, with few differences noted between intracranial and gonadal germinomas.206 For details about GCTs, see Chapters 16 and Chapter 18.
Hematopoietic and Lymphoid Neoplasms LYMPHOMA (WHO GRADE III OR IV)
Primary CNS lymphomas grow within the CNS parenchyma (see Table 20-9) and have a diffuse, invasive margin. These lymphomas are nearly always of B-cell origin (Fig. 20-56; see Fig. 20-3).1,207-209 Some of these B-cell lymphomas afflict immunosuppressed patients,
Tumors of the Nervous System
813
gene rearrangement); and diffuse large B-cell immunoblastic lymphoma (monoclonal; Fig. 20-57). EpsteinBarr virus (EBV) can be identified in most cases of posttransplant lymphoma.208 Lymphomatoid Granulomatosis
Lymphoid granulomatosis is a lymphoproliferative disease that resembles vasculitis and neoplasm. It is an EBV-related process, so the identification of EBV by ISH or immunoperoxidase aids the diagnosis. Lymphomatoid granulomatosis can progress to lymphoma, and claims have been made that all lymphomatoid granulomatosis is lymphoma. CNS involvement is usually seen in conjunction with pulmonary disease.3 Figure 20-56 Primary brain lymphoma. Hematoxylin nuclear counterstain reveals malignant lymphocytes with clumped chromatin and huge nucleoli. The immunohistochemical stain is L26, a B-lymphocyte marker. Malignant lymphocytes were negative for T-cell and macrophage markers (not shown). From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Intravascular Lymphoma
The CNS is one of the sites of predilection for intravascular lymphoma, a large B-cell lymphoma. The neoplastic cells fill the blood vessel lumina, and the clinical presentation mimics vasculitis. Neoplastic cells are positive for CD20. LEUKEMIA
such as those with AIDS and other immunocompromised conditions.210-212 With few exceptions, AIDSrelated lymphomas have poor outcomes.211,213 Primary CNS lymphomas of T-cell origin are rare.214 In paraffin sections, CD20 and CD79a B-cell markers and CD3-ε and CD45RO T-cell markers should be used along with LCA.13 Because primary CNS lymphoma invades CNS parenchyma, responses to CNS markers such as GFAP must be interpreted with extreme caution. The nuclear counterstain identifies the nonneoplastic nuclei of gliosis in GFAP-positive cells intermixed among lymphoma cells. Monoclonal staining for B-cell (or rarely T-cell) markers helps distinguish lymphoma from CNS inflammation, which is polyclonal (see Fig. 20-6, B-C), and from nonlymphoid neoplasms. However, many lymphomas contain polyclonal reactive lymphoid elements, which may be recognized from their smaller size and benign nuclei.3 The aforementioned markers are sufficient for most primary CNS lymphomas (see Fig. 20-56). For details about subclassifying these and other lymphomas, see Chapter 5, “Immunohistology of Hodgkin Lymphoma,” and Chapter 6, “Immunohistology of Non-Hodgkin Lymphoma.” In patients with peripheral lymphoma, secondary involvement of the CNS can occur with a frequency that has been estimated at 5% to 29% of patients.3 Secondary involvement may occur anywhere, but it often involves the meninges. Posttransplant Lymphoproliferative Disorders
Following organ transplantation and associated immunosuppression, a range of posttransplant lymphoproliferative disorders can develop. Histologic, IHC, and gene rearrangement studies can distinguish among lymphoid or plasmacytic hyperplasia (polyclonal with no immunoglobulin gene rearrangement); atypical hyperplasia or B-cell hyperplasia and lymphoma (monoclonal with
The diagnosis of leukemia within the craniospinal vault is usually established by cytologic examination of CSF.215 Terminal intraparenchymal CNS hemorrhages reflect blast crises that lead to leukocyte counts greater than 300,000/mm3, which results in intravascular leukostasis. Cerebral vasculitis is rarely associated with leukemia. Focal masses of leukemic cells (chloromas) in the meninges may be heralded by peripheral eosinophilia.13 HISTIOCYTOSIS
Histiocytosis occurs predominantly in children and young adults (see Table 20-7).3 The CNS is often involved secondary to bony or systemic involvement, often by Langerhans cell histiocytosis (LCH). NonLangerhans types of histiocytosis also occur (Fig. 20-58).216,217 Although any region of brain or meninges may be affected, the parasellar region is particularly susceptible.218 A typical lesion is firm because of the collagen fibers mixed with histiocytes and inflammatory cells, and new lesions are less fibrotic than older ones. Langerhans cells are S-100 protein positive. The literature suggests that CD1a has good sensitivity in this disease, however, its sensitivity is not quite 100%; therefore a panel of markers is recommended (Table 20-11). Lack of structural GFAP in cellular fibrils distinguishes histiocytes from astrocytes and from ependymal and subependymal cells. Electron microscopy identifies the Birbeck granules of LCH.8,10,13 Less common histiocytoses can be differentiated by their IHC and morphology. Erdheim-Chester disease of the CNS has characteristic Touton giant cells that are CD68 positive and CD1a negative.208 Rosai-Dorfman disease, or sinus histiocytosis with massive lymphadenopathy, can be seen with or without systemic disease. It is characterized by emperipolesis, which is the engulfment by macrophages of intact leukocytes. The
814
Immunohistology of the Nervous System
A
C macrophages are CD68 positive, S-100 protein positive, and CD1a negative (see Table 20-11).
Miscellaneous Intracranial or Spinal Masses HEMANGIOBLASTOMA, CAPILLARY HEMANGIOBLASTOMA (WHO GRADE I)
Capillary hemangioblastoma resembles an endocrine neoplasm (Fig. 20-59). It has close juxtaposition of
Figure 20-58 Histiocytosis. This specimen from a mass within the brain parenchyma contains S-100–positive cells. The immunohistochemical stain highlights intracellular leukocytes seen in RosaiDorfman disease.
B
Figure 20-57 Posttransplant lymphoma. This diffuse B-cell lymphoma is CD20-positive (A) and mixed with nonneoplastic CD68positive macrophages (B). Note the malignant nuclei in the CD20-positive cells and the nonmalignant nuclei in the CD68positive cells revealed by hematoxylin. The in situ probe for Epstein-Barr virus was positive (C) in this lymphoma that arose in this 33-year-old woman’s cerebellum 2 years after kidney transplantation.
capillary and stromal cells (see Table 20-8) and occasionally shows secretory granules or expresses erythropoietin.219 Its pink, vacuolated stromal cells often contain NSE, which is present in neuroendocrine cells.220 No gland of origin has been found. Because the hemangioblastoma is nonfibrillar, it should not resemble an astrocytoma. However, the resemblance may occur for two reasons: sampling and artifact. Cerebellar hemangioblastomas are often cystic, with the actual neoplasm embedded somewhere in the wall of the cyst as a mural nodule. Biopsy specimens of the cyst wall may show conspicuously GFAP-positive gliosis (see the “Gliosis vs. Glioma” section later in this chapter). Sampling a hemangioblastoma can reveal GFAPpositive cells.221 Some of these cells are reactive astrocytes, which are common near the periphery of the tumor. However, others are stromal cells common in the cellular and angioglioma variants of hemangioblastoma, which either take up GFAP from adjacent reactive astrocytes or express their own GFAP.222 To minimize confusion, the central portion of the solid tumor should be sampled. Epithelioid hemangioblastoma can resemble paraganglioma223 or renal cell carcinoma (RCC). Hemangioblastomas have more capillaries and much less chromogranin A than paragangliomas.224 Compared with RCC, hemangioblastoma has a more uniform distribution of nuclear chromatin, absence of necrosis, small nucleoli, and intimate arrangement of capillaries and stromal cells (see Fig. 20-59, A). This arrangement
Tumors of the Nervous System
815
TABLE 20-11 Histiocytoses that Affect the Central Nervous System Compared with Macrophages CD68 (KP1)
S-100
CD1a
Birbeck Granules (EM)
Foamy epithelioid, multinucleated giant cells
+
–
–
–
Erdheim-Chester
Touton giant cells
+
S*
–
–
Rosai-Dorfman
Emperipolesis
+
+
–
–
Langerhans histiocytosis
Reniform nuclei, eosinophilic cytoplasm
+
+
+
+
Disease
Characteristic Histology
Macrophage
Modified from McKeever PE, Boyer PJ: The brain, spinal cord, and meninges. In Mills SE, Carter D, Greenson, JK, et al (eds): Sternberg’s diagnostic surgical pathology, ed 4. New York, 2004, Lippincott Williams & Wilkins; pp 399-506. Key to staining results: +, almost always strong, diffuse positivity; S, sometimes, many positive. *S-100 protein has been positive in some, but not all, cases of Erdheim-Chester disease. EM, Electron microscopy.
is accentuated by FVIII for vessels, contrast to RCC, for NSE, and they Fig. 20-59, B-C).
staining with CD31, CD34, antior NSE for stromal cells.220 In hemangioblastomas tend to stain do not stain for EMA or CK (see
Beyond Immunohistochemistry: Anatomic Molecular Diagnostic Applications
Many hemangioblastomas are associated with von Hippel-Lindau (VHL) disease, which should be considered in patients with more than one tumor or a hemangioblastoma in an unusual location.221 Two hemangioblastomas, one hemangioblastoma and a
A
Figure 20-59 Cerebellar hemangioblastoma. A, Contrastenhanced tumor in the right cerebellar hemisphere of an elderly woman contains a mixture of many capillaries and vacuolated cells (hematoxylin and eosin). B, The cerebellar hemangioblastoma was positive for neuron-specific enolase (NSE). The antiNSE stains stromal cells and highlights their clear, round lipid vacuoles. C, The specimen is negative for epithelial membrane antigen (EMA). A single, brown, EMA-positive plasma cell confirms the integrity of this stain. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004. Courtesy of Dr. Roger A. Hawkins, Greenville, PA.
family history of VHL disease, or one hemangioblastoma and another tumor seen in VHL make the clinical diagnosis of VHL disease. Tumors other than hemangioblastoma seen in VHL are endolymphatic sac tumors, broad ligament or epididymal cystadenoma, pancreatic cysts or tumors, pheochromocytoma, and renal cysts or carcinoma. RCC is a frequent cause of death in VHL patients. The VHL gene is on the short arm of chromosome three (3p25-26). An inherited somatic mutation in this tumor suppressor gene creates the chance for a single mutation in the other allele to produce a tumor, following Knudson’s hypothesis. To some extent, VHL
B
C
816
Immunohistology of the Nervous System
mutations at certain sites correlate with the propensity of a family to have type 1 (without pheochromocytoma), type 2A (with pheochromocytoma and RCC), type 2B (with pheochromocytoma but without RCC), or type 2C VHL disease (with only pheochromocytoma).225 Some sporadic hemangioblastomas are associated with VHL gene abnormalities.226 Antibodies to the VHL gene product have been produced. These may aid the diagnosis of clear cell carcinomas and metastatic RCC.227 CRANIOPHARYNGIOMA (WHO GRADE I)
This tumor is found within or above the sella turcica. The epithelial appearance of craniopharyngioma is distinctive. A properly sampled craniopharyngioma, including a sample of the solid mass associated with cystic craniopharyngiomas, is difficult to confuse with other brain tumors because of its characteristic epithelium, which may be adamantinomatous, keratinizing, or both (Table 20-12; see also Table 20-2).3 Three of four craniopharyngiomas calcify, a feature that helps to distinguish them from metastatic carcinoma, which rarely calcifies in brain and is rarely as differentiated as craniopharyngioma. Poor sampling of cystic craniopharyngioma may yield a few epithelial cells of unknown origin. In such cases, lack of cytokeratins 8 and 20 favor craniopharyngioma over epithelial cysts common in the same location.228 Craniopharyngiomas express more highmolecular-weight keratin than most carcinomas metastatic to brain (Fig. 20-60).
Figure 20-60 Most craniopharyngiomas are obvious from their structural features, as is this adamantinomatous craniopharyngioma. Nonetheless, its immunoreactivity for high-molecular-weight keratin 903 distinguishes it even further and emphasizes the cytoplasm of shrunken epithelial cells (stellate reticulum).
Confusion can arise from sampling of only the intensely GFAP-positive gliotic margin of a craniopharyngioma, which may closely resemble a pilocytic astrocytoma. The highly reactive and fibrillar gliosis that surrounds a craniopharyngioma may be distinguished from that of pilocytic astrocytoma on the basis of the even spacing between GFAP-positive cells, the lower cellularity, and the lack of microcysts in the former (see the “Gliosis” section earlier in this chapter). If a craniopharyngioma nearby is suspected, keratin IHC analysis may reveal epithelial cells in the gliosis.229
TABLE 20-12 Differential Diagnosis of a Cyst with Wall Lined by Epithelium Differential Features Diagnosis
Structures
Antibody*
Locations†
Cystic craniopharyngioma
Wall of adamantinomatous or incompletely keratinized squamous epithelium; cyst contains “motor oil”
Cytokeratin (+)
Suprasellar, sellar
Ependymal cyst
Columnar epithelium usually ciliated
GFAP (+)
Spinal cord, brain
Colloid cyst
Fibrous wall lined by inner ciliated and/or nonciliated simple columnar epithelium; cyst contains colloid and cell “ghosts”
Cytokeratin (+), EMA
Third ventricle
Dermoid cyst
Epidermoid cyst features plus adnexa of skin; cyst contains sebum, squames, and hair
Keratin (+)
Midline cerebellum, fourth ventricle, skull, spinal dura, cauda equina
Epidermoid cyst
Fibrous wall lined by inner keratinizing stratified squamous epithelium; cyst contains waxy squames
Keratin (+)
CP angle, temporal lobe, spinal dura, pineal gland, sella, brainstem, CNS
Enterogenous cyst
Lining as above; cyst contains mucin, rests on collagen
Cytokeratin (+), EMA
Spinal cord
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *Key to staining results: +, almost always strong, diffuse positivity. † Most common or most specific location is listed first. CNS, Central nervous system; CP, cerebellopontine; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein.
Tumors of the Nervous System
Metastatic Tumors (WHO Grade IV) CARCINOMA
Relevant to the CNS and meninges, important characteristics of carcinoma are its distinctively epithelial structure (Fig. 20-61, A; see Table 20-2) and the overwhelming predominance of metastatic over primary carcinomas. Metastatic carcinomas are described in detail in Chapter 8, “Immunohistology of Metastatic Carcinomas of Unknown Primary.” Rare primary brain carcinomas occur in the choroid plexus, from GCTs of the pineal and suprasellar regions, and from cysts.3,230 This section emphasizes how to distinguish between carcinomas (see Fig. 20-61, B-D) and various primary intracranial tumors (see Fig. 20-2). Metastatic carcinoma uncommonly produces neoplastic meningitis. Although its clinical features resemble those of inflammatory meningitis, cytologic examination of the CSF distinguishes the two.210,230-232 Many carcinomas metastatic to the CNS and meninges are adenocarcinomas that form acini and produce mucin. Others are small cell or undifferentiated carcinoma. The histologic hallmarks of carcinoma
817
metastatic to the CNS are an epithelial appearance and a distinct tumor margin with CNS parenchyma. Distinct epithelial borders and lack of fibrillar cytoplasmic processes contrast with the pattern of glioblastoma. Within CNS parenchyma, few neoplasms other than glioblastoma or anaplastic glioma show the intensity of nuclear pleomorphism, profuse mitotic abnormalities, or spontaneous tumor necrosis present in metastatic carcinomas. Carcinomas metastatic to the CNS stain abundantly for CAM5.2 (see Fig. 20-61, C), less commonly for 34βE12, and often for EMA. These features and the lack of GFAP (see Fig. 20-57, B) together distinguish carcinomas from gliomas.3 In distinguishing carcinomas from gliomas, the pathologist should avoid the use of AE1/AE3 anti-CK, which cross-reacts with GFAP (see Fig. 20-18). Another common mistake is to interpret gliosis trapped by advancing carcinoma as a GFAP-positive neoplasm (see Figs. 20-28 and 20-29, B). Meningiomas usually contain focal EMA, but so do many carcinomas. Meningiomas rarely contain CAM5.2 except for secretory meningiomas in proximity to their secretory globules. These tumors can often be distinguished from carcinoma on the basis of their focal CK staining (see Fig. 20-50, C). Most meningiomas have
A
B
C
D
Figure 20-61 A, Epithelioid cells in this tumor from the parietal lobe of an elderly woman show distinct borders between cells (hematoxylin and eosin). These particular epithelioid cells are pleomorphic and contain malignant nuclear features. The algorithm in Figure 20-2 offers a way to dissect the differential diagnosis of a brain tumor composed of epithelioid cells. Nearby sections of the tumor shown in A are negative for glial fibrillary acidic protein (B), highly positive for CAM5.2 cytokeratin (C), and negative for S-100 protein (D). The tumor was also positive for epithelial membrane antigen and negative for chromogranin A (not shown). The diagnostic path in Figure 20-2 leads to carcinoma. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
818
Immunohistology of the Nervous System
more diffuse and more intense vimentin staining than carcinomas, and their CK/vimentin ratio is either zero or is at least lower than that of carcinomas. Importantly, all but the most malignant meningiomas lack the abundant and often abnormal mitotic spindles found in metastatic carcinomas. RCC metastasis to brain must be distinguished from hemangioblastoma and oligodendroglioma. These neoplasms contain clear cytoplasm and distinct cell borders (see Fig. 20-1).95 Presence of EMA and CK distinguishes RCC from cerebellar hemangioblastoma and oligodendroglioma (see the sections on hemangioblastoma and oligodendroglioma earlier in this chapter).3 Small cell carcinoma can be very difficult to distinguish from lymphoma and medulloblastoma or PNET (see Fig. 20-3). EMA or CK is expressed abundantly by small cell carcinoma, infrequently and focally by medulloblastoma, but not by brain lymphoma. Although either small cell carcinoma or medulloblastoma may express synaptophysin, S-100 protein, or NSE, stains for these substances are valuable discriminators for the trained eye; they accentuate the fibrillarity of medulloblastoma in contrast to the epithelioid cells of small cell carcinoma. As more precise IHC markers of carcinomas that originate in different organs have become available, the estimation of the primary origin of a carcinoma in brain has become realistic.233,234 Thyroid transcription factor 1 (TTF-1) is positive in some lung and many thyroid carcinomas and is negative in most other carcinomas.235 A panel of immunostains for brain should include CAM5.2, CK7, CK20, CEA, and TTF-1. In the right clinical setting, include ERBB2, estrogen receptor (ER) and progesterone receptor (PR), or prostate-specific antigen (PSA). The interpretation must be made in accordance with the histologic features. Most breast and lung carcinomas in brain are undifferentiated are or adenocarcinoma, which are positive for CAM5.2 and CK7 and negative for CK20. Breast cancers are often ERBB2 or ER positive. Some lung adenocarcinomas are TTF-1 positive, and such positivity markedly reduces the likelihood of a nonpulmonary primary. A negative TTF-1 result is less useful. Colon carcinoma is typically CK20 and CEA positive and CK7 and TTF-1 negative. The diagnosis of gastric and pancreatobiliary carcinoma is more challenging, because these show variable CK7 and CK20 expression but are often CEA positive. Virtually all prostate carcinomas are positive for CAM5.2 and PSA. Prostate carcinomas are found in vertebrae more often than in CNS. Most RCCs are positive for CAM5.2 and negative for CK20, and a few are positive for CK7. TTF-1 is negative. The large, clear cells of RCC and its tendency to hemorrhage aid identification. MELANOCYTIC NEOPLASMS
Metastatic melanoma is the most common melanocytic tumor encountered in the nervous system (see Fig. 20-54), and its histologic features are variable (see Tables 20-2, 20-7, and 20-8). Melanomas, described in Chapter 7, are often strongly positive for S-100 protein
(see Fig. 20-54, C), a marker of low specificity in brain, because it is also seen in many CNS tumors.236 Human melanoma black 45 (HMB-45) and tyrosinase are the markers recommended as most likely to discriminate melanoma from other brain tumors (see Fig. 20-54, B). Rare meningiomas, schwannomas, ependymomas, neuroblastomas, and PNETs contain melanin,78 and they can be identified from their individually described features. Primary melanomas confined to the craniospinal vault are rare, and most arise from meningeal melanocytes. They are often found in the meninges, where they may infiltrate the CNS via the perivascular space. Primary melanocytomas are less malignant3 and occur in the Meckel space and elsewhere.
Cysts of the Nervous System Cysts differ from tumors in their lack of a solid nodule of tissue. This simple fact is critical to distinguishing glial cysts from gliomas and epithelial cysts from cystic craniopharyngiomas. Cysts specific to nervous tissue are emphasized here.3 Others are described in their primary chapters.
Glial, Simple, and Pineal Cysts and Wall of Syrinx The common denominator of four cysts of various locations and obscure etiologies—glial cyst, simple cyst, pineal cyst, and wall of syrinx—is that the wall is lined only by gliosis (Table 20-13). Histologic characteristics of these cysts are those of gliosis: highly GFAP-positive stellate cells are uniformly spaced, with vast tangles of GFAP-positive astrocytic processes between them.237 Sometimes only the passage of time proves such cysts not to be associated with low-grade astrocytomas.3
Neuroepithelial Cyst and Ependymal Cyst Both neuroepithelial and ependymal cysts have an epithelioid surface that is positive for S-100 protein and GFAP, resting on a fibrillary glial base that is also positive for these two antibodies.238 These cysts often occur near a ventricle (see Table 20-12) and rarely cause aseptic meningitis.13
Colloid Cyst Location is a key feature of colloid cyst, more precisely referred to as colloid cyst of the third ventricle (see Table 20-12). Its location in the third ventricle, usually near the choroid plexus and foramen of Monro, helps distinguish the colloid cyst from other cysts that superficially resemble it but that occur in different locations. This cyst’s simple columnar and cuboidal epithelium, which may be flattened to squamous epithelium, often contains a mixture of ciliated and nonciliated cells.239 Motile and sensory cilia suggest olfactory and respiratory epithelium.240 These cells are positive for CK and EMA,
Dementias
819
TABLE 20-13 Differential Diagnosis of a Cyst with Wall of Fibrillar Cells Differential Features Diagnosis
Structures
Antibody*
Locations†
Cavitary gliosis
Wall of gliosis
GFAP in glial filaments (+), S-100 (+)
Cerebrum, CNS
Abscess
Wall of granulation tissue, fibrosis (Table 20-7), inflammation and gliosis, purulent contents
Collagen (+), reticulin (+), L26 (S), A6 (S), LCA, κ and λ Ig, α-ACT, KP1 (S), microorganisms
Basal frontal and temporal lobes, CNS
Cystic astrocytoma
Wall of pilocytic astrocytoma
GFAP (+), S-100 (+)
Cerebellum, CNS
Hemangioblastoma
Wall of gliosis, mural nodule of hemangioblastoma (Table 20-8)
Factor VIII (S), CD31(S), NSE (S); Wall: GFAP (+)
Cerebellum, CNS
Glial, simple, and pineal cysts, wall of syrinx
Wall of gliosis, Rosenthal fibers
GFAP in glial filaments (+), S-100 (+), alpha B–crystalline
Pineal gland, cerebellum, spinal cord, brainstem
Meningeal cyst
Wall of dura, arachnoid; syncytial cells
Collagen (S), EMA (S)
Spinal epidural surface
Modified from McKeever PE, Blaivas M: The brain, spinal cord, and meninges. In Sternberg SS (ed): Diagnostic surgical pathology, ed 2. New York, 1994, Raven Press; pp 409-492. *Key to staining results: +, almost always strong, diffuse positivity; S, sometimes positive; R, rare cells may be positive. † Most common or most specific location is listed first. α-ACT, Alpha-antichymotrypsin; CNS, central nervous system; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; Ig, immunoglobulin; LCA, leukocyte common antigen; NSE, neuron-specific enolase.
and cyst contents are predominantly carboxymucins, rendering them positive for PAS and Alcian blue.241
Dermoid Cyst Dermoid cysts are frequently midline cysts, possibly arising from embryonic inclusions of skin at the time of closure of the neural groove (see Table 20-12). They occur between the cerebellar hemispheres and in the fourth ventricle, lumbosacral region of the cord, and skull. These cysts may involve CNS, meninges, or both.78,80 Ruptured dermoid cysts can cause sterile meningitis and inflammation that resembles an abscess. Identification of squamous epithelial cells with CK or cholesterol clefts within the inflammation offers clues to its true cause.
Epidermoid Cyst Epidermoid cysts are more common in lateral than midline sites, but they have been found in many different locations (see Table 20-12). Common locations are the cerebellopontine angle, around the pons, near the sella, within the temporal lobe, in the diploë, and in the spinal canal.78 Carcinoma rarely arises within an epidermoid cyst.230
Enterogenous Cyst Enterogenous cysts occur throughout the craniospinal vault. Such a cyst is lined by columnar epithelium, which secretes mucus (see Table 20-12). The epithelium resembles intestinal epithelium or, more rarely, bronchial epithelium. It is immunoreactive for keratin and EMA, and some reactivity for CEA and S-100 protein has been noted.242
Meningeal Cyst A cyst located in the posterior or lateral epidural space in the spinal canal lined only by fibrous tissue that resembles dura and lacks arachnoid membrane is a meningeal cyst or diverticulum (see Table 20-13). A subdural or subarachnoid cyst that has a thinner wall than the epidural cyst and that protrudes toward brain or spinal cord is an arachnoid cyst. Reactivity for vimentin, progesterone receptors, and EMA is common. This immunoreactivity resembles that of arachnoid granulations and meningiomas.243 Other cysts have variable thickness and are more difficult to categorize.
Dementias Dementia, a progressive and persistent alteration and decline of the normal cognitive state, has numerous causes that include degenerative, infectious, inflammatory, demyelinating, cerebrovascular, neoplastic, and toxic-metabolic diseases. This section deals principally with the most common degenerative diseases (see the sections on infectious diseases, spongiform encephalopathies, and cerebrovascular diseases earlier in this chapter and the “CNS Demyelination” section later in the chapter). Rare diseases are covered in a recent text.12 Specimens intended to determine the etiology of dementia may be submitted along with note of the clinical suspicion of Alzheimer disease or CJD. Any biopsy specimen being evaluated for the etiology of dementia, however, should be processed as if it were CJD until proved otherwise.8 In processing, the specimen is split, and a third is sent for Western blot.9 The rest is fixed in 10% formalin; a third of that specimen, which must include a portion of cerebral cortex, is
820
Immunohistology of the Nervous System
treated with either neat formic acid or 10% formalin plus 20% bleach for primary processing, and the remaining fixed tissue is labeled “CJD HAZARD” and locked up in fixative without formic acid or bleach. The pathologist should avoid placing any portion of the fresh specimen in bleach without formalin, which would dissolve the tissue. A small portion of cortex is held in glutaraldehyde, in case EM is needed later. The neat formic acid or formalin-bleach solution inactivates the infective agent and provides tissue preservation adequate to screen for CJD with H&E and GFAP. GFAP is a very stable antigen that resists oxidation. The pathologist should look for vacuoles in neurons and gray matter on H&E-stained specimens and for substantial stellate gliosis on GFAP-stained specimens. If these features are not present, the remaining portions of the specimen fixed optimally without bleach can then be processed and stained as needed to investigate other diagnostic possibilities (see Table 20-3). Neurons are lost in all dementias. Neuronal loss is more difficult to assess than other findings in most biopsy specimens. The assessment requires quantitation more suited to morphometry than to the interpretative eye, and anatomic variation is present in neuronal density. Neuronal loss causes gliosis, which is easier to appreciate than loss of neurons after staining. Control tissue, consisting of age- and locationmatched cerebral cortex obtained from autopsy specimens, provides a valuable baseline for assessing various abnormalities peculiar to dementias. This control is particularly important for assessing cytoplasmic vacuoles, minimal gliosis, and numbers of neurons.
Alzheimer Disease Minimal criteria necessary to diagnose Alzheimer disease (AD) have been established.239-246 These criteria refer to counts of argyrophilic plaques and neurofibrillary tangles (NFTs) in microscopic fields under a ×10 or ×20 objective. Chief among the microscopic criteria for AD is the number of argyrophilic plaques (see Table 20-3). Our approach is to count the number of plaques in a case and compare it with the number illustrated in a credible series.245 The diagnosis of AD requires clinical input, and it should not be attempted on biopsy material without the clinical certainty of dementia. Neither argyrophilic plaques nor NFTs are adequately demonstrated for enumeration by H&E stains. Bielschowsky silver stain is recommended for staining both argyrophilic plaques and NFTs (Fig. 20-62).71 Thioflavin S, excited by blue light suitable for fluorescein fluorescence, also reveals these structures.127 NFTs are located in neurons and are composed of bihelical filaments7,127 that are now being detected immunohistochemically by staining of their protein constituents, tau and ubiquitin (Table 20-14).3,247 NFTs are also intensely stained by Alz-50, a monoclonal antibody raised against a brain with AD.248 Antisera to amyloid will detect the amyloid plaques seen in silver stains and will also detect congophilic angiopathy when present.249 AD is the most common “tauopathy,” a neurodegenerative disease that shows abnormal accumulations of
Figure 20-62 Alzheimer disease. Two plaques at opposite corners of this specimen contain dark, twisted neurites. Although argyrophilic plaques key to the diagnosis stain with ubiquitin and other immunohistochemical stains, the Bielschowsky silver stain is still the gold standard for diagnosing this disease. The patient, a middleaged woman, had displayed progressive dementia for at least 3 years and satisfied the Consortion to Establish a Registry for Alzheimer Disease (CERAD) criteria for diagnosis.240
tau protein. Other tauopathies include Pick disease, progressive supranuclear palsy, cortical basal degeneration, argyrophilic grain disease, and frontotemporal dementia with parkinsonism linked to chromosome 17.
Multiinfarct Vascular Dementia Cerebral ischemic injury is a common cause of dementia in older adults.13,250 Combination cases of vascular dementia and AD are also quite frequent. Vascular causes of dementia include subcortical vascular dementia, multiinfarct dementia, ischemic dementia, cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and leukoaraiosis (see the “Cerebrovascular Diseases” section earlier in this chapter).3
Lewy Body Diseases Diffuse cortical Lewy body disease (DCLBD) is a very common disease associated with dementia.251 Consensus criteria have been developed and validated for the clinical and autopsy diagnosis of DCLBD, and a staging system has been proposed.252 The hallmark lesion, the Lewy body, is accompanied by neuronal loss and gliosis. In comparison to the Lewy bodies of the brainstem in Parkinson disease, cortical Lewy bodies are difficult to recognize in H&E-stained sections. IHC for ubiquitin, p62, or α-synuclein should be done, and the presence of round brown Lewy bodies in cortical neurons should be sought (see Table 20-14); the cingulate gyrus is a good place to find them.252
Frontotemporal and Other Dementias If IHC does not reveal histologic features of AD and DCLBD, the pathologist should consider the frontotemporal dementia group of diseases. Subclassification
Demyelination
821
TABLE 20-14 Inclusions in Immunophenotypes of Neurodegenerative Diseases Inclusion
Ubiquitin (HAR)*
Tau (HAR)*
α-Synuclein (Formic Acid)*
β-Amyloid (Formic Acid)*
AD tangles
+
+
–
–
AD neuritic plaque
+ Neurites
+ Neurites
S Plaque
+ Plaque
Lewy bodies
+
–
+
–
Pick bodies
+
+
–
–
Frontotemporal dementia or motor neuron disease
+
–
–
–
Multiple system atrophy
+ GCIs
S GCIs
+ GCIs
–
†
Modified from McKeever PE, Boyer PJ: The brain, spinal cord, and meninges. In Mills SE, Carter D, Greenson, JK, et al (eds): Sternberg’s diagnostic surgical pathology, ed 4. New York, 2004, Lippincott Williams & Wilkins; pp 399-506. Key to staining results: +, almost always strong, diffuse positivity; S, sometimes positive. *Preferred antigen retrieval technique. † Labeling of Pick bodies and neurites has reported with proteinase K antigen retrieval. AD, Alzheimer disease; GCIs, glial cytoplasmic inclusions; HAR, heat antigen retrieval, microwave in citrate buffer.
includes Pick disease, corticobasal degeneration, progressive supranuclear palsy, frontotemporal dementia with parkinsonism linked to chromosome 17, and frontotemporal lobar degeneration with or without motor neuron disease.253 Most forms of frontotemporal dementia manifest vacuolation of the superficial cortical layers, ballooned neurons, and gliosis. Specific inclusions include silver stain–positive and tau-positive Pick bodies in Pick disease and ubiquitin-positive cytoplasmic inclusions that are tau and α-synuclein negative in frontotemporal lobar degeneration (see Table 20-14). Additional causes of dementia are Lafora disease, neuronal ceroid lipofuscinosis, adrenoleukodystrophy, and others. Key histologic features of these diseases are outside the scope of this chapter and are covered in other references.3,12,15
Demyelination
of white matter, parallel NF-positive axons are straight and long. Secondary demyelination is the loss of myelin secondary to loss of axons. Axonal trophic factors sustain myelin. When the axon is severed or not sustained by its neuron of origin, the axon and then its myelin degenerates. This degeneration can happen secondary to infarcts or trauma or to toxic, metabolic, or degenerative nervous system diseases. In contrast to primary demyelination, straight and long NF-positive axons are not seen in secondary demyelination. Acutely, the axons crumble into short pieces, and in a few days, they are eaten by macrophages and disappear. NF stains thus distinguish secondary from primary demyelination.
Central Nervous System Demyelination In the CNS, the major demyelinating disorder is multiple sclerosis (MS), which should be differentiated
Unless progressive multifocal leukoencephalopathy (PML) is found in a biopsy specimen, demyelinating diseases are usually investigated clinically and at autopsy (see Table 20-3). Other viral disorders known to cause demyelination are HIV, cytomegalovirus (CMV), EBV, and varicella zoster. If the lesion was induced by a virus, amphophilic inclusions may be found, particularly at the periphery of the lesion. IHC analysis, ISH, and PCR assay are available for detecting many of these viruses.42,43,254,255 These are discussed in the “Organisms” section earlier in this chapter.
Primary and Secondary Demyelination Primary demyelination affects only the myelin, and primary demyelinating lesions are characterized histologically by destruction of myelin and by abundant, foamy, KP1-positive macrophages that contain myelin debris and lipid droplets. Within the lesion, NF-positive axons are spared (Fig. 20-63). In longitudinal sections
Figure 20-63 Primary demyelination with preservation of brown neurofilament-positive axons. Some axons are swollen and are called spheroids. Lipid-laden macrophages and gliosis are not stained brown in this section of brain biopsy specimen, but their pale gray features are still evident.
Immunohistology of the Nervous System
from other disorders with a similar histologic lesion appearance and relapsing and remitting clinical course.7 Acute MS lesions, in addition to plentiful foamy macrophages with increased proteases, have perivascular LCA-positive lymphocytes, EMA-positive and immunoglobulin-positive plasma cells, variable GFAPpositive gliosis, and less endothelial CD34 immunostaining. The macrophages stain positively for class II major histocompatibility complex antigens (human leukocyte antigen DR [HLA-DR; Ia]). They contain myelin debris related to phagocytosis. Oligodendroglia are usually seen only at the periphery of lesions. Acute plaques can show blood-brain barrier leakage from vessel wall damage with intramural complement on smooth muscle cells and infiltration by HLA-DR– positive macrophages.13 Macrophages can accumulate in an active region of white matter demyelination to the extent that they mimic oligodendroglioma and hemangioblastoma (see Fig. 20-1).95,256,257 KP1 is used to confirm the presence of macrophages. Oligodendrogliomas have fewer cytoplasmic vacuoles, more central nuclei, and Leu-7 and S-100 protein immunoreactivity, features that distinguish them from lipid-laden macrophages. Hemangioblastomas have more FVIII- and CD31-reactive capillaries and less Leu-7 (CD57) than oligodendrogliomas. Lipid-laden macrophages in a region of demyelination can be distinguished from neoplasia on the basis of the histochemical or EM demonstration of small, round globules of phagocytosed myelin.71 One of the best stains for distinguishing early necrosis from demyelination is the combined NF and Luxol fast blue with hematoxylin counterstain.
Peripheral Nervous System Demyelination In peripheral nerve, KP1 (CD68)-positive macrophages engulf myelin and cluster around endoneurial vessels.258 T lymphocytes that are immunoreactive with CD3, CD4, and CD8 are abundant in the endoneurium, and B lymphocytes are virtually absent in chronic inflammatory demyelinating polyneuropathy (CIDP).259 T lymphocytes are prominent in both CIDP and Guillain-Barré syndrome (Fig. 20-64).258 Primary and secondary peripheral nervous system (PNS) demyelinations are best distinguished by non-IHC methods that include teased fiber preparations and EM.
Epilepsy Complex partial seizure disorder, previously called temporal lobe epilepsy, may involve the temporal, frontal, parietal, or occipital lobes.260-264 Approximately 80% of complex partial seizures originate in the temporal lobe, thus the most common surgical procedure for intractable epilepsy is temporal lobectomy.260 Staining for neurons and for GFAP, with the use of age-matched temporal lobe autopsy control tissue, is recommended for detection and neuroanatomic localization of the most common abnormalities, neuronal loss and resulting
100 90 80 70 Positive %
822
60 50 40 30 20 10 0 CIDP
GBS
Epineurial
CIDP
GBS
Endoneurial
Figure 20-64 Locations of T lymphocytes among sural nerve biopsy specimens from 13 cases of chronic inflammatory demyelinating polyneuropathy (CIDP) and 22 cases of Guillain-Barré syndrome (GBS). Data from Schmidt B, Toyka KV, Keifer R, et al: Inflammatory infiltrates in sural nerve biopsies in Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy. Muscle Nerve 1996;19:474-487.
GFAP-positive gliosis (Fig. 20-65 through 20-67; see Table 20-3).3 A remarkably effective new stain for highlighting layers of neurons is NeuN, which highlights pyramidal neurons in the hippocampus, making it easy to find regions of neuronal loss (see Fig. 20-66). Neuronal loss triggers gliosis. Both are common in the hippocampus, which is vulnerable to hypoxia (see Fig. 20-65). The large pyramidal neurons are very sensitive,261 and among these, neurons in the Sommer sector of cornu ammonis region 1 (CA1) are most sensitive, whereas neurons in CA2 are least sensitive. Neuronal markers, particularly synaptophysin, often are depleted in mirror-image staining to the (increased) GFAP. Surgical fragmentation of some specimens confounds identification of these regions (see Fig. 20-65), but CA4 is obviously enclosed by the series of crowded, small, synaptophysin-positive round neurons of the dentate fascia. Other regions may be evident by their relationship to CA4, the opening of the dentate fascia, and the widening layers of neurons at the subiculum (see Fig. 20-65). NF and synaptophysin stains aid recognition of neuroanatomic regions in these fragments. Loss of pyramidal and granular cell neurons in the hippocampus (see Fig. 20-66), large numbers of corpora amylacea (see Fig. 20-67), deposits of hemosiderin pigment in perivascular macrophages, and inflammation, focal meningeal fibrosis, calcification, and ferrugination of large pyramidal neurons can be seen in the hippocampus.3 Cytoarchitectural studies of gray and white matter in resected temporal neocortex may reveal features of neuronal dysgenesis, such as neuronal ectopia and clustering and subpial gliosis.13,262,263 Deep or surface electrodes are occasionally placed within the region of the future surgical resection to monitor and evaluate seizure activity. The surgeon should inform the pathologist if such electrodes have been used, because they leave a trail of encephalomalacia with chronic inflammation of A6-positive and
Epilepsy
id oro
Ch
xus
ple
CA1
CA3
CA2
id Larg e pyram
Inferior horn lateral ventricle
al
CA4 ate ent
neu
cia
fas
D
m
iculu
rons
Sub
A
B
CA rg e
s
3
ur on
La
xus Choroid ple CA2
823
pyr amidal ne
CA4 ascia
ate f
CA1 rg La
Dent
e
p
yr am idal neurons
C
m
iculu
Sub
D
Figure 20-65 Two ways the cornu ammonis (CA) of the hippocampus can be received. A, Diagram of an en bloc surgical resection or autopsy block provides an intact view of the large pyramidal neurons and dentate fascia composed of small, crowded neurons with round nuclei. Adjacent brain (top of diagram) dorsal to the CA may be present in an autopsy specimen but not in a surgical specimen. Lateral temporal lobe tissue (left of diagram) may be present in either specimen. B, These structures can be seen in the actual autopsy specimen of normal hippocampus oriented the same as the diagram and stained for neurons with Nissl stain. C, Diagram depicting a fragmented surgical specimen still shows recognizable regions, such as CA4 enclosed by dentate fascia and CA1 between ependymal cells, lining ventricle and unbroken line of small neurons in dentate fascia. D, An actual surgical specimen and slightly larger fragment than the diagram shows loss of neurons particularly prominent in CA1 and CA4.
A
B
Figure 20-66 This intact specimen was resected from a young woman with lifelong medically refractory temporal lobe seizures. A, Hematoxylin and eosin stain highlights hemorrhage related to the resection in the cornu ammonis (CA), or Ammon’s horn. B, NeuN stain for neurons highlights near total loss of neurons in the portion of CA1 most sensitive to hypoxia, the Sommer sector (arrow).
824
Immunohistology of the Nervous System
Cortical Malformations
Figure 20-67 Pyramidal cell layer of cornu ammonis region 1 of hippocampus in a patient with decades of partial complex seizures. Two large, clear, glial fibrillary acidic protein (GFAP)–negative neurons can be seen near a vessel at the left side of the field. Other neurons have died. Although numerous brown, GFAP-positive fibrils of gliosis are evident, the astrocytes are quiescent and have scarce brown cytoplasm around their nuclei. Round, fuzzy, lightpurple bodies the size of nuclei are called corpora amylacea and are common in such specimens. This neuronal loss and gliosis happened many years ago.
L26-positive T and B lymphocytes, KP1-positive macrophages, and hemorrhage in the surgical specimen.263 Surface electrodes may also cause focal meningitis. A variety of clinically unsuspected pathologic entities are found in individual specimens. Stereotactic resection of cerebral lesions in partial epilepsy may yield vascular malformations and glial neoplasms. Primary intracerebral tumors that manifest as medically refractory epilepsy are usually low-grade gliomas, mixed tumors with glial or neuronal components or both, hamartomas, or dysembryoplastic neuroepithelial tumors (see Figs. 20-15, A; 20-25, A; and 20-36).264,265
A
Malformations of the cerebral architecture are seen by the autopsy pathologist, pediatric pathologist, and surgical pathologist. Neuronal markers such as NeuN, synaptophysin, and NF supplement H&E, S-100, and GFAP in finding these abnormal regions of cortex. They also classify the abnormal cells.3 Microdysgenesis is a term for lesions noted only microscopically: this includes 1) focal S-100 protein– positive oligodendroglial clusters, which are often interspersed with NF-positive ganglion cells in gray or white matter; 2) oligodendroglial hyperplasia that is too small and well-differentiated to be oligodendroglioma; and 3) increased numbers of normally scarce neurons in the white matter. Cortical dysplasia consists of disrupted cortical architecture and abnormally enlarged cells, some with IHC staining of neurons, some with GFAP-positive astrocytes, and some individual cells that stain for both glial and neuronal markers.79 Cortical tubers, seen with tuberous sclerosis, are cortical dysplasias. Other dysplastic abnormalities are described in the “Neuronal Tumors” section earlier in this chapter.
Pitfalls in Diagnosis Is It Really Negative? The simple question, whether a specimen is really “negative” for the marker in question, must be asked to avoid misinterpretation. Whenever possible, the pathologist should include a positive control for the marker, as described earlier, within the same block as the lesion to be identified (Fig. 20-68); a positive control from a different block does not work as well. If this control tissue does not stain, the lesion cannot be evaluated with the IHC stain.
B
Figure 20-68 Importance of internal tissue control for immunohistochemistry. Tiny tissue fragment at the edge of two sections of this tumor specimen and at the corners of the two illustrations is brain. Brain tissue, which always makes S-100 protein, is the internal positive control. Two procedures were used to test for S-100 protein. A, One procedure does not stain the tumor, but neither does it stain the brain. B, The second procedure stains the brain fragment dark brown and also stains the tumor. Only the second procedure is a reliable stain for S-100, and it reveals the tumor to be S-100 positive. Subsequent human melanoma black 45 staining suggested metastatic melanoma, and ultimately, the cutaneous primary tumor was found. From McKeever PE: New methods of brain tumor analysis. In Mena H, Sandberg G [eds]: Dr. Kenneth M. Earle memorial neuropathology review. Washington, DC: Armed Forces Institute of Pathology, 2004.
Pitfalls in Diagnosis
825
Are the Cells of Interest Positive?
Gliosis vs. Glioma
A good nuclear counterstain is essential to correct interpretation of IHC stain responses. The counterstain distinguishes reactive, neoplastic, and necrotic cells and also defines neuroanatomic relationships. Necrotic cells have nuclear pyknosis, karyorrhexis, and karyolysis. Necrotic regions generate a false-positive response to IHC staining, so both features need to be recognized to avoid misinterpretation. Reactive astrocytosis is associated with many brain tumors. Nuclear features of GFAP-positive cells must be examined to distinguish reactive from neoplastic astrocytes (see Fig. 20-29). The immunoreactivity in closest proximity to the definitive nuclei should be checked for correct identification of these cells. This rule has general application to other markers (see Figs. 20-19, B, and 20-56). Neoplastic cells have atypical or pleomorphic nuclei. Their chromatin patterns are often distinctive (see Fig. 20-14, B), and glioma nuclei are crowded. WHO grade III and higher gliomas and other malignancies show mitoses (see Figs. 20-18, C, and 20-29, A). It is important to know the characteristics of the antigen being localized. Like all intermediate filament components, histones, and many membrane components, structural proteins tend to remain exactly where they belong in their original cellular compartment (e.g., cytoplasm, nucleus, membrane) after excision, application of saline and formalin, dehydration, and embedding. Small or water-soluble molecules that include proteins and peptides may move around during a pathologic process, such as invasion or inflammation, or later during specimen handling and processing. S-100 protein is a small, acidic molecule that sometimes moves around (Fig. 20-69). The localization of such molecules requires careful interpretation.
The question of whether a CNS lesion represents gliosis or glioma is shorthand for the more realistic question: Does this gliotic brain also contain glioma cells? This question most commonly arises when viewing slightly to moderately hypercellular brain tissue. Four useful stains for distinguishing glioma cells are H&E, IDH1 mutant, MIB-1, and p53 (Fig. 20-70). H&E and GFAP are listed high, because they reveal structure and topography (S&T), the primary use of a stain (see Figs. 20-14, 20-15, 20-18, and 20-25). GFAP finds glial cells, but interpretation relies on S&T, as described below. IDH1 mutant reveals abnormal molecules, and results can be controlled with tissues of known mutation status. MIB-1 and p53 are a bit more tricky, because they seek to quantitate differences in expression of normal molecules using tools that are semiquantitative at best, and they require more standardization than the others. Distinguishing gliosis from glioma can be most difficult (see Fig. 20-12, A).3 Diffuse gliomas infiltrate brain tissue and stimulate their own gliosis, which compounds the problem. Swollen GFAP-positive cells can occur in gliosis and glioma (Table 20-15). Features that distinguish glioma cells from gliosis and normal parenchyma include individual cell variation in GFAP staining, nuclear hyperchromasia (see Fig. 20-14, B), nuclear cluster formation, nuclear molding (see Fig. 20-16), mitoses, and calcifications. Mitoses suggest not only that the tumor is a glioma but also that it has a high grade of malignancy. Abnormal variations in size and shape of glial nuclei are common in margins of gliomas. In astrocytomas, ependymomas, and astrocytic gliomas, GFAP stain is ideal for highlighting glioma cells; it delineates the cytoplasm in both gliosis and glioma and facilitates interpretation of these cells. High nuclear to low brown cytoplasmic ratios typify gliomas.
A
B
Figure 20-69 Sometimes we may wish we had not tried a stain. Both S-100 protein stains are on the same tumor. Neither the primary nor any region of the spinal metastasis of this lung carcinoma stained with S-100 protein (A), except regions near robustly S-100–positive spinal nerve roots (B). This suggests the possibility that this small, acidic molecule may have leaked from damaged nerve (arrows) to adjacent carcinoma. This odd pattern of localization suggests restraint in interpretation of the original S-100 protein status of the lung carcinoma.
826
Immunohistology of the Nervous System
A
B
C
D
Figure 20-70 Gliosis vs. glioma. A, Glioma cells are elusive on hematoxylin and eosin stain. B, Their true nature is revealed by the new immunohistochemical marker for IDH1 mutant, which stains the cytoplasm of these glioma cells with the mutation. Vessels and normal and reactive glial cells are negative. C, Proliferation measured by MIB-1 is only slightly above reactive brain, but notice that at least three brown, MIB-1–positive nuclei are larger and oddly shaped compared with most other nuclei. D, Larger, oddly shaped, and molding nuclei overexpress p53. This series illustrates roles of positive staining, structural interpretation of staining, and corroboration of features to arrive at the diagnosis of low-grade glioma in this specimen.
Mitotic spindles with chromosomes near each other and surrounded by scarce cytoplasm (see Fig. 20-29, A) are exceptionally rare in gliosis (see Fig. 20-29, B). Reactive astrocytes have much lower nuclear to cytoplasmic ratios, abundant dark-brown stellate processes, and spaces between them so that they resemble trees in an
apple orchard (see Fig. 20-5). IHC markers of proliferation, such as MIB-1, may help distinguish neoplastic glia from gliosis at these margins. Gliomas expand, but gliosis contracts; however, this important feature is difficult to confirm without serial radiographs and in situ observation of the confluence of
TABLE 20-15 Types of Swollen Glial Fibrillary Acidic Protein–Positive Cells Lesion
Relationship of GFAP+ Cells
Intracellular Distribution of GFAP
Reactive gliosis
Spaced apart
Gemistocytic astrocytoma Oligodendroglioma with microgemistocytes
Nuclear Features
Other Tissue Elements
Low nuclear/cytoplasmic ratios; very fibrillar; longest cell process
Smooth, oval, fine chromatin
Neurofilament-positive axons are normally spaced.
Collide, clump
Some long processes
Pleomorphic, elongated, hyperchromatic
Neurofilament-positive axons are spread apart.
Collide, clump
Shortest processes; mixed with negative oligodendroglia
Smooth, round, hyperchromatic
Neurofilament-positive axons are spread apart.
GFAP, Glial fibrillary acidic protein.
Pitfalls in Diagnosis
the actual mass and its margin to exclude the possibility of gliosis around another tumor. Granular calcifications scattered among hypercellular glia distinguish glioma from gliosis and normal white matter. Caution is required not to overinterpret calcifications associated with neurons and neuropil within 0.5 cm of a large mass. Although microcysts, calcifications, and mitoses are important diagnostic features of gliomas, they are not seen in every case and are uncommon within the margins of gliomas that invade CNS parenchyma. Another important feature that distinguishes the margin of a glioma from gliosis is cellular density (see Table 20-4). Some gliomas exhibit uneven distribution of cellular density (see Fig. 20-13). Others obscure the junction between gray matter and white matter. Still others spawn secondary structures of Scherer, and the most distinctive are subpial and perineuronal gliomatosis; these secondary structures are collections of neoplastic glia beneath the pia or around neurons.3 GFAP stain highlights secondary structures from astrocytomas; secondary structures of oligodendrogliomas show much less synaptophysin than the neuropil of gray matter and are highlighted as lightly staining clusters of cells around neurons or vessels.61 Glioma nuclei frequently touch and even indent one another (see Fig. 20-16 and Table 20-15), even among scattered clusters of cells in the diffuse margins of gliomas. Gliosis, however, consists of evenly spaced astrocytes with high GFAP immunoreactivity (see Fig. 20-5). This even spacing is best seen with anti-GFAP staining at low magnification. The low nuclear to cytoplasmic ratio of gliosis is seen at high magnification. Because the nuclei of gliomas are more pleomorphic than normal or gliotic CNS parenchyma, nuclear pleomorphism and hyperchromasia—seen best with a good nuclear counterstain, such as hematoxylin—help identify a glioma (see Figs. 20-11, 20-12, 20-14, 20-16, and 20-18).5,92 New advances in staining glioma cells have significantly improved the pathologist’s ability to distinguish between gliosis and glioma. Reactive astrocytes do not overexpress p53 protein sufficiently to appear to show positive nuclei. More than 50% of astrocytomas overexpress p53, and they can be identified by their positive nuclei, even when they are individual cells at the margin of a glioma mixed with gliosis. Unfortunately, a negative result with p53 does not exclude the possibility of glioma. MIB-1 requires standardization in individual laboratories to be useful for distinguishing gliomas of low cellular density from gliosis. One way to do this is to keep a log of these two entities stained in your laboratory and counted by you or your associates, and look for counts of gliomas and gliosis that do not overlap. Demyelination may be confused with neoplastic disease, because it produces abundant gliosis.257 Large cells with short chromosomes spread apart in their cytoplasm mimic mitotic activity in a glioma.256 If numerous lipid-laden KP1-positive macrophages are encountered within parenchyma and around
827
vessels, demyelinating disease should be considered. Appropriate stains for myelin, NF stain for axons (see Fig. 20-2), and features described in this section should be considered in the interpretation (see Figs. 20-6, A; 20-14, A-B; and 20-63).
Infiltrating Versus Noninfiltrating Cells Noninfiltrating gliomas are often surgically resectable, a characteristic that places a premium on distinguishing them from infiltrating gliomas. This distinction can be difficult in fragmented or incomplete specimens. After the possibility of neoplastic neurons has been ruled out (see the “Neuronal Tumors” section earlier in this chapter), neuroanatomic brain tissue constituents are useful markers for the distinction of infiltrating and noninfiltrating gliomas. Axons run close together in parallel in white matter tracts and also are identifiable in gray matter. Noninfiltrating gliomas leave these axons unmolested (see Fig. 20-13, B), whereas infiltrating gliomas spread them apart (see Fig. 20-13, A) and cause some to swell. Synaptophysin carpets the neuropil of gray matter with tiny brown dots. Synaptophysinnegative glioma cells either stop abruptly or invade this fine carpet.61 In specimens that lack discernible brain tissue, information is still available after the tumor has been stained. If axons of nonneoplastic origin are found in the glioma, it is infiltrative (see Fig. 20-16). This feature distinguishes infiltrating gliomas from noninfiltrating gliomas, which have more discrete margins with brain tissue.
Abscess Versus Neoplasm Entities such as granulomas (Table 20-5), histiocytosis (Tables 20-7 and 20-11), and fibrous tumors (Table 20-8) that produce collagen can be confused with the wall of an abscess (see Fig. 20-7). If abscess is suspected during the biopsy procedure, sterile tissue should be sent to microbiology for culture. A negative culture is better than a missed opportunity to find something treatable. Failing culture, stains for microorganisms may help. Gliomas, sarcomas, and desmoplastic neoplasms and various cysts with collagenous walls may simulate abscesses (see Figs. 20-33, B; 20-37, A; and 20-42). These tumors may be distinguished by the lack of an inflammatory component and the presence of a neoplastic component. More problematic are cysts that have ruptured and exuded material foreign to the CNS, such as colloid or squamous epithelial cells. If this material is not detectable on H&E staining within the inflammatory reaction, IHC stains for cyst wall material, such as CK stains for epithelial cells, assist the interpretation.229 These other lesions are sterile in situ and do not stain for microorganisms, as an abscess would (see Fig. 20-2). In reoperations and primary cases, leukocytes and histiocytes can produce considerable MIB-1 staining that either obscures or mimics a glioma. It becomes important to see whether a glioma cell population with significant proliferative activity is present. Serial
828
Immunohistology of the Nervous System
sections of MIB-1 sandwiched between glial markers, such as GFAP and S-100 protein, can test this. Dual stains of brown MIB-1 plus red GFAP on the same section can be extremely useful in these situations.
Dysplasia Versus Neoplasm The distinction between cortical dysplasia and gangliocytic neoplasms can be difficult. Presented here are some guidelines for using IHC to distinguish dysplasia from neoplasm. Dysplasias consist of abnormal architecture, such as too many or too few layers of neurons, ectopic neurons, or abnormally large or too small neurons or glia. They may be large, but they do not grow. Neuronal neoplasms typically manifest more than just architectural abnormalities. Their cytologic abnormalities include binucleation, large and bizarre nuclei, and hyperchromatism. These become evident with NF or synaptophysin stain and a good hematoxylin counterstain. They are easiest to recognize when the suspected lesion is compared with a normal sample of the same region of brain from another case or from autopsy material. Neoplasms have cellular proliferation. The MIB-1 stain measures their proliferation potential, many of which show no mitoses. On stains that I have observed, dysplasia generally falls below a 3% maximal MIB-1 LI, and neoplasia falls above 3%. Sampling and laboratory techniques can affect this cutoff.
Summary As in other organ systems, it is imperative that the H&E stain be used as the starting and ending points for investigative diagnostic issues in neuropathology. This philosophy is especially crucial in neuropathology, in which morphologic overlap is abundant between nonneoplastic and neoplastic conditions.
Acknowledgments The following colleagues provided particularly valuable assistance. Drs. Philip Boyer, Mila Blaivas, Jeanne Bell, Larry Junck, and Ricardo Lloyd provided key citations. I thank Ms. Elizabeth Wawrzaszek, Ms. Dianna Banka, and Ms. Peggy Otto for their skill and patience in preparing this chapter. Mr. Mark Deming and Ms. Elizabeth Horn Walker carefully prepared the illustrations. Immunohistologists and histopathologists in the University of Michigan Medical Center Pathology Laboratories prepared the fine slides. The work described in this chapter is supported in part by NIH CA68545 and CA47558 grants awarded by the U.S. Public Health Service. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. McKeever PE: New methods of brain tumor analysis. In Rushing EJ, Sandberg GD, editors: Dr. Kenneth M. Earle Memorial Neuropathology Review, Washington, D.C., 2008 (updated 2012), Armed Forces Institute of Pathology. 2. Louis DN, Ohgaki H, Wiestler OD, et al, editors: World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of the Nervous System, Lyon, 2007, IARC Press. 3. McKeever PE, Boyer P: The brain, spinal cord, and meninges. In Mills SE, Carter D, Greenson JK, et al, editors: Sternberg’s Diagnostic Surgical Pathology, ed 4, Philadelphia, 2004, Lippincott Williams & Wilkins, pp 400–503. 4. Firlik KS, Martinez AJ, Lunsford LD: Use of cytological preparations for the intraoperative diagnosis of stereotactically obtained brain biopsies: A 19-year experience and survey of neuropathologists. J Neurosurg. 91:454–458, 1999. 5. McKeever PE: Laboratory methods in brain tumor diagnosis. In Nelson JS, Mena H, Parisi J, et al, editors: Principles and Practice of Neuropathology, ed 2, New York, 2003, Oxford University Press, pp 272–297. 6. Kepes JJ, Moral LA, Wilkinson SB, et al: Rhabdoid transformation of tumor cells in meningiomas: A histologic indication of increased proliferative activity: Report of four cases. Am J Surg Pathol. 22:231–238, 1998. 7. Graham DI, Lanto PL, editors: Greenfield’s neuropathology, ed 7, New York, 2002, Edward Arnold. 8. Garcia JH, Budka H, McKeever PE, et al, editors: Neuropathology: The diagnostic approach, Philadelphia, 1997, Mosby. 9. Castellani RJ, Parchi P, Madoff L, et al: Biopsy diagnosis of Creutzfeldt-Jakob disease by Western blot: A case report. Hum Pathol. 28:623–641, 1997. 10. Mrak RE: The big eye in the 21st century: The role of electron microscopy in modern diagnostic neuropathology. J Neuropathol Exp Neurol. 61:1027–1039, 2002. 11. Dickson DW, Bergeron C, Chin SS, et al: Office of Rare Diseases neuropathologic criteria for corticobasal degeneration. J Neuropathol Exp Neurol. 61:935–946, 2002. 12. Dickson DW, editor: Neurodegeneration: The molecular pathology of dementia and movement disorders, Basel, 2003, ISN Neuropath Press. 13. McKeever PE: Immunohistology of the nervous system. In Dabbs D, editor: Diagnostic Immunohistochemistry, ed 2, Philadelphia, 2006, Churchill Livingstone, pp 746–816. 14. Sundgren PC, Fan X, Weybright P, et al: Differentiation of recurrent brain tumor versus radiation injury using diffusion tensor imaging in patients with new contrast-enhancing lesions. Magn Reson Imaging. 24:1131–1142, 2006. 15. Ellison D, Love S, Chimelli L, et al: Neuropathology, Amsterdam, 2003, Elsevier. 16. McKeever PE, Balentine JD: Macrophage migration through the brain parenchyma to the perivascular space following particle ingestion. Am J Pathol. 93:153–164, 1978. 17. Danton GH, Dietrich WD: Inflammatory mechanisms after ischemia and stroke. J Neuropathol Exp Neurol. 62:127–136, 2003. 18. McKeever PE, Fligiel SEG, Varani J, et al: Products of cells cultured from gliomas. IV: Extracellular matrix proteins of gliomas. Int J Cancer. 37:867–874, 1986. 19. Thomson RB Jr, Bertram H: Laboratory diagnosis of central nervous system infections. Infec Dis Clin North Am. 15:1047– 1071, 2001. 20. Park do Y, Kim JY, Chi KU, et al: Comparison of polymerase chain reaction with histopathologic features for diagnosis of tuberculosis in formalin-fixed, paraffin-embedded histologic specimens. Arch Pathol Lab Med. 127:326–330, 2003. 21. Kaufman L, Standard PG, Jalbert M, et al: Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent antibodies. J Clin Microbiol. 35:2206– 2209, 1997. 22. Kainer MA, Reagan DR. Nguyen DB, et al: Fungal infections associated with contaminated methylprednisolone in Tennessee. N Engl J Med. 367(23):2194–2203, 2012. 23. Jentzen J, Shaller N: Report of a series of autopsy cases involving Exserohilum rostratum contamination of methylprednisolone. Am J Forensic Med Pathol. (in press).
828.e1
24. Guarner J, Greer PW, Bartlett J, et al: Congenital syphilis in a newborn: An immunopathologic study. Mod Pathol. 12:82–87, 1999. 25. Shankar SK, Ravi V, Suryanarayana V, et al: Immunoreactive antigenic sites of Cysticercus cellulosae relevant to human neurocysticercosis—Immunocytochemical localization using human CSF as source of antibody. Clin Neuropathol. 14:33–36, 1995. 26. Diogo CM, Mendonca MC, Savino W, et al: Immunoreactivity of a cytokeratin-related polypeptide from adult Schistosoma mansoni. Int J Parasitol. 24:727–732, 1994. 27. Schwartz MA, Selhorts JB, Ochs AL, et al: Oculomasticatory myorrhythmia: A unique movement disorder occurring in Whipple’s disease. Ann Neurol. 20:677–683, 1986. 28. Cadavid D, Barbour AG: Neuroborreliosis during relapsing fever: Review of the clinical manifestations, pathology, and treatment of infections in humans and experimental animals. Clin Infect Dis. 26:151–164, 1998. 29. Cassady KA, Whitley RJ: Pathogenesis and pathophysiology of viral infections of the central nervous system. In Scheld WM, Whitley RJ, Durack DT, editors: Infections of the Central Nervous System, ed 2, Philadelphia, 1997, Lippincott-Raven, pp 7–22. 30. Fleming KA: Analysis of viral pathogenesis by in situ hybridization. J Pathol. 166:95–96, 1992. 31. Mrak RE, Young L: Rabies encephalitis in humans: Pathology, pathogenesis and pathophysiology. J Neuropathol Exp Neurol. 53:1–10, 1994. 32. McQuaid S, Cosby SL, Koffi K, et al: Distribution of measles virus in the central nervous system of HIV-seropositive children. Acta Neuropathol. 96:637–642, 1998. 33. Gray F, Chretien F, Vallat-Decouvelaere AV, et al: The changing pattern of HIV neuropathology in the HAART era. J Neuropathol Exp Neurol. 62:429–444, 2003. 34. Nath A, Sinai AP: Cerebral toxoplasmosis. Curr Treat Options Neurol. 5:3–12, 2003. 35. Zimmer C, Daeschlein G, Patt S, et al: Strategy for diagnosis of Toxoplasma gondii in stereotactic brain biopsies. Stereotact Funct Neurosurg. 56:66–75, 1991. 36. Rhodes RH: Histopathology of the central nervous system in the acquired immunodeficiency syndrome. Hum Pathol. 18:636– 643, 1987. 37. Persons DL, Moore JA, Fishback JL: Comparison of polymerase chain reaction, DNA hybridization, and histology with viral culture to detect cytomegalovirus in immunosuppressed patients. Mod Pathol. 4:149–152, 1991. 38. Vazeux R, Cumont M, Girard PM, et al: Severe encephalitis resulting from coinfections with HIV and JC virus. Neurology. 40:944–948, 1990. 39. Daley CL, Small PM, Schecter GF, et al: An outbreak of the tuberculosis with accelerated progression among persons infected with the human immunodeficiency virus. N Engl J Med. 4:231–235, 1992. 40. Feraru ER, Aronow HA, Lipton RB: Neurosyphilis in AIDS patients: Initial CSF VDRL may be negative. Neurology. 40:541– 543, 1990. 41. Martinez AJ, Visvesvara GS: Free-living, amphizoic and opportunistic amebas. Brain Pathol. 7:583–598, 1997. 42. Prayson RA, Estes ML: Stereotactic brain biopsy for diagnosis of progressive multifocal leukoencephalopathy. South Med J. 86:1381–1394, 1993. 43. Hulette CM, Downey BT, Burger PC: Progressive multifocal leukoencephalopathy: Diagnosis by in situ hybridization with a biotinylated JC virus DNA probe using an automated histomatic code-on slide stainer. Am J Surg Pathol. 15:791–797, 1991. 44. Hulette CM, Earl NL, Crain BJ: Evaluation of cerebral biopsies for the diagnosis of dementia. Arch Neurol. 49:28–31, 1992. 45. Capellari S, Vital C, Parchi P, et al: Familial prion disease with a novel 144-bp insertion in the prion protein gene in a Basque family. Neurology. 49:133–141, 1997. 46. Bruce ME, Will RG, Ironside JW, et al: Transmissions to mice indicate that “new variant” CJD is caused by the BSE agent. Nature. 389:498–501, 1997. 47. Kovacs GG, Head MW, Hegy I, et al: Immunohistochemistry for the prion protein: Comparison of different monoclonal antibodies in human prior disease subtypes. Brain Pathol. 12:1–11, 2002.
828.e2
Immunohistology of the Nervous System
48. Ironside JW, Sutherland K, Bell JE, et al: A new variant of Creutzfeldt-Jakob disease: Neuropathological and clinical features. Cold Spring Harb Symp Quant Biol. 50:523–527, 1996. 49. Vinters HV, Secor DL, Pardridge WM, et al: Immunohistochemical study of cerebral amyloid angiopathy. III: Widespread Alzheimer A4 peptide in cerebral microvessel walls colocalizes with gamma trace in patients with leukoencephalopathy. Ann Neurol. 28:34–42, 1990. 50. Chen ST, Chen SD, Hsu CY, et al: Progression of hypertensive intracerebral hemorrhage. Neurology. 39:1509–1514, 1989. 51. Kittner SJ, Sharkness CM, Sloan MA, et al: Infarcts with a cardiac source of embolism in the NINDS stroke data bank: Neurologic examination. Neurology. 42:299–302, 1992. 52. Miettinen M, Lindenmayer AE, Chaubal A: Endothelial cell markers CD31, CD34, and BNH9 antibody to H- and Y-antigens—evaluation of their specificity and sensitivity in the diagnosis of vascular tumors and comparison with von Willebrand factor. Mod Pathol. 7:82–90, 1994. 53. Takahashi A, Ushiki T, Abe K, et al: Cytoarchitecture of periendothelial cells in human cerebral venous vessels as compared with the scalp vein: A scanning electron microscopic study. Arch Histol Cytol. 57:331–339, 1994. 54. Lanthier S, Lottie A, Michaud J, et al: Isolated angiitis of the CNS in children. Neurology. 56:837–842, 2001. 55. Jennekens FG, Kater L: The central nervous system in systemic lupus erythematosus. Parts 1 and 2. Rheumatology. 41:605–630, 2002. 56. McKelvie PA, Collins S, Thyagarajan D, et al: Meningoencephalomyelitis with vasculitis due to varicella zoster virus: A case report and review of the literature. Pathology. 34:88–93, 2002. 57. Markus HS, Martin RJ, Simpson MA, et al: Diagnostic strategies in CADASIL. Neurology. 59:1134–1138, 2002. 58. Fleetwood IG, Steinberg GK: Arteriovenous malformations. Lancet. 359:863–873, 2002. 59. Hoya K, Asai A, Sasaki T: Expression of myosin heavy chain isoforms by smooth muscle cells in cerebral arteriovenous malformations. Acta Neuropathol. 105:455–461, 2003. 60. McKeever PE: Insights about brain tumors gained through immunohistochemistry and in situ hybridization of nuclear and phenotypic markers. J Histochem Cytochem. 46:585–594, 1998. 61. McKeever PE: Neurofilament (NF) and synaptophysin stains reveal diagnostic and prognostic patterns of interaction between normal and neoplastic tissues. Presented at the annual meeting of the Histochemical Society; Bethesda, MD: March 24, 2000. 62. Shi S-R, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry: Past, present, and future. J Histochem Cytochem. 45:327–343, 1997. 63. McKeever PE, Ross DA, Strawderman MS, et al: A comparison of the predictive power for survival in gliomas provided by MIB-1, bromodeoxyuridine and proliferating cell nuclear antigen with histopathologic and clinical parameters. J Neuropathol Exp Neurol. 7:798–805, 1997. 64. McKeever PE, Junck L, Strawderman MS, et al: Proliferation index is related to patient age in glioblastoma. Neurology. 56:1216–1108, 2001. 65. McKeever PE, Strawderman MS, Yamini B, et al: MIB-1 proliferation index predicts survival among patients with grade II astrocytoma. J Neuropathol Exp Neurol. 57:931–936, 1998. 66. Hsu DW, Louis DN, Efird JT, et al: Use of MIB-1 (Ki-67) immunoreactivity in differentiating grade II and grade III gliomas. J Neuropathol Exp Neurol. 56:857–865, 1997. 67. Aboussekhra A, Wood RD: Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA. Exp Cell Res. 221:326–332, 1995. 68. Schiffer D, Cavalla P, Migheli A, et al: Apoptosis and cell proliferation in human neuroepithelial tumors. Neurosci Lett. 195:81– 84, 1995. 69. Taniguchi K, Wakabayashi T, Yoshida T, et al: Immunohistochemical staining of DNA topoisomerase II alpha in human gliomas. J Neurosurg. 91:477–482, 1999. 70. Chronwall BM, McKeever PE, Kornblith PL: Glial and nonglial neoplasms evaluated on frozen section by double immunofluorescence for fibronectin and glial fibrillary acidic protein. Acta Neuropathol. 59:283–287, 1983.
71. McKeever PE, Balentine JD: Histochemistry of the nervous system. In Spicer SS, editor: Histochemistry in Pathologic Diagnosis, New York, 1987, Marcel-Dekker, pp 871–957. 72. Reifenberger G, Louis DN: Oligodendroglioma: Toward molecular definitions in diagnostic neuro-oncology. J Neuropathol Exp Neurol. 62:111–126, 2003. 73. van den Bent MJ, Carpentier AF, Brandes AA, et al: Adjuvant procarbazine, lomustine, and vincristine improves progressionfree survival but not overall survival in newly diagnosed anaplastic oligodendrogliomas and oligoastrocytomas: A randomized European Organisation for Research and Treatment of Cancer phase III trial. J Clin Oncol. 24(18):2715–2722, 2006. 74. Ichimura K: Molecular pathogenesis of IDH mutations in gliomas. Brain Tumor Pathology. 29:131–139, 2012. 75. Cheng Y, Pang JC, Ng HK, et al: Pilocytic astrocytomas do not show most of the genetic changes commonly seen in diffuse astrocytomas. Histopathology. 37:437–444, 2000. 76. Korshunov A, Golanov A, Timirgaz V: Immunohistochemical markers for intracranial ependymoma recurrence. An analysis of 88 cases. J Neurol Sci. 177:72–82, 2000. 77. Rodas RA, Fenstermaker RA, McKeever PE, et al: Correlation of intraluminal thrombosis in brain tumor vessels with postoperative thrombotic complications. J Neurosurg. 89:200–205, 1998. 78. Rubinstein LJ: Tumors of the central nervous system, Washington, D.C., 1972, Armed Forces Institute of Pathology. 79. Burger PC, Scheithauer BW, Vogel FS: Surgical Pathology of the Nervous System and Its Coverings, ed 4, New York, 2002, Churchill Livingstone. 80. Burger PC, Scheithauer BW: AFIP Atlas of Tumor Pathology: Tumors of the Central Nervous System, 4th Series, Washington, D.C., 2007, American Registry of Pathology & Armed Forces Institute of Pathology. 81. McKeever PE, Blaivas M, Gebarski SS: Sellar tumors other than adenomas. In Thapar K, Kovacs K, Scheithauer BW, et al, editors: Diagnosis and Management of Pituitary Tumors, Totowa, NJ, 2001, Humana Press, pp 387–447. 82. Hayostek C, Shaw EG, Scheithauer BW, et al: Astrocytomas of the cerebellum: A comparative clinicopathologic study of pilocytic and diffuse astrocytomas. Cancer. 72:856–869, 1993. 83. Forsyth PA, Shaw EG, Scheithauer BW, et al: 51 cases of supratentorial pilocytic astrocytomas: A clinicopathologic, prognostic, and flow cytometric study. Cancer. 72:1335–1342, 1993. 84. Coakley KJ, Huston J, Scheithauer BW, et al: Pilocytic astrocytomas: Well-demarcated magnetic resonance appearance despite frequent infiltration histologically. Mayo Clin Proc. 70:747–751, 1995. 85. Clark GB, Henry JM, McKeever PE: Cerebral pilocytic astrocytoma. Cancer. 56:1128–1133, 1985. 86. Hitotsumatsu T, Iwaki T, Fukui M, et al: Distinctive immunohistochemical profiles of small heat shock proteins (heat shock protein 27 and alpha B-crystallin) in human brain tumors. Cancer. 77:352–361, 1996. 87. Camby I, Lefranc F, Titeca G, et al: Differential expression of S100 calcium-binding proteins characterizes distinct clinical entities in both WHO grade II and III astrocytic tumours. Neuropathol Applied Neurobiol. 26:76–90, 2000. 88. Biegel JA: Genetics of pediatric central nervous system tumors. J Pediatr Hematol Oncol. 19:492–501, 1997. 89. Rao RD, James CD: Altered molecular pathways in gliomas: An overview of clinically relevant issues. Semin Oncol. 31:595–604, 2004. 90. Bigner SH, Schrock E: Molecular cytogenetics of brain tumors. J Neuropathol Exp Neurol. 56:1173–1181, 1997. 91. Ho DM, Wong TT, Hsu CY, et al: MIB-1 labeling index in nonpilocytic astrocytoma of childhood: A study of 101 cases. Cancer. 82:2459–2466, 1998. 92. McKeever PE, Blaivas M, Nelson JS: Diagnosis of nervous system tumors by light microscopic methods. In Garcia JH, Budka H, McKeever PE, et al, editors: Neuropathology: The Diagnostic Approach, Philadelphia, 1997, CV Mosby, pp 193–218. 93. Kimura N, Watanabe M, Date F, et al: HMB-45 and tuberin in hamartomas associated with tuberous sclerosis. Mod Pathol. 10:952–959, 1997.
References 94. Levy RA, Allen R, McKeever P: Pleomorphic xanthoastrocytoma presenting with massive intracranial hemorrhage. AJNR. 17:154–156, 1996. 95. Gokden M, Roth KA, Carroll SL, et al: Clear cell neoplasms and pseudoneoplastic lesions of the central nervous system. Semin Diagn Pathol. 14:253–269, 1997. 96. Powell SZ, Yachnis AT, Rorke LB, et al: Divergent differentiation in pleomorphic xanthoastrocytoma: Evidence for a neuronal element and possible relationship to ganglion cell tumors. Am J Surg Pathol. 20:80–85, 1996. 97. Kaulich K, Blaschke B, Numann A, et al: Genetic alterations commonly found in diffusely infiltrating cerebral gliomas are rare or absent in pleomorphic xanthoastrocytomas. J Neuropathol Exp Neurol. 61:1092–1099, 2002. 98. Martinez-Diaz H, Kleinschmidt-DeMasters BK, Powell SZ, et al: Giant cell glioblastoma and pleomorphic xanthoastrocytoma show different immunohistochemical profiles for neuronal antigens and p53 but share reactivity for class III beta-tubulin. Arch Pathol Lab Med. 127:1187–1191, 2003. 99. Zhu Y, Guignard F, Zhao D, et al: Early inactivation of p53 tumor suppressor gene cooperating with NF1 loss induces malignant astrocytoma. Cancer Cell. 8:119–130, 2005. 100. Idbaih A, Boisselier B, Sanson M, et al: Tumor genomic profiling and TP53 germline mutation analysis of first-degree relative familial gliomas. Cancer Genet Cytogenet. 176:121–126, 2007. 101. Li YJ, Sanson M, Hoang-Xuan K, et al: Incidence of germ-line p53 mutations in patients with gliomas. Int J Cancer. 64:383– 387, 1995. 102. Broniscer A, Ke W, Fuller CE, et al: Second neoplasms in pediatric patients with primary central nervous system tumors. Cancer. 100:2246–2252, 2004. 103. Krutilkova V, Trkova M, Fleitz J, et al: Identification of five new families strengthens the link between childhood choroid plexus carcinoma and germline TP53 mutations. Eur J Cancer. 41:1597– 1603, 2005. 104. Rutherford J, Chu CE, Duddy PM, et al: Investigations on a clinically and functionally unusual and novel germline p53 mutation. Br J Cancer. 86:1592–1596, 2002. 105. Tokunaga A, Onda M, Matsukura N, et al: Li-Fraumeni syndrome. Nippon Rinsho. 53:2797–2802, 1995. 106. Figarella-Branger D, Gambarelli D, Dollo C, et al: Infratentorial ependymomas of childhood: Correlation between histologic features, immunohistological phenotype, silver nucleolar organizer region staining values and post-operative survival in 16 cases. Acta Neuropathol. 82:208–216, 1991. 107. Mason WP, Krol GS, DeAngelis LM: Low-grade oligodendroglioma responds to chemotherapy. Neurology. 46:203–207, 1996. 108. Yong WH, Chou D, Ueki K, et al: Chromosome 19q deletions in human gliomas overlap telomeric to D19S219 and may target a 425 kb region centromeric to D19S112. J Neuropathol Exp Neurol. 54:622–626, 1995. 109. Dehghani F, Schachenmayr W, Laun A, et al: Prognostic implication of histopathological, immunohistochemical and clinical features of oligodendrogliomas: A study of 89 cases. Acta Neuropathol. 95:493–504, 1998. 110. Coons SW, Johnson PC, Pearl DK: The prognostic significance of Ki-67 labeling indices for oligodendrogliomas. Neurosurgery. 41:878–884, 1997. 111. van den Bent MJ, Dubbink HJ, Marie Y, et al: IDH1 and IDH2 mutations are prognostic but not predictive for outcome in anaplastic oligodendroglial tumors: a report of the European Organization for Research and Treatment of Cancer Brain Tumor Group. Clin Cancer Res. 16(5):1597–1604, 2010. 112. Cairncross G, Berkey B, Shaw E, et al: Phase III trial of chemotherapy plus radiotherapy compared with radiotherapy alone for pure and mixed anaplastic oligodendroglioma: Intergroup RTOG 9402. J Clin Oncol. 24:2707–2714, 2006. 113. Vogelbaum MA, Berkey B, Peereboom D, et al: RTOG 0131: Phase II trial of pre-irradiation and concurrent temozolomide in patients with newly diagnosed anaplastic oligodendrogliomas and mixed anaplastic oligodendrogliomas: Relationship between 1p/19q status and progression-free survival. J Clin Oncol. 24:1517, 2006.
828.e3
114. Perry A, Fuller CE, Banerjee R, et al: Ancillary FISH analysis for 1p and 19q status: Preliminary observations in 287 gliomas and oligodendroglioma mimics. Front Biosci. 8:a1–a9, 2003. 115. Fuller CE, Schmidt RE, Roth KA, et al: Clinical utility of fluorescence in situ hybridization (FISH) in morphologically ambiguous gliomas with hybrid oligodendroglial/astrocytic features. J Neuropathol Exp Neurol. 62:1118–1128, 2003. 116. Takekawa Y, Sawada T: Vascular endothelial growth factor and neovascularization in astrocytic tumors. Pathol Int. 48:109–114, 1998. 117. Paulus W, Peiffer J: Intratumoral histologic heterogeneity of gliomas: A quantitative study. Cancer. 64:442–447, 1989. 118. Davenport RD, McKeever PE: Ploidy of endothelium in high grade astrocytomas. Anal Quant Cytol Histol. 9:25–29, 1987. 119. McKeever PE, Zhang K, Nelson JS, et al: Type IV collagen messenger RNA localizes within cells of abnormal vascular proliferations of glioblastoma and sarcomatous regions of gliosarcoma. J Histochem Cytochem. 41:1124, 1993. 120. McKeever PE, Davenport RD, Shakui P: Patterns of antigenic expression in cultured glioma cells. Crit Rev Neurobiol. 6:119– 147, 1991. 121. Horiguchi H, Hirose T, Kannuki S, et al: Gliosarcoma: An immunohistochemical, ultrastructural and fluorescence in situ hybridization study. Pathol Int. 48:595–602, 1998. 122. Boerman RH, Anderl K, Herath J, et al: The glial and mesenchymal elements of gliosarcomas share similar genetic alterations. J Neuropathol Exp Neurol. 55:973–981, 1996. 123. Schröck E, Thiel G, Lozanova T, et al: Comparative hybridization of human malignant gliomas reveals multiple amplification sites and nonrandom chromosomal gains and losses. Am J Pathol. 144:1203–1218, 1994. 124. McKeever PE, Dennis TR, Burgess AC, et al: Chromosomal breakpoint at 17q11.2 and insertion of DNA from three different chromosomes in a glioblastoma with exceptional glial fibrillary acidic protein expression. Cancer Genet Cytogenet. 87:41–47, 1996. 125. Liu L, Ichimura K, Pettersson EH, et al: Chromosome 7 rearrangements in glioblastomas: Loci adjacent to EGFR are independently amplified. J Neuropathol Exp Neurol. 57:1138–1145, 1998. 126. Hegi ME, Diserens AC, Gorlia T, et al: MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 352:997–1003, 2005. 127. Oberc-Greenwood MA, McKeever PE, Kornblith PL, et al: A human ganglioglioma containing paired helical filaments. Hum Pathol. 15:834–838, 1984. 128. Wirnsberg GH, Becker H, Ziervogel K, et al: Diagnostic immunohistochemistry of neuroblastic tumors. Am J Surg Pathol. 15:49–57, 1992. 129. Laeng RH, Scheithauer BW, Altermatt HJ: Anti-neuronal nuclear autoantibodies, types 1 and 2: Their utility in the study of tumors of the nervous system. Acta Neuropathol. 96:329–339, 1998. 130. Goldbart A, Cheng ZJ, Brittian KR, et al: Intermittent hypoxia induces time-dependent changes in the protein kinase B signaling pathway in the hippocampal CA1 region of the rat. Neurobiol Dis. 14:440–446, 2003. 131. Felix I, Bilbao JM, Asa SL, et al: Cerebral and cerebellar gangliocytomas: A morphological study of nine cases. Acta Neuropathol. 88:246–251, 1994. 132. Lindboe CF, Helseth E, Myhr G: Lhermitte-Duclos disease and giant meningioma as manifestations of Cowden’s disease. Clin Neuropathol. 14:327–330, 1995. 133. Daumas-Duport C, Varlet P, Bacha S, et al: Dysembryoplastic neuroepithelial tumors: Nonspecific histological forms—a study of 40 cases. J Neurooncol. 41:267–280, 1999. 134. Wolf HK, Buslei R, Blumcke I, et al: Neural antigens in oligodendro and dysembryoplastic neuroepithelial tumors. Acta Neuropathol. 94:436–443, 1997. 135. Guo SP, Zhang F, Li QL, et al: Papillary glioneuronal tumor— Contribution to a new tumor entity and literature review. Clin Neuropathol. 27:72–77, 2008. 136. Gelpi E, Preusser M, Czech T, et al: Papillary glioneuronal tumor. Neuropathology. 27:468–473, 2007.
828.e4
Immunohistology of the Nervous System
137. Pimentel J, Resende M, Vaz A, et al: Rosette-forming glioneuronal tumor: Pathology case report. Neurosurgery. 62:E1162– E1163, 2008. 138. Rodriguez FJ, Scheithauer BW, Robbins PD, et al: Ependymomas with neuronal differentiation: A morphologic and immunohistochemical spectrum. Acta Neuropathol. 113:313–324, 2007. 139. Perry A, Scheithauer BW, Macaulay RJ, et al: Oligodendrogliomas with neurocytic differentiation. A report of 4 cases with diagnostic and histogenetic implications. J Neuropathol Expl Neurol. 61:947–955, 2002. 140. Rout P, Santosh V, Mahadevan A, et al: Desmoplastic infantile ganglioglioma—Clinicopathological and immunohistochemical study of four cases. Childs Nerv Syst. 18:463–467, 2002. 141. Figarella-Branger D, Pellissier JF, Daumas-Duport C, et al: Central neurocytomas: Critical evaluation of a small-cell neuronal tumor. Am J Surg Pathol. 16:97–109, 1992. 142. Tong CY, Ng HK, Pang JC, et al: Central neurocytomas are genetically distinct from oligodendrogliomas and neuroblastomas. Histopathology. 37:160–165, 2000. 143. Albrecht S, Rouah E, Becker LE, et al: Transthyretin immunoreactivity in choroid plexus neoplasms and brain metastases. Mod Pathol. 4:610–614, 1991. 144. Kubo S, Ogino S, Fukushima T, et al: Immunocytochemical detection of insulin-like growth factor II (IGF-II) in choroid plexus papilloma: A possible marker for differential diagnosis. Clin Neuropathol. 18:74–79, 1999. 145. Kepes JJ, Collins J: Choroid plexus epithelium (normal and neoplastic) expresses synaptophysin: A potentially useful aid in differentiating carcinoma of the choroid plexus from metastatic papillary carcinomas. J Neuropathol Exp Neurol. 58:398–401, 1999. 146. Varga Z, Vajtai I: Prognostic markers in the histopathological diagnosis of tumors of the choroid plexus. Orv Hetil. 139:761– 765, 1998. 147. Kamaly-Asl ID, Shams N, Taylor MD: Genetics of choroid plexus tumors. Neurosurg Focus. 20(1):E10, 2006. 148. Mena H, Rushing EJ, Ribas JL, et al: Tumors of pineal parenchymal cells: A correlation of histological features, including nucleolar organizer regions, with survival in 35 cases. Hum Pathol. 26:20–30, 1995. 149. Hasselblatt M, Blumcke I, Jeibmann A, et al: Immunohistochemical profile and chromosomal imbalances in papillary tumours of the pineal region. Neuropathol Appl Neurobiol. 32:278–283, 2006. 150. Buffenoir K, Rigoard P, Wager M, et al: Papillary tumor of the pineal region in a child: case report and review of the literature. Childs Nerv Syst. 24:379–384, 2008. 151. Roberts RO, Lynch CF, Jones MP, et al: Medulloblastoma: A population-based study of 532 cases. J Neuropathol Exp Neurol. 50:134–144, 1991. 152. Gilhuis HJ, Anderl KL, Boerman RH, et al: Comparative genomic hybridization of medulloblastomas and clinical relevance: Eleven new cases and a review of the literature. Clin Neurol Neurosurg. 102:203–209, 2000. 153. Tong CY, Hui AB, Yin XL, et al: Detection of oncogene amplifications in medulloblastomas by comparative genomic hybridization and array-based comparative genomic hybridization. J Neurosurg Spine. 100:187–193, 2004. 154. Goldberg-Stern H, Gadoth N, Stern S, et al: The prognostic significance of glial fibrillary acidic protein staining in medulloblastoma. Cancer. 68:568–573, 1991. 155. Katsetos CD, Herman MM, Frankfurter A, et al: Cerebellar desmoplastic medulloblastomas: A further immunohistochemical characterization of the reticulin-free pale islands. Arch Pathol Lab Med. 113:1019–1029, 1989. 156. Freyer DR, Hutchinson RJ, McKeever PE: Primary primitive neuroectodermal tumor of the spinal cord associated with neural tube defect. Pediatr Neurosci. 15:181–187, 1989. 157. Mobley BC, Roulston D, Shah GV, et al: Peripheral PNET/Ewing sarcoma in the craniospinal vault: Case report and review. Hum Pathol. 37:845–853, 2005. 158. Burnett ME, White EC, Sih S: Chromosome arm 17p deletion analysis reveals molecular genetic heterogeneity in supratentorial and infratentorial primitive neuroectodermal tumors of the central nervous system. Cancer Genet Cytogenet. 97:25–31, 1997.
159. Rorke LB, Packer R, Biegel J: Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood. J Neurooncol. 24:21–28, 1995. 160. Biegel JA: Molecular genetics of atypical teratoid/rhabdoid tumor. Neurosurg Focus. 20:E11, 2006. 161. Nishihira Y, Tan CF, Hirato J, et al. A case of congenital supratentorial tumor: Atypical teratoid/rhabdoid tumor or primitive neuroectodermal tumor? Neuropathol. 27:551–555, 2007. 162. Khoddami M, Becker LE: Immunohistochemistry of medulloepithelioma and neural tube. Pediatr Pathol Lab Med. 17:913– 925, 1997. 163. Mork SJ, Rubinstein LJ: Ependymoblastoma: A reappraisal of a rare embryonal tumor. Cancer. 55:1536–1542, 1985. 164. Salvati M, Artico M, Lunardi P, et al: Intramedullary meningioma: Case report and review of the literature. Surg Neurol. 37:42–45, 1992. 165. Meis JM, Ordonez NG, Bruner JM: Meningiomas: An immunohistochemical study of 50 cases. Arch Pathol Lab Med. 110:934– 937, 1986. 166. Perry A, Scheithauer BW, Nascimento AG: The immunophenotypic spectrum of meningeal hemangiopericytoma: A comparison with fibrous meningioma and solitary fibrous tumor of meninges. Am J Surg Pathol. 21:1354–1360, 1997. 167. Carneiro SS, Scheithauer BW, Nascimento AG, et al: Solitary fibrous tumor of the meninges: A lesion distinct from fibrous meningioma: A clinicopathologic and immunohistochemical study. Am J Clin Pathol. 106:217–224, 1996. 168. Kepes JJ: Meningiomas: biology, pathology, and differential diagnosis, Chicago, 1982, Year Book Medical. 169. Lattes R, Bigotti G: Lipoblastic meningioma: “Vacuolated meningioma.” Hum Pathol. 22:164–171, 1991. 170. Ito H, Kawano N, Yada K, et al: Meningiomas differentiating to arachnoid trabecular cells: A proposal for histological subtype “arachnoid trabecular cell meningioma.” Acta Neuropathol. 82:327–330, 1991. 171. Kulah A, Ilcayto R, Fiskeci C: Cystic meningiomas. Acta Neurochir (Wien). 111:108–113, 1991. 172. Winek RR, Scheithauer BW, Wick MR: Meningioma, meningeal hemangiopericytoma (angioblastic meningioma), peripheral hemangiopericytoma, and acoustic schwannoma: A comparative immunohistochemical study. Am J Surg Pathol. 13:251–261, 1989. 173. Kobata H, Kondo A, Iwasaki K, et al: Chordoid meningioma in a child. J Neurosurg. 88:319–323, 1998. 174. Probst-Cousin S, Villagran-Lillo R, Lahl R, et al: Secretory meningioma: Clinical, histologic, and immunohistochemical findings in 31 cases. Cancer. 79:2003–2015, 1997. 175. Alameda F, Lloreta J, Ferrer MD, et al: Clear cell meningioma of the lumbo-sacral spine with chordoid features. Ultrastruct Pathol. 23:51–58, 1999. 176. Cerdá-Nicolás M, López-Ginés C, Peydró-Olaya A, et al: Histologic and cytogenetic patterns in benign, atypical, and malignant meningiomas: Does correlation with recurrence exist? Int J Surg Pathol. 2:301–310, 1995. 177. Perry A, Scheithauer BW, Stafford SL, et al: “Rhabdoid” meningioma: An aggressive variant. Am J Surg Pathol. 22:1482–1490, 1998. 178. Kobayashi S, Haba R, Hirakawa E, et al: Cytology and immunohistochemistry of anaplastic meningiomas in squash preparations: A report of two cases. Acta Cytol. 39:118–124, 1995. 179. Perry A, Scheithauer BW, Stafford SL, et al: “Malignancy” in meningiomas: A clinicopathologic study of 116 patients, with grading implications. Cancer. 85:2046–2056, 1999. 180. Prayson RA: Malignant meningioma: A clinicopathologic study of 23 patients including MIB-1 and p53 immunohistochemistry. Am J Clin Pathol. 105:719–726, 1996. 181. Hatiboglu MA, Cosar M, Iplikcioglu AC, et al: Sex steroid and epidermal growth factor profile of giant meningiomas associated with pregnancy. Surg Neurol. 69:356–362, 2008. 182. Wolfsberger S, Doostkam S, Boecher-Schwarz HG, et al: Progesterone-receptor index in meningiomas: Correlation with clinico-pathological parameters and review of the literature. Neurosurg Rev. 27:238–245, 2004. 183. Buccoliero AM, Gheri CF, Castiglione F, et al: Merlin expression in secretory meningiomas: Evidence of an NF2-independent
References pathogenesis? Immunohistochemical study. Appl Immunohistochem Mol Morphol. 15:353–357, 2007. 184. Hanemann CO: Magic but treatable? Tumours due to loss of merlin. Brain. 131:606–615, 2008. 185. Begnami MD, Rushing EJ, Santi M, et al: Evaluation of NF2 gene deletion in pediatric meningiomas using chromogenic in situ hybridization. Int J Surg Pathol. 15:110–115, 2007. 186. Fletcher CD, Unni KK, Mertens F, editors: World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of Soft Tissue and Bone, Lyon, 2002, IARC Press. 187. Probst-Cousin S, Rickert CH, Gullotta F: Factor XIIIaimmunoreactivity in tumors of the central nervous system. Clin Neuropathol. 17:79–84, 1998. 188. Tihan T, Viglione M, Rosenblum MK, et al: Solitary fibrous tumors in the central nervous system: A clinicopathologic review of 18 cases and comparison to meningeal hemangiopericytomas. Arch Pathol Lab Med. 127:432–439, 2003. 189. Miettinen MM, el-Rifai W, Sarlomo-Rikala M, et al: Tumor sizerelated DNA copy number changes occur in solitary fibrous tumors but not in hemangiopericytomas. Mod Pathol. 10:1194– 1200, 1997. 190. Persson S, Kindblom LG, Angervall L: Classical and chondroid chordoma: A light-microscopic, histochemical, ultrastructural and immunohistochemical analysis of the various cell types. Pathol Res Pract. 187:828–838, 1991. 191. Paulus W, Slowik F, Jellinger K: Primary intracranial sarcomas: Histopathological features of 19 cases. Histopathology. 18:395– 402, 1991. 192. Powell HC, Marshall LF, Igneizi RJ: Post-irradiation pituitary sarcoma. Acta Neuropathol. 39:165–167, 1977. 193. McKeever PE, Blaivas M, Sima AAF: Neoplasms of the sellar region. In Lloyd RV, editor: Surgical Pathology of the Pituitary Gland, Philadelphia, 1993, Saunders, pp 141–210. 194. Ariza A, Kim JH: Kaposi’s sarcoma of the dura mater. Hum Pathol. 19:1461–1463, 1988. 195. Rushing EJ, Mena H, Smirniotopoulos JG: Mesenchymal chondrosarcoma of the cauda equina. Clin Neuropathol. 14:150–153, 1995. 196. Matsukado Y, Yokota A, Marubayashi T: Rhabdomyosarcoma of the brain. J Neurosurg. 43:215–221, 1975. 197. Scheithauer BW, Woodruff JM, Erlandson RA: Tumors of the Peripheral Nervous System, Washington, DC, 1999, American Registry of Pathology. 198. Geddes JF, Sutcliffe JC, King TT: Mixed cranial nerve tumors in neurofibromatosis type 2. Clin Neuropathol. 14:310–313, 1995. 199. Hayashi K, Hoshida Y, Horie Y, et al: Immunohistochemical study on the distribution of alpha and beta subunits of S-100 protein in brain tumors. Acta Neuropathol. 81:657–663, 1991. 200. Warren C, James LA, Ramsden R, et al: Identification of recurrent regions of chromosome loss and gain in vestibular schwannomas using comparative genomic hybridisation. J Med Genet. 40:802–806, 2003. 201. Thaxton C, Lopera J, Bott M, et al: Neuregulin and laminin stimulate phosphorylation of the NF2 tumor suppressor in Schwann cells by distinct protein kinase A and p21-activated kinase-dependent pathways. Oncogene. 27:2705–2715, 2008. 202. Flaiz C, Utermark T, Parkinson DB, et al: Impaired intercellular adhesion and immature adherens junctions in merlin-deficient human primary schwannoma cells. GLIA. 56:506–515, 2008. 203. von Deimling A, Krone W, Menon AG: Neurofibromatosis type 1: Pathology, clinical features and molecular genetics. Brain Pathol. 5:153–162, 1995. 204. Storlazzi CT, Von Steyern FV, Domanski HA, et al: Biallelic somatic inactivation of the NF1 gene through chromosomal translocations in a sporadic neurofibroma. Int J Cancer. 117:1055–1057, 2005. 205. Valdez R, McKeever P, Finn WG, et al: Composite germ cell tumor and B-cell non-Hodgkin’s lymphoma arising in the sella turcica. Hum Pathol. 33:1044–1047, 2002. 206. Nakagawa Y, Perentes E, Ross GW, et al: Immunohistochemical differences between intracranial germinomas and their gonadal equivalents: An immunoperoxidase study of germ cell tumours with epithelial membrane antigen, cytokeratin, and vimentin. J Pathol. 156:67–72, 1988.
828.e5
207. Garvin AJ, Spicer SS, McKeever PE: The cytochemical demonstration of intracellular immunoglobulin: In neoplasms of lymphoreticular tissue. Am J Pathol. 82:457–478, 1976. 208. Jaffee ES, Harris NL, Stein H, et al, editors: World Health Organization Classification Of Tumors: Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues, Lyon, 2001, IARC Press. 209. Lai R, Rosenblum MK, DeAngelis LM: Primary CNS lymphoma: A whole-brain disease? Neurology. 59:1557–1562, 2002. 210. Davenport RD, O’Donnell LJ, Schnitzer B, et al: Non-Hodgkin’s lymphoma of the brain following Hodgkin’s disease: An immunohistochemical study. Cancer. 67:440–443, 1991. 211. Morgello S: Pathogenesis and classification of primary central nervous system lymphoma: An update. Brain Pathol. 5:383–393, 1995. 212. Carbone A: AIDS-related non-Hodgkin’s lymphomas: From pathology and molecular pathogenesis to treatment. Hum Pathol. 33:392–404, 2002. 213. Kadan-Lottick NS, Skluzacek MC, Gurney JG: Decreasing incidence rates of primary central nervous system lymphoma. Cancer. 95:193–202, 2002. 214. Ferracini R, Bergmann M, Pileri S, et al: Primary T-cell lymphoma of the central nervous system. Clin Neuropathol. 14:125– 129, 1995. 215. An-Foraker SH: Cytodiagnosis of malignant lesions in cerebrospinal fluid. Review and cytohistologic correlation. Acta Cytol. 29:286–290, 1985. 216. Deodhare SS, Ang LC, Bilbao JM: Isolated intracranial involvement in Rosai-Dorfman disease: A report of two cases and review of the literature. Arch Pathol Lab Med. 122:161–165, 1998. 217. Adle-Biassette H, Chetritt J, Bergemer-Fouquet AM, et al: Pathology of the central nervous system in Chester-Erdheim disease: Report of three cases. J Neuropathol Exp Neurol. 56:1207–1216, 1997. 218. McKeever P, Lloyd RV: Tumors of the pituitary region. In Garcia JH, Budka H, McKeever PE, editors: Neuropathology: The Diagnostic Approach, Philadelphia, 1997, CV Mosby, pp 219–262. 219. Tachibana O, Yamashima T, Yamashita J: Immunohistochemical study of erythropoietin in cerebellar hemangioblastomas associated with secondary polycythemia. Neurosurgery. 28:24–26, 1991. 220. Feldenzer JA, McKeever PE: Selective localization of gammaenolase in stromal cells of cerebellar hemangioblastomas. Acta Neuropathol. 72:281–285, 1987. 221. Rubio A, Meyers SP, Powers JM, et al: Hemangioblastoma of the optic nerve. Hum Pathol. 25:1249–1251, 1994. 222. McComb RD, Eastman PJ, Hahn FJ, et al: Cerebellar hemangioblastoma with prominent stromal astrocytosis: Diagnostic and histogenetic considerations. Clin Neuropathol. 6:149–154, 1987. 223. Silverstein AM, Quint DJ, McKeever PE: Intraductal paraganglioma of the thoracic spine. Am J Neuroradiol. 11:614–616, 1990. 224. Lloyd RV: Immunohistochemical localization of chromogranin in polypeptide hormone producing cells and tumors. In Lechago J, Kameya T, editors: Endocrine Pathology Update, Philadelphia, 1990, Field and Wood. 225. Abbott MA, Nathanson KL, Nightingale S, et al: The von Hippel-Lindau (VHL) germline mutation V84L manifests as early-onset bilateral pheochromocytoma. Am J Med Genet A. 140:685–690, 2006. 226. Vortmeyer AO, Huang SC, Koch CA, et al: Somatic von HippelLindau gene mutations detected in sporadic endolymphatic sac tumors. Cancer Res. 60:5963–5965, 2000. 227. Lin F, Shi J, Liu H, et al: Immunohistochemical detection of the von Hippel-Lindau gene product (pVHL) in human tissues and tumors: A useful marker for metastatic renal cell carcinoma and clear cell carcinoma of the ovary and uterus. Am J Clin Pathol. 129:592–605, 2008. 228. Xin W, Rubin MA, McKeever PE: Differential expression of cytokeratins 8 and 20 distinguishes craniopharyngioma from Rathke cleft cyst. Arch Pathol Lab Med. 126:1174–1178, 2002. 229. McKeever PE, Spicer SS: Pituitary histochemistry. In Spicer SS, editor: Histochemistry in Pathologic Diagnosis, New York, 1987, Marcel-Dekker, pp 603–645.
828.e6
Immunohistology of the Nervous System
230. Wong SW, Ducker TB, Powers JM: Fulminating parapontine epidermoid carcinoma in a four-year-old boy. Cancer. 37:1525– 1531, 1976. 231. Weller M, Stevens A, Sommer N, et al: Tumor cell dissemination triggers an intrathecal immune response in neoplastic meningitis. Cancer. 69:1475–1480, 1992. 232. Bigner SH, Johnston WW: The cytopathology of cerebrospinal fluid. II: Metastatic cancer, meningeal carcinomatosis and primary central nervous system neoplasms. Acta Cytol. 25:461–479, 1981. 233. DeYoung BR, Wick MR: Immunohistologic evaluation of metastatic carcinomas of unknown origin: An algorithmic approach. Semin Diagn Pathol. 17:184–193, 2000. 234. Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases. Med Pathol. 13:962–972, 2000. 235. Srodon M, Westra WH: Immunohistochemical staining for thyroid transcription factor-1: A helpful aid in discerning primary site of tumor origin in patients with brain metastases. Hum Pathol. 33:642–645, 2002. 236. Cochran AJ, Wen DR: S-100 protein as a marker for melanocytic and other tumors. Pathology. 17:340–345, 1985. 237. Rushing EJ, Mena J, Ribas JL: Primary pineal parenchymal lesions: A review of 53 cases. J Neuropathol Exp Neurol. 50:364, 1991. 238. Coca S, Martinez A, Vaquero J, et al: Immunohistochemical study of intracranial cysts. Histol Histopathol. 8:651–654, 1993. 239. Ho KL, Garcia JH: Colloid cysts of the third ventricle: Ultrastructural features are compatible with endodermal derivation. Acta Neuropathol. 83:605–612, 1992. 240. McKeever PE, Brissie NT: Scanning electron microscopy of neoplasms removed at surgery: surface topography and comparison of meningioma, colloid cyst, ependymoma, pituitary adenoma, schwannoma and astrocytoma. J Neuropathol Exp Neurol. 36:875–896, 1977. 241. McKeever PE, Hall BJ, Spicer SS: The origin of colloid cysts of the third ventricle. J Neuropathol Exp Neurol. 37:658, 1978. 242. Bejjani GK, Wright DC, Schessel D, et al: Endodermal cysts of the posterior fossa: Report of three cases and review of the literature. J Neurosurg. 89:326–335, 1998. 243. Go KG, Blankenstein MA, Vroom TM, et al: Progesterone receptors in arachnoid cysts: An immunocytochemical study in 2 cases. Acta Neurochirurg. 139:349–354, 1997. 244. Mirra SS, Heyman A, McKeel D, et al: The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II: Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology. 41:479–486, 1991. 245. Mirra SS, Hart MN, Terry RD: Making the diagnosis of Alzheimer’s disease. A primer for practicing pathologists. Arch Patho Lab Med. 117:132–144, 1993. 246. National Institute on Aging: Reagan Institute Working Group on diagnostic criteria of the neuropathological assessment of Alzheimer’s disease: Consensus recommendations for the postmortem diagnosis of Alzheimer’s disease. Neurobiol Aging. 18:S1–S2, 1997. 247. Feany MB, Dickson DW: Neurodegenerative disorders with extensive tau pathology: A comparative study and review. Ann Neurol. 40:139–148, 1996. 248. Dwork AJ, Liu D, Kaufman MA, et al: Archival, formalin-fixed tissue: Its use in the study of Alzheimer’s type changes. Clin Neuropathol. 17:45–49, 1998.
249. Lue LF, Brachova L, Civin WH, et al: Inflammation, A beta deposition, and neurofibrillary tangle formation as correlates of Alzheimer’s disease neurodegeneration. J Neuropathol Exp Neurol. 55:1083–1088, 1996. 250. Jellinger KA: Vascular-ischemic dementia: An update. J Neurol Transm Suppl. 62:1–23, 2002. 251. McKeith IG, Ballard CG, Perry RH, et al: Prospective validation of consensus criteria for the diagnosis of dementia with Lewy bodies. Neurology. 54:1050–1058, 2000. 252. Braak H, Del Tredici K, Rub U, et al: Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging. 24:197– 211, 2003. 253. McKahann GM, Albert MS, Grossman M, et al: Clinical and pathological diagnosis of frontotemporal dementia: report of the Work Group on Frontotemporal Dementia and Pick’s Disease. Arch Neurol. 58:1803–1809, 2001. 254. Wanschitz J, Hainfellner JA, Simonitsch I, et al: Non-HTLV-I associated pleomorphic T-cell lymphoma of the brain mimicking post-vaccinal acute inflammatory demyelination. Neuropathol Appl Neurobiol. 23:43–49, 1997. 255. Tachikawa N, Goto M, Hoshino Y, et al: Detection of Toxoplasma gondii, Epstein-Barr virus, and JC virus DNAs in the cerebrospinal fluid in acquired immunodeficiency syndrome patients with focal central nervous system complications. Intern Med. 38:556–562, 1999. 256. Zagzag D, Miller DC, Kleinman GM, et al: Demyelinating disease versus tumor in surgical neuropathology: Clues to a correct pathologic diagnosis. Am J Surg Pathol. 17:537–545, 1993. 257. Reith KG, Di Chiro G, Cromwell LD, et al: Primary demyelinating disease simulating glioma of the corpus callosum. J Neurosurg. 55:620–624, 1981. 258. Schmidt B, Toyka KV, Kiefer R, et al: Inflammatory infiltrates in sural nerve biopsies in Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy. Muscle Nerve. 19:474– 487, 1996. 259. Matsumuro K, Izumo S, Umehara F, et al: Chronic inflammatory demyelinating polyneuropathy: Histological and immunopathological studies on biopsied sural nerves. J Neurol Sci. 127:170– 178, 1994. 260. Babb TL, Brown WJ: Pathological findings in epilepsy. In Engel J, editor: Surgical Treatment of the Epilepsies, New York, 1987, Raven Press, pp 511–540. 261. Bruton CJ: The Neuropathology of temporal lobe epilepsy. In Russel G, Marley E, Williams P, editors: Maudsley Monographs. No. 31, London, 1988, Oxford Press, pp 1–94. 262. Prayson RA, Frater JL: Rasmussen encephalitis: A clinicopathologic and immunohistochemical study of seven patients. Am J Clin Pathol. 117:776–782, 2002. 263. Frater JL, Prayson RA, Morris III HH, et al: Surgical pathologic findings of extratemporal-based intractable epilepsy: A study of 133 consecutive reactions. Arch Pathol Lab Med. 124:545–549, 2000. 264. Volk EE, Prayson RA: Hamartomas in the setting of chronic epilepsy: A clinicopathologic study of 13 cases. Hum Pathol. 28:227–232, 1997. 265. Smith DF, Hutton JL, Sandemann D, et al: The prognosis of primary intracerebral tumours presenting with epilepsy: The outcome of medical and surgical management. J Neurol Neurosurg Psychiatry. 54:915–920, 1991.
C H A P T E R 2 1
IMMUNOCYTOLOGY SARA E. MONACO, DAVID J. DABBS
Overview 829 Use of Immunocytology 829 Immunocytology Techniques 829 Interpretation and Limitations of Immunocytology 836 Applications of Immunocytology 837 Theranostic Applications of Immunocytology 851 Tumor of Unknown Primary 852 Summary 853
Overview Immunocytology, or immunocytochemistry (ICC), involves the use of ICC in diagnostic cytopathology. The application of immunostains in cytologic cases has expanded and improved our ability to make definitive and accurate diagnoses beyond prior special stains. As seen in surgical pathology, a great deal of progress and expansion has occurred over the years in immunocytology, which has led to an increased number of available antibodies and subsequently an increased use of antibodies in cytopathology.1-4
Use of Immunocytology The many roles of immunostains in cytopathology are summarized in Box 21-1. These include the ability to determine the cell type, such as differentiating lymphoid and epithelial cells by using CD45 (leukocyte common antigen [LCA]) and cytokeratin, respectively. In addition, immunostains help provide site-specific markers to determine the tissue of origin in a metastatic carcinoma and help to subclassify poorly differentiated malignancies.1-7 Immunostains for infectious etiologies also exist and can help determine the type of bacterial, fungal, viral, or other infection.8 Immunostains such as Ki-67 also help to grade certain malignancies, particularly neuroendocrine and mesenchymal tumors. Finally,
an increasing role and greater interest in using immunostains to provide predictive and prognostic information for patients has emerged, and these roles will be addressed in greater detail in this chapter.
Immunocytology Techniques Specimen Collection Both aspiration and exfoliative specimens can be used for ICC by utilizing a variety of cell preparations that include cytospins, smears or imprints, liquid-based cytology specimens (e.g. ThinPrep [Hologic, Bedford, MA] specimens), and cell blocks (Figs. 21-1 and 21-2).9-11 The initial cytomorphologic evaluation helps to determine the need for ICC and other ancillary studies to reach a definitive diagnosis and exclude other entities in the differential diagnosis. Once the conventional Romanowsky or Papanicolaou stains have been examined, a differential diagnosis is generated, and the specimen is appropriately triaged; this procedure includes selection of the optimal cell preparation for immunostains and other ancillary studies, which can help answer important questions and support the diagnosis. Cytospins, smears, and imprint cytology slides may be air-dried or alcohol-fixed before staining (see Fig. 21-2).9-11 The most important feature is a thin, even layer of cells with minimal obscuring factors to avoid problems interpreting the stain results. Direct smears are helpful for ICC when no other material is available; however, the background artifacts can obscure interpretation of the immunostains, and limited material is available for a panel of markers.12 Cytospins are also helpful when there is no cell block or other material available, and given the limited material needed, several cytospin slides could be generated from a small sample for a panel of immunostains. In all of these specimens, prior staining or alcohol fixation may affect the results, and this should be considered.8 Liquid-based cytology or thin-layer techniques can also be used for ICC and have achieved good immunostaining results by using the proprietary solutions CytoLyt and PreservCyt (Cytyc, Marlboro, MA) with or without processing on the ThinPrep processor.13-17 Even with long-term storage in PreservCyt, immunostaining is crisp, the background is cleaner than with direct smears, 829
830
Immunocytology
Box 21-1 USE OF IMMUNOCYTOLOGY IN CYTOPATHOLOGY • Determine cell type (e.g. lymphocyte vs. epithelial cell). • Determine tissue of origin by using site-specific markers, such as use of thyroid transcription factor 1 in metastatic carcinoma to support a pulmonary or thyroid origin. • Subclassify the type of infection by using immunostains for viruses, such as herpes simplex virus and cytomegalovirus, and so on. • Subclassify or subtype tumors (e.g., subclassify non–small cell carcinoma as adenocarcinoma or squamous cell carcinoma). • Grade malignancies, such as with Ki-67 in neuroendocrine and mesenchymal neoplasms. • Provide prognostic and predictive information (e.g., ERBB2 in breast carcinoma).
and immunoreactivity is stable.16 It is crucial that the immunostaining pattern is validated to the standard formalin protocol to ensure optimum results, and appropriate PreservCyt fixed controls should be used. A recent study showed that immunostaining for hormone receptors and ERBB2 on alcohol-fixed ThinPrep specimens was not affected by the fixation and correlated well with the results on formalin-fixed paraffinembedded (FFPE) tissue.17 However, some antibodies may be affected by the alcohol fixation.8,18 Cell blocks are all-purpose cellular material that can be used for special stains and for ICC (see Figs. 21-1 and 21-2). Similar to their paraffin-embedded histology specimen counterparts, cell blocks can withstand the processing protocols. In addition to the obvious advantage in studying the tissue architecture, additional tissue sections can be cut for ICC. Ten percent neutral phosphate-buffered formalin (NBF) is used for fixation of tissue fragments. Interpretation of the results and storage of the specimen are easier, and antigen preservation is unlimited; for these reasons, cell blocks are the superior method for immunohistochemistry (IHC)
Figure 21-1 Formalin-fixed, paraffin-embedded cell block. A cell block is similar to a tissue block in surgical pathology and can be used to generate numerous slides for immunocytochemistry.
ICC on aspirate smear
ICC on cell block
ICC on ThinPrep
ICC on cytospin
Figure 21-2 Examples of immunocytochemistry (ICC) on different cytologic preparations. Immunostaining has been performed with success on various cytology specimens, including aspirate smears, cytospins, ThinPrep or liquid-based cytology specimens, and cell block sections. A familiarity with the advantages and disadvantages of these preparations for ICC is important in cytopathology.
for cytologic specimens. Cell block specimens can be collected directly into formalin or an alternate solution (e.g., RPMI salt solution, CytoLyte, other preservative); treated with a commercial thrombin-plasma agent to organize a clot, or treated with HistoGel to create a pellet; and then fixed in 10% formalin and processed in a manner similar to that of small tissue biopsies. The main disadvantage of this method is lack of sufficient material for processing and lack of a sufficient number of the cells of interest within the block for immunostaining.8 A fully automated rapid cell block (RCB) system introduced by Cytyc (Cellient; Hologic, Bedford, MA) increases overall cellularity in the resulting sections with decreased time and a necessity for fewer reagents to make a cell block.19 A proprietary tissue cassette and filter assembly designed to capture tissue fragments also permits them to be positioned in a plane for microtomy (Fig. 21-3). Small aliquots of xylene, alcohol, and paraffin are rapidly drawn through the sample to produce a broad, uniform layer of cells embedded in paraffin. The RCB produces a cell block in 15 minutes from residual ThinPrep vials or other specimens and can be used for a variety of gynecologic and respiratory tissues, fine needle aspiration (FNA) biopsies, body fluids, and other materials. If formalin is not used as the primary fixative, the alternate fixative must be validated against the formalin-fixed specimen, and controls must be used that have been fixed in the alternate material. In addition, the cost of the equipment can be a limiting factor for some institutions. The use of cytoscrape cell blocks (SCBs) is another technique to prepare cell blocks, especially from stained FNA smears.20 This technique is useful when cell groups are obscured by clotted blood, or when overlapping cell clusters interfere with the cytologic details and make interpretation difficult. The method involves previously stained smears that contain thick material that are de-coverslipped in xylene. The slides are passed through two changes of absolute alcohol and water.
Immunocytology Techniques
A
B
C
D
831
Figure 21-3 A to D, Cellient automated cell block system. A, Tissue cassette with filter: sample collected on cassette. B, Tissue cassette with filter: sample embedded in paraffin. C, Additional layering of paraffin during processing in finishing station. D, Finishing station for easy sectioning.
Papanicolaou-stained smears are destained by 1% acid alcohol, whereas Romanowsky-stained smears are destained by 2% glacial acetic acid. Then the smears are thoroughly rinsed in running tap water for 2 hours. Slides are carefully scraped with a scalpel blade, and the scraped material is meticulously transferred with a forceps in 3% molten agar to form a small button. After the agar solidifies, it is wrapped in Whatman filter paper No. 1 and put in a tissue cassette. The scraped material is refixed in a histologic fixative, such as Bouin’s fluid or formal saline, for 5 to 6 hours and is routinely processed to make a paraffin wax block. Sections of 5 µm are cut and stained with hematoxylin and eosin. A study that compared SCBs with conventional cell blocks
found that cytomorphologic details are equally superior in both types of specimen samples. An added advantage with this method is that additional panels of immunostains can be performed on SCBs, particularly when repeat FNA is not feasible.20 Although cell blocks are commonly and routinely utilized in nongynecologic cytopathology, they can also be helpful in cervicovaginal cytology.21 The utility of cell blocks prepared from residual material from Pap tests has been reported to be helpful for morphologic evaluation and ICC and for molecular studies.21-23 Cell blocks can be useful for identifying a high-grade squamous intraepithelial lesion (HSIL) and glandular abnormalities in addition to differentiating between atrophy
832
Immunocytology
and metaplasia. The diagnostic sensitivity and specificity of cell blocks from the residual samples have been shown to range from 86% to 100%.22 Another important use of cell blocks is for performing ICC when the Pap test interpretation is equivocal. A few papers have demonstrated the performance of IHC stains on cell blocks. A study that looked at the utility of p16 on cell blocks obtained from the residual samples found that the sensitivity of this immunostain was as high as 85% in Pap tests with a diagnosis of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); another similar study that used p16 and MIB-1 (Ki-67) found the concordance of Pap test and cell block interpretation to be as high as 85%,21-23 thus it is important to recognize the growing importance of cell block preparations in cervicovaginal cytology. Some cytologic preparations are not suitable for ICC. This category includes filter preparations, because the filter can detach from the slide and absorb the immunologic reagents and the chromogen, leading to high background staining. In addition, fluids with abundant mucoid material, necrosis, blood, or high protein content may have a precipitate that prevents penetration of reagents, and thus these specimens may benefit from an additional washing step or from the use of an alternative preparation for ICC, such as a ThinPrep specimen that can help to minimize obscuring blood.
Fixation Some of the important prerequisites for optimal ICC results include well-prepared material—that is, a thin and uniform spread of cells with adequate fixation— minimal obscuring factors (e.g., necrosis, blood, mucus, and proteinaceous material), and a reproducible qualitycontrolled method of ICC.1-4 Wet fixation in alcohol (WFA) must be performed without delay, because air drying may result in distortion and false-positive results. An air-dried smear (ADS) is often more cellular than an alcohol-fixed slide, because some material often floats off the slide when it is alcohol fixed. Such airdried slides must be fixed immediately before performing ICC, and the types of preferred fixatives vary among cytopathologists and different institutions. Cold acetone, formalin, CytoLyt, and 95% alcohol are commonly used fixatives.5,6 Of all the specimens and fixatives, FFPE cell blocks provide an optimal specimen for ICC because of better specimen standardization, and thus they are commonly used in most institutions.8 Suthipintawong and Leong and colleagues9,12 advocated post fixation of air-dried smears after testing different fixatives. Although physiologic saline and 96% alcohol with rehydration in normal saline for 30 minutes are the best fixatives, the cytomorphology is crisper, and less background staining is seen with the latter. A study by Fulciniti and associates10 has shown that formalin postfixed air-dried smears are reliable and better than the standard wet-fixed smears. The study proposed that the slow dehydration and short rehydration might contribute to a superior interpretation of the results. Our experience showed similar results with preparations
that were relatively free of background blood and mucus.11 The antibodies used with postfixation are listed in Table 21-1. An important point to keep in mind is that certain antibodies—such as S-100 protein, HepPar-1, estrogen receptor (ER), and gross cystic disease fluid protein 15 (GCDFP-15)—may be leached from alcohol fixatives and render false-negative results.8,18 For instance, ER staining on FNA smears by using different methods has shown that destaining the slides with alcohol before immunocytostaining significantly reduces the number of cells with positive nuclear staining.18 The available anti-ER antibodies (SP1, ID5, 6F11) perform best with 10% NBF for hormone fixation, as do other immunostains. In addition, S-100 protein can show false-negative or decreased staining with alcohol fixation, as shown in the case of malignant melanoma involving the biliary tract in Figure 21-4. An example that illustrates the importance of fixation is the evaluation of breast carcinoma. ERs and progesterone receptors (PRs) and overexpression of ERBB2 are important prognostic and predictive markers of breast carcinoma. The 2010 American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) guideline recommendations for hormone receptor testing highlighted the importance of an accurate and reproducible assay with optimal tissue handling.24 The need for standardizing preanalytic variables is now well recognized, particularly tissue fixation, including the time to fixation (i.e., cold ischemia time), type of fixative, and duration of tissue fixation.25,26 Underfixation of tissue cannot be repaired and is the least desirable result of handling tissues in the anatomic pathology laboratory. No amount of antigen retrieval can resurrect a tissue that is underfixed. Antigens will be lost, and false negatives will abound in such situations, regardless of the quality of instrumentation. Overfixation, on the other hand, may result in a specimen needing changes in antigen retrieval, antibody titer, and detection systems.27,28 The optimal result is to have a standard fixation time, ideally one that is unique for every antigen the pathologist is attempting to detect. For example, as per the ASCO/CAP guidelines, the recommended minimum fixation in 10% NBF for hormone receptors is no less than 6 hours and no more than 72 hours.24 Routinely processed cytologic specimens fixed in formalin can also be used to assess the receptor status and HER2 protein by IHC.29
Standardization Issues The history of standardization attempts is long, and it has been well discussed by Clive Taylor, beginning with his transactions on the Biological Stain Commission and continuing into detailed discussions about the “total test concept” of the IHC tests (see also Chapter 1).30,31 The Biologic Stain Commission was one of the original agencies to oversee the special stains used in the anatomic pathology laboratory. The agencies that followed include the Clinical Laboratory Standards Institute (CLSI, previously NCCLS), the Food and Drug Administration (FDA), and commissions set up by professional
Immunocytology Techniques
833
TABLE 21-1 Antibodies Used with Postfixation Technique Antibody Type
Clone/Code
Producer
Dilution
Antigen Retrieval
CK7
NCL-CK7 OVTL
Novocastra
1 : 80
MW
CK20
NCL-CK20 543
Novocastra
1 : 100
MW
AE1/AE3
NCL-AE1-AE3
Novocastra
1 : 100
MW
EMA
NCL-EMA
Novocastra
1 : 400
None
Vimentin
NCL-VIM V9
Novocastra
1 : 100
MW
Smooth muscle actin
NCL-SMA
Novocastra
1 : 80
None
Desmin
NCL-DES-DER II
Novocastra
1 : 100
Trypsin
Neurofilaments
NCL-NF68-DA2
Novocastra
1 : 200
MW
Estrogen receptor
1D5
Dako
1 : 35
MW pH 9
Progesterone receptor
SP1
Ventana
1 : 50
MW
HER2
4B5
Ventana
1 : 80
MW pH 9
Ki-67
NCL-Ki-67-MM1
Novocastra
1 : 200
MW
Cyclin D1
P2D11F11
Novocastra
1 : 50
MW
E-cadherin
NCH-38
Dako
1 : 100
HCB
EGFR
E30
Dako
1 : 50
Proteinase K
CD3
PS1
Novocastra
1 : 200
MW
CD4
4B2
Novocastra
1 : 30
MW
CD5
4C7
Novocastra
1 : 50
MW
CD8
CD5/54/F6
Dako
1 : 50
MW pH 9
CD10
SS2/36
Dako
1 : 40
None
CD15
C3D-1
Dako
1 : 50
MW pH 9
CD20
NCL-CD20-L26
Novocastra
1 : 50
MW
CD30
Ber-H2
Dako
1 : 40
MW pH 9
CD45
X16-99
Novocastra
1 : 20
MW
CD56
CD564
Novocastra
1 : 100
MW
CD246
ALK1
Dako
1 : 50
MW pH 9
Myeloperoxidase
Polyclonal
Dako
1 : 500
MW
S-100
Polyclonal
Dako
1 : 400
MW
NSE
BBS/NC/VI-H14
Dako
1 : 250
MW
HMB-45
HMB-45
Novocastra
1 : 60
Trypsin
Calcitonin
M3509
Dako
1 : 80
None
mCEA
M7072
Dako
1 : 50
MW
Epithelial Markers
Mesenchymal Markers
Prognostic Markers
CD Markers
Neuroendocrine Markers
CK, Cytokeratin; EGFR, epidermal growth factor receptor; EMA, epithelial membrane antigen; HCB, hot citrate buffer bath treatment; HMB-45, human melanoma black 45; mCEA, monoclonal carcinoembryonic antigen; MW, microwave open treatment; NSE, neuronspecific enolase.
834
Immunocytology
A
B
C
D
Figure 21-4 A to D, Bile duct brushing with metastatic malignant melanoma. The ThinPrep and cell block show a dyscohesive malignant population with increased nuclear/cytoplasmic ratios, hyperchromasia, and binucleation (A and B, original magnification ×400; inset, original magnification ×600). Immunostains performed on residual material from the ThinPrep specimen revealed that the tumor cells only showed weak, focal staining for S-100 protein (C, original magnification ×400) but were strongly positive for human melanoma black 45 (HMB-45) (D, HMB-5 immunostain, original magnification ×400) and negative for cytokeratin and leukocyte common antigen. The decreased staining for S-100 may be due to fixation in the alcohol-based fixative.
organizations, including CAP. The emphasis of all these organizations has been on consistent, quality assay components for IHC. The consequence of all this work has resulted in the package inserts that accompany various IHC reagents. A study by members of the Ad Hoc Committee on Immunohistochemistry Standardization recommended formalin fixative as the standard for IHC testing, along with a minimum of 6 hours fixative time for prognostic/ predictive markers, including ER, PR, and ERBB2.24,30 These guidelines have been incorporated by the ASCO/ CAP guidelines for breast cancer hormone receptor testing24,32 and follow the simple fact that all clinically validated studies on ER, PR, and ERBB2 antibodies have been performed on FFPE tissue. Alcohol fixatives may result in spurious results for certain antibodies such as S-100, HepPar-1, hormone receptors, and ERBB2; thus alcohol fixative is not recommended for evaluation of these markers.8,18 Although alcohol fixatives can be used for other antibodies, it is imperative for the laboratory director to validate protocols and use appropriate
alcohol-based controls if alcohol fixation of cytologic specimens is used. FFPE cell blocks are the preferred samples for IHC. In our laboratory, we have successfully extended our standardization protocols for surgical specimens to cytology specimens.
Rehydration and Storage Air-dried slides and partially fixed air-dried slides can be rehydrated in normal saline to optimize immunoreactivity as well as cytomorphology.33-35 Chan and Kung35 found that the optimal time for rehydration is less than 1 minute, provided that the air-drying time did not exceed 30 minutes. This procedure may be used when cytomorphology is critically important, because air-dried slides can sit for up to 1 week at room temperature and still be used for ICC, provided they are fixed immediately before use as already described.12 Slides for ICC, whether air dried or fixed, can be stored at −70° C for at least 1 month and still maintain immunoreactivity.9
Immunocytology Techniques
Antigen Retrieval One of the important methods through which standardization of IHC can be achieved is antigen retrieval. An ideal antigen-retrieval technique is considered to maintain formalin as a standard fixative for both morphology and IHC.36 High-temperature heating is the most crucial step in this methodology to retrieve the antigens masked by formalin fixation. However, simple methods such as immersing in water or an NaOHmethanol solution will yield dramatic retrieval results.37 Although use of metal ions with zinc and lead in the antigen retrieval solutions has shown improved results in many studies,38 their environmental toxicity has given way to alternatives such as citrate, Tris, urea, and ethylenediamine tetraacetic acid (EDTA). Antigen retrieval has been widely used for detection of ER, PR, MIB-1, p53, Bcl-2, retinoblastoma gene (pRB), and some cluster designation (CD) markers. Although the literature on the subject of use of antigen retrieval on cytology specimens is still evolving, some important studies have successfully shown that antigen retrieval can be applied to these specimens for a wide range of antibodies.38 Results are satisfactory and are comparable to their tissue specimen counterparts. Sherman and colleagues39 have shown a method of “cell transfer” that can facilitate immunostaining on small samples. In a study that compared the effects of heat (with a pressure cooker) on air-dried versus alcohol-fixed smears for cytokeratins AE1/AE3 (5D3), 7, and 20; neurofibrillary protein (NFP); synaptophysin; ER (clone ID5) and PR (clone 1294); and vimentin, it was found that ADS results improved with antigen retrieval. Furthermore, alcohol-fixed smears can be successfully immunostained without antigen retrieval or with mild heat-induced antigen retrieval (HIER) by citrate buffer.40 Miller and associates41 found that FNA biopsy material studied through tissue transfer to adhesive slides followed by antigen retrieval using the same conditions as for routine paraffin sections substantially improved the ease of interpretation, especially in the air-dried smears.
Controls Positive and negative controls must be performed with each test sample. Tissue controls are typically used in most laboratories for convenience, but the ideal control should be a comparably fixed cytology sample.42 If tissue controls are used, the pathologist must exercise caution in interpretation, because a different set of artifacts is present in tissue compared with cytology samples. Cytologic controls can be obtained on a daily basis from the surgical pathology bench with the use of aspirates or direct imprints. It is more practical to use cells on the cytology slide as a positive or negative internal control, depending on the antibody and cells present. In a meta-analysis of the use of controls in cytopathology articles, only 13% of articles described the use of positive and negative controls run on identically prepared samples. This finding suggests that there should be more stringent documentation of
835
appropriate controls in cytopathology on par with that seen in surgical pathology.42
Specimens of Limited Quality ICC can be hampered by a limited quantity of the specimen, which happens more often in cytopathology than surgical pathology. Some of the ways to overcome limited specimens include appropriately triaging the material for the studies needed, cutting blank slides from a cell block up front to avoid trimming the block, use of alternate material if available, appropriate selection of the important studies needed, and performance of multiplex staining for ICC. For example, in scenarios of scant cellularity on a cell block, aspirate smears or other material may be utilized (see Fig. 21-2). In addition, dual immunolabeling may be performed on the same slide instead of on separate slides, or even more antibodies may be multiplexed on the same slide, which will maximize the immunomarkers that can be used on smaller amounts of material. Some of the ways to maximize the cytologic material available for ICC are summarized in Box 21-2. In one study, Abendroth and Dabbs11 described a double-labeling method to address the problem of limited material when more than one antibody is required to make a diagnosis. Immunostaining was performed on these slides with and without a preceding decolorization step, and the results were similar with good immunostain results. Background staining was more of a problem on the air-dried, unfixed cases. Thus recently stained or archived cytology slides can be used for ICC studies, which include commonly used
Box 21-2 WAYS TO MAXIMIZE TISSUE FOR IMMUNOSTAINING IN CYTOLOGY SPECIMENS • Educate those who obtain the biopsies about the importance of procuring sufficient tissue. • Select the optimal type of specimen (exfoliative or aspiration cytology). • Ensure rapid on-site assessment by cytologists for adequacy assessment and appropriate triage. • Select appropriate fixative and processing by avoiding certain fixatives for certain antibodies. • Acquire additional dedicated material for ancillary studies, such as dedicated passes for cell block. • Establish, validate, and standardize laboratory protocol for processing and evaluation of samples. • Cut extra blank slides up front in an effort to conserve tissue and triage the blank slides for the appropriate stains. • Limit immunoperoxidase staining panels. • Use alternative material in cases with insufficient tissue in the cell block (e.g., aspirate smear material). • Use alternative procedures in cases of scant material, such as dual immunostaining or multiplex immunostaining. • Optimize and assess specimen processing with appropriate quality assurance.
836
Immunocytology
antibodies such as CAM5.2, AE1/AE3, 34βE12, carcinoembryonic antigen (CEA), desmin, HHF-35 (muscle-specific actin), vimentin, CD20, and CD45RO.11 Dual staining has also been shown in other studies in which more than one antibody, typically a nuclear and cytoplasmic or membranous stain, is used on the same slide as opposed to separate slides.43 Cytologic material can also be used in a variation of the dual immunolabeling technique.11,44 Cytology slides that were subjected to an immunoperoxidase test and produced a negative result can also be subjected to another immunoperoxidase test by using a different antibody. It is imperative to use positive and negative controls with both test runs. For example, a poorly differentiated tumor found to be negative for LCA can then be tested with cytokeratin to determine whether it is an epithelial tumor. Antibodies that have been used with this method include CAM5.2, AE1/AE3, 34βE12, GCDFP-15, vimentin, CD20, CD45RO, muscle-specific actin (HHF-35), desmin, CEA, and S-100.44 Sherman and associates39 described the cell-transfer technique for use with limited cytologic samples. In this technique, cells are lifted off the slide by redissolving them in a new mounting medium. The medium can be removed from the slide, cut into pieces, and reapplied to slides for individual antibody studies. The results of immunostains are generally good, although Hunt and colleagues45 described some decreased staining with this method. In the future, multiplex staining with multiple antibodies on a single slide may help to minimize the tissue needed for ICC and maximize the results.
Interpretation and Limitations of Immunocytology As in surgical pathology, the morphologic study of the specimen in conjunction with the clinical and radiologic findings is important to arrive at an accurate diagnosis. Without correlation with the other findings, immunostaining alone cannot establish a final diagnosis. A plethora of antibodies are available for use in cytology, and none of them is specific for its intended target. Antibodies may be polyclonal or monoclonal, may vary in sensitivity and specificity, or may be dependent on certain types of fixative for proper immunoreactivity. In addition, the differentiation of many tumors exhibits a wide spectrum. For these reasons, it is important to use and to document the known positive and negative controls for different antibodies.42 In general, a negative result by itself is of lesser value than a positive result, unless the cytology specimen itself contains known positive and negative control cells. In addition, a panel of multiple antibodies is typically used that includes markers known to be positive and negative in the entity to maximize sensitivity and specificity. The pattern of immunostaining will depend on the presence of neoplastic cells, the location of staining (e.g., cell membrane, cytoplasm, or nucleus), proper fixation, background staining, and antibody concentration.
Immunostaining for any antigen is rarely uniform in nature. Heterogeneity of immunostaining is the rule, rather than the exception, and it is proper to state the pattern, cell localization, and distribution of positive and negative immunostaining relative to normal cells that may be present in the sample.2,3,8,12 False-positive immunostaining results can be a result of a multitude of factors that include interpretation errors and technical errors.8,12 First and foremost, the cytopathologist must not mistake normal or reactive cellular elements for neoplastic cells, especially in samples in which neoplastic cells are limited. Air drying of the slide during any step in the immunoperoxidase procedure results in nonspecific antibody binding that can be misinterpreted as positive findings. Necrotic, poorly preserved, and crushed cells must be avoided, because nonspecific binding may yield a false-positive result. Care must be taken not to overinterpret the cellular background that is generally seen with polyclonal antibodies. This nonspecific binding to stromal elements can be avoided by carefully assessing the patterns of staining of normal, neoplastic, and stromal elements. Antibodies may cross-react with nonlesional components, or the antibody used may not be as specific as advertised. Only experience and comfort with the antibody in use will satisfy the observer’s interpretation in these situations. Improper fixation and incomplete blocking of endogenous peroxidase or biotin activity are well-known sources of false-positive results. Proper blocking with commercial blocking products should eliminate this problem. Furthermore, the pathologist must avoid interpreting immunostains on the edge of the tissue section, in the setting of uneven staining with high background, and with folded or obscured tissue, all of which could lead to false positive results (Fig. 21-5). Finally, appropriate chromagens should be selected in pigmented tumors, such as malignant melanoma, because of the fact that the brown melanin pigment in the tumor may be misinterpreted as a positive result if a brown chromagen is used (see Fig. 21-4). This is the reason for the use of red chromagens in cases of pigmented tumors to avoid false-positive results. False-negative interpretations are also potentially multifactorial in nature. Improper fixation that results in denaturation or masking of antigen may completely escape the attention of the observer. The pathologist can eliminate this problem only by ascertaining the working dilution of each antibody and ensuring quality fixation. Antibody concentration is a critical determinant, because concentrations that are too low or too high can cause false-negative and false-positive results, respectively. Insufficient antigen retrieval or even decolorization of specimens can cause false-negative results. The type of fixation is critical for antibody performance. The best example of false-negative results occurs for certain antibodies when a sample is fixed in alcohol, and this result has been reported with antibodies to S-100 protein, GCDFP-15, and others (see Fig. 21-4).8,18 Additional issues in interpretation of ICC occurs with obscuring inflammation, necrosis, excess bacteria, crystalluria, hematuria, poor preservation of cells, and poor cellularity (<50 cells), which may cause the test to
Applications of Immunocytology
A
B
C
D
837
Figure 21-5 A to D, Technical artifacts and pitfalls in interpretation of immunocytochemistry include uneven staining and high background staining of red blood cells (A, original magnification ×400) and edge artifact, where the specimen on the edge shows darker, more nonspecific staining than the other cells (B, original magnification ×400). Furthermore, the cell block sections may have floaters or folded tissue obscuring the sample (C and D, original magnification ×400), which makes interpretation difficult.
fail or become uninterpretable. In addition, failure of the controls, high background staining, uneven staining, obscuring folded tissue or floaters, and other technical variables can also make the interpretation difficult (see Fig. 21-5). In these scenarios, analysis of the problem and repetition of the stains can be helpful.
Applications of Immunocytology Effusion Cytology Effusion cytology is one of the most challenging areas of cytopathology, wherein ICC serves as a valuable adjunct tool in definitive interpretation. Immunostains are critical to distinguish reactive mesothelial cells from
carcinomas that involve body cavity fluids, because mesothelial cells can show significant atypia that mimics adenocarcinomas and other malignancies. In the absence of these studies, the sensitivity for the detection of malignant cells has been reported to be as low as 40%.46 Normal fluid specimens contain mesothelial cells, which can exhibit a spectrum of cytomorphologic features that include changes that can mimic malignancy and features that can mimic bland-appearing neoplasms; this makes it difficult to recognize the foreign (i.e., neoplastic) population, thus ICC plays an important role in the identification of reactive mesothelial cells from adenocarcinoma. Several studies have highlighted and proposed panels of antibodies to resolve this issue. These studies indicate that combining the cytomorphologic evaluation with the results of a panel of antibodies
838
Immunocytology
that comprises both mesothelial and adenocarcinoma markers can significantly improve diagnostic accuracy.46-48 ICC can be performed on a variety of preparations from fluid specimens, such as cell blocks, direct smears, cytospins, and liquid-based cytology preparations (see Fig. 21-2). Storage of effusions at 4° C gives a satisfactory IHC outcome when a delay in processing of the samples is anticipated.49 In our experience, optimal results are achieved when ICC is performed on 10% NBF cell-block material, because the immunoreactivity pattern may be changed by different fixation methods.
Mesothelial Markers CALRETININ
A 29-kD calcium-binding protein that is a member of the elongation factor (EF) family of proteins, calretinin is thought to play a role in the cell cycle and is helpful in confirming cells of mesothelial origin, but it does not distinguish benign or reactive mesothelial cells from malignant mesothelial cells (i.e., mesothelioma; Fig. 21-6). Gotzos and colleagues50 were the first to report its expression by epithelioid mesotheliomas and epithelioid components of mixed mesotheliomas, and they reported negative results in adenocarcinoma. These results were later confirmed by studies that showed only rare focal positivity, primarily as a cytoplasmic blush, in a variety of carcinomas that included 10% to 30% of adenocarcinomas.51,52 The sensitivity of calretinin to distinguish reactive mesothelial cells from adenocarcinoma cells approaches 100%, and the specificity is as high as 80%. Interpretation of this marker is based on finding strong nuclear and cytoplasmic positivity in the cells of interest (see Fig. 21-6, C).53,54 Ordoñez55 demonstrated a difference in sensitivities of the human recombinant antibodies from Zymed and a guinea pig calretinin from Chemicon. The Zymed antibody sensitivity was 100% compared with a Chemicon sensitivity of 74%. Focal, weak staining of adenocarcinoma was seen in 4% and 9%, respectively. Nagel and colleagues56 had a similar experience with the study of cytospins. They found that the sensitivity for mesothelial cells was 93%, with 5% of tumor cells immunostaining. In a study by Yaziji and associates,57 calretinin was one of three antibodies, along with Bg8 and MOC-31, identified in the evaluation of a 12-antibody panel, which was cited to be the most efficient panel for distinguishing epithelioid mesothelioma from adenocarcinoma. HBME1
The mesothelial cell clone HBME1 is an antibody against cultured mesothelial cells and recognizes an antigen on the microvillus surface. HBME1 has been used as a part of the panel of ICC to distinguish adenocarcinoma from mesothelial cells. Interpretation of this stain is complex compared with calretinin. The staining pattern of mesothelial cells shows a thick, bushy membrane pattern and a thin membrane or cytoplasmic staining of adenocarcinoma. A gradient in the staining
pattern was observed in malignant mesothelioma versus adenocarcinoma. The specificity of HBME1 as a mesothelial marker approaches 80%.54 Calretinin tends to be a preferred marker over HBME1 because of its superior sensitivity and specificity in most studies. CYTOKERATIN 5/6
Keratin 5 was found to be a constituent of mesothelioma but not lung adenocarcinoma in a few early studies,58,59 which was confirmed later in larger series that reported only focal staining in less than 10% of adenocarcinomas.60,61 Diffuse cytoplasmic staining is seen in almost all mesothelial cells, including mesotheliomas, in addition to squamous cell carcinomas.61 The sensitivity and specificity of this stain in distinguishing malignant mesothelioma from adenocarcinoma in pleural effusions is 90% to 100%.61 However, this marker is of no value in differentiating mesothelial cells from metastatic pulmonary squamous cell carcinoma, because squamous cells are also positive for CK5/6. It is important to keep in mind that certain breast carcinomas, particularly tumors with a basal phenotype, express CK5/6 as well. Given these data, CK5/6 is only useful for differentiating epithelioid malignant mesothelioma and reactive mesothelial cells from adenocarcinoma. Mesothelial cells are also positive for CK7, however, this cytokeratin is also positive in many metastatic adenocarcinomas and is therefore not helpful in making the distinction between mesothelial cells and metastatic adenocarcinoma (see Fig. 21-6, E). WILMS TUMOR GENE 1
The Wilms tumor gene, WT1, is a tumor suppressor gene present on chromosome 11, and it is a helpful mesothelial marker in that it shows nuclear positivity (see Fig. 21-6, D). It was first reported as a candidate for the main gene in the development of Wilms tumor.62,63 The IHC expression of WT1 can be seen in a variety of neoplasms, such as serous tumors of the ovary, leukemia, desmoplastic small round cell tumors, and others.63 In addition, a majority of epithelioid mesotheliomas and a small percentage of sarcomatoid mesotheliomas express WT1, which represents an effective marker for mesotheliomas and reactive mesothelial cells in cell block preparations, and this can aid in the distinction of Wilms tumor from pulmonary adenocarcinoma. However, in recent years, WT1 has emerged as a highly sensitive and specific marker for carcinomas of müllerian origin, therefore the pathologist should exercise caution when a metastatic carcinoma is of the ovarian serous type, because strong nuclear positivity will be seen in serous tumors and mesothelial cells.64 D2-40
D2-40 was initially reported to be a marker for lymphatic endothelium but was found to be expressed by benign and neoplastic mesothelial cells. As with WT1, D2-40 is a sensitive marker, and in recent studies, D2-40
Applications of Immunocytology
A
B
C
D
E
F
839
Figure 21-6 A to F, Malignant mesothelioma with immunostain profile. The fluid specimen shows numerous large, cohesive clusters of mesothelial cells with atypia in the cytospin and cell block sections (A and B, original magnification ×400). Immunostains performed on the cell block showed positivity for calretinin (C, original magnification ×400), Wilms tumor 1 (D, original magnification ×400), cytokeratin 7 (E, original magnification ×400), and cytokeratin 5/6 but was negative for BerEP4 and MOC-31 (F, original magnification ×400).
840
Immunocytology
was found to be a highly sensitive and specific marker (up to 100%) for malignant mesothelioma. Therefore it can be used in a panel to distinguish mesothelioma or mesothelial cells from pulmonary carcinomas in effusion cytology specimens.65 However, D2-40 expression has also been reported in approximately 50% of ovarian carcinomas, 30% of lung carcinomas, and 30% of breast carcinomas. In addition, the pattern of staining in the ovarian tumors was strong and was similar to the staining seen in mesothelial cells, leading some to believe that it is not a useful stain, particularly if an ovarian tumor is a consideration, analogous to WT1.66 GLUT-1
GLUT-1, a member of the family of glucose transporter isoforms (GLUT), facilitates the entry of glucose into cells and is expressed in a variety of malignancies. Immunohistochemically, GLUT-1 expression is confirmed by membranous staining, sometimes with cytoplasmic staining. Red blood cells in the sample can serve as a helpful internal positive control. GLUT-1 has been reported to be a sensitive and specific IHC marker that can help to differentiate reactive mesothelial cells from malignant mesothelioma. It cannot, however, discriminate between malignant mesothelioma and other malignancies.67,68 Thus ICC is required to confirm that the malignant population is mesothelial in origin, in addition to using GLUT-1 as a proposed “malignancy marker.” In a comparison of GLUT-1 with fluorescence in situ hybridization (FISH) detection of the CDKN2A (formerly p16) gene deletion in mesothelioma, the deletion was found to be more sensitive and specific as a malignancy marker than immunostaining with GLUT-1.69
Nonmesothelial (Adenocarcinoma) Markers The accurate diagnosis of adenocarcinoma in body-fluid cytology can be maximized with the use of an immunopanel to demonstrate negative staining for mesothelial markers and positive staining for adenocarcinoma markers (e.g., Bg8, MOC-31, BerEP4, CEA, or B72.3), as seen in Figures 21-7 and 21-8. MOC-31
MOC-31 is a commercially available monoclonal antibody that recognizes an epithelial-associated transmembrane glycoprotein of 40 kD (see Fig. 21-7, C). Squamous cell carcinomas, adenocarcinomas, and small cell carcinomas show a membranous staining pattern, whereas mesothelial cells are typically negative. Although the utility of this marker in distinguishing adenocarcinoma from mesothelial cells has been reported by few studies,70-72 the sensitivity and specificity of this marker in these studies has been between 80% and 90% and 100%, respectively.73 MOC-31 expression in mesothelial cells is usually less than 15% and tends to be weak, focal, and cytoplasmic.57,73
BerEP4
BerEP4 is a monoclonal antibody that reacts with two glycoproteins on the surface of epithelial cells, as well as in the cytoplasm, and does not react with mesothelial cells to a significant degree (Figs. 21-7, B, and 21-8, D).74-77 The role of BerEP4 in the detection of epithelial malignancies is reported in several studies in the literature, with a sensitivity of more than 90% and a specificity of 90%.71-79 BerEP4 has the advantage of high sensitivity and ease of interpretation because of the high percentage of tumor cells stained, characteristic membranous pattern, and lack of cross-reaction with background inflammatory cells; however, isolated mesothelial cells can show some nonspecific focal staining. In addition, some adenocarcinomas tend to be negative with BerEP4; this includes renal cell carcinomas, in which about 50% cases have been reported to be BerEP4 negative.80 Thus in these scenarios, other markers should be used. MONOCLONAL CARCINOEMBRYONIC ANTIGEN
Carcinoembryonic antigen (CEA) is a 180-kD glycoprotein that is 50% carbohydrate, and many antibodies to a variety of epitopes are available. CEA is a highly sensitive marker for adenocarcinomas and is therefore used occasionally in effusion cytology because of its ability to distinguish adenocarcinoma from mesothelial cells. A substantial number of adenocarcinomas stain strongly for CEA, which can support the diagnosis.74-76 A negative result does not exclude adenocarcinoma, because various percentages of carcinomas are negative for CEA, including some breast carcinomas.76 Generally, mesothelial cells do not stain, but weak peripheral CEA staining of some reactive mesothelial cells has been reported, and occasional rare cases of benign effusions show strong reactivity, thus the use of CEA is best with a panel of other markers to maximize sensitivity and specificity.76 TUMOR-ASSOCIATED GLYCOPROTEIN AND B72.3
The monoclonal antibody (mAb) B72.3, which is directed against tumor-associated glycoprotein 72 (TAG-72), is associated with 95% overall recognition for adenocarcinomas on paraffin-embedded effusions, particularly those with origin in the lung, whereas reactive mesothelial cells are negative.74 Only rare benign effusions have been reported to show positivity.81 However, the sensitivity of detecting adenocarcinoma with B72.3 is lower than with other stains, with a sensitivity of approximately 70% to 80%.81 Thus a combination of B72.3 and BerEP4 or MOC-31 can help to maximize sensitivity and specificity. CD15 AND LeuM1
CD15, or Lewis X antigen, can be identified with the LeuM1 antibody. Monoclonal antibodies to LeuM1 (CD15 granulocyte antigen) and BMA/070 (CD16 natural killer antigen) did not react with mesothelial
Applications of Immunocytology
A
B
C
D
841
F
E Figure 21-7 A to F, Metastatic high-grade serous carcinoma in a peritoneal fluid specimen (original magnification ×400). The cell block shows papillary clusters of malignant cells with surrounding lacunae or clear spaces on the cell block (A); cells stained positive for BerEP4 (B), MOC-31 (C), and Pax-8 (D) and showed patchy positivity for estrogen receptor (E). F, The tumor cells stained negative for calretinin.
842
Immunocytology
A
B
C
D
E
F
Figure 21-8 A to F, Metastatic adenocarcinoma of breast origin in pleural fluid (original magnification ×400). The cytospin, ThinPrep, and cell block specimens showed cells with nuclear enlargement, prominent nucleoli, and glassy cytoplasm (A to C). Given the resemblance of these tumor cells to mesothelial cells, in a so-called “mesothelioma pattern,” immunocytochemistry is important to exclude reactive or neoplastic mesothelial cells. The cells stained positive for BerEP4 (D) and estrogen receptor (E), whereas they were negative for calretinin and Wilms tumor 1 (F).
Applications of Immunocytology
cells, although they stained carcinoma cells.74 However, LeuM1 has a sensitivity of 29% in identifying adenocarcinoma cells, and false-positive staining of malignant mesothelioma has been reported as a result of high hyaluronic acid content.74 CD15 is no longer a popular adenocarcinoma marker because of its low sensitivity and the emergence of superior markers, such as BerEP4 and MOC-31.
Site-Specific and Other Markers LUNG MARKERS Thyroid Transcription Factor 1
Thyroid transcription factor 1 (TTF-1) is a homeodomaincontaining transcription factor that is relatively selectively expressed in pulmonary adenocarcinomas, thyroid tumors, and small cell carcinomas (pulmonary and extrapulmonary) with relatively high sensitivity and specificity. TTF-1 is a highly sensitive and specific immunomarker for distinguishing metastatic pulmonary from extrapulmonary adenocarcinoma in effusion cytology specimens, with a sensitivity of 88.2% and specificity of 100%.81-83 Anti–TTF-1 can be used as a reliable component of an antibody panel to support the diagnosis of adenocarcinoma of pulmonary origin in patients who come to medical attention with metastatic adenocarcinoma in serous fluid with an unknown primary site (Fig. 21-9).81 In cytologic preparations, TTF-1 is a highly selective marker for pulmonary adenocarcinoma and also can have a role in the distinction between pulmonary adenocarcinoma and mesothelioma.82,83 It is important to keep in mind that TTF-1 is expressed in a subset of neuroendocrine carcinomas (ovary, breast), in carcinoid tumors of lung origin, and in most thyroid neoplasms.82 Thus although TTF-1 is still regarded as a sensitive and specific marker for adenocarcinoma of primary pulmonary origin, TTF-1 immunoreactivity can occur in a subset of tumors of other origins, which again emphasizes the importance of an immunopanel. Napsin A
Napsin A is a proteinase involved in the maturation of surfactant protein B and has been shown to be positive in more than 80% of lung adenocarcinoma, with a higher specificity than TTF-1.84 Thus cytoplasmic positivity of napsin A can be supportive evidence of an adenocarcinoma of the lung, and it is particularly helpful in cases with equivocal TTF-1 staining.85 One of the only nonpulmonary adenocarcinomas reported to be napsin A positive is renal cell carcinoma.86 However, given that it is a newer marker, napsin A has not been extensively investigated in nonpulmonary adenocarcinomas. Furthermore, the cytoplasmic staining pattern can be more difficult to interpret than the nuclear staining of TTF-1. p63/p40
One nuclear marker for squamous cell carcinomas, p63, has been utilized as the primary marker for identifying
843
squamous cell carcinoma because of its high sensitivity, which approaches 100%. The main limitation is the low specificity as a result of staining reported in 16% to 65% of lung adenocarcinomas.84,87 A relatively new marker for the identification of squamous cell carcinomas is p40, an antibody that recognizes a p63 isoform. The advantage of p40 is that it appears to be as sensitive as p63 but shows superior specificity in that some adenocarcinomas and lymphomas that show some p63 positivity tend to be negative for p40.87 The lung markers—TTF-1, p63/p40, napsin A, and others—are particularly helpful in those tumors that are poorly differentiated and show only subtle features of an adenocarcinoma or squamous cell carcinoma, in that immunostains have been shown to refine these tumors into a particular subtype in 65% to 75% of cases.84,85 GASTROINTESTINAL MARKERS CDX2
CDX2 is a homeobox domain–containing transcription factor that is important in the development and differentiation of the intestine and is expressed in colorectal carcinoma (Fig. 21-10, C). Studies have shown that strong nuclear staining of CDX2 is a specific and sensitive marker to detect gastrointestinal (GI) malignancies in cytology samples and to differentiate them from reactive mesothelial cells in exfoliative cytology.88 CDX2 is also seen in mucinous tumors with GI differentiation that originate in the lung or ovary.88-91 In addition, a high frequency of CDX2 expression has been noted in midgut and foregut carcinoid tumors, making it a marker of neuroendocrine tumors of midgut origin as well.92 CDX2 may also be expressed in cervical, endometrial, and ovarian adenocarcinomas, and differential CK7/20 expression may be of additional aid in these situations.93,94 LYMPHOID MARKERS
When considering a lymphoid neoplasm, a variety of antibodies are available that can help in making a diagnosis of lymphoma.1,2,4,95 These include LCA in addition to B-cell markers, such as the commonly utilized immunomarkers CD20 and CD79a, and T-cell markers, such as CD3 and CD5. A wide variety of immunostains is also available for further subclassification of lymphoid neoplasms. When considering a Hodgkin lymphoma, immunostains such as CD30, CD15, MUM-1, Pax-5, and LCA can be helpful to confirm the presence of Reed-Sternberg cells. In addition, if a B-cell non-Hodgkin lymphoma is a consideration, immunostains are helpful to determine whether the tumor is of germinal center origin (e.g., CD10, Bcl-6, MUM-1) and whether it has a high proliferation index (i.e., with Ki-67) in addition to looking for CD20 expression, which can help select patients who may respond to anti-CD20 therapies, as described later in the chapter (Fig. 21-11). Immunostains are most helpful in lymphoid neoplasms when used in conjunction with flow cytometry, which allows for better analysis of discrete populations and provides
844
A
Immunocytology
B
Figure 21-9 A and B, Adenocarcinoma of lung origin (original magnification ×400). A, The cell block demonstrates tissue fragments with malignant cells, showing prominent nucleoli and nuclear pleomorphism in a vague glandular arrangement. B, The thyroid transcription factor 1 stain performed on the cell block shows strong nuclear staining.
A
B
C
D
Figure 21-10 A to D, Metastatic mucinous adenocarcinoma of the rectum (original magnification ×400). The aspirate smears and cell block sections show neoplastic vacuolated cells in a background of abundant mucinous material (A and B). Immunostains performed on the cell block reveal that the tumor cells are positive for CDX2 (C) and cytokeratin 20 (D), confirming the diagnosis.
Applications of Immunocytology
A
B
C
D
E
F
845
Figure 21-11 A to F, B-cell Lymphoma with CD20 positivity. The aspirates in this case were abundantly cellular and showed a dyscohesive population of cells with crush artifact (A and B, Diff-Quik stain and hematoxylin and eosin; original magnification ×400 and ×200, respectively). C, Immunostains showed that the tumor cells were positive for CD20, confirming the B-cell origin. D, The cells were predominantly negative for CD3. E, The tumor cells were also positive for CD10, confirming their germinal center origin. F, They showed a high proliferation index with Ki-67 (original magnification ×200 for C to F).
846
Immunocytology
more quantitative data. However, flow cytometry alone cannot evaluate for important markers such as cyclin D1, Ki-67, Epstein-Barr virus latent membrane protein (EBV-LMP), anaplastic lymphoma kinase (ALK), and other markers available for ICC that help in reaching a definitive and accurate diagnosis in many cases. In fluid cytology, CD3 and CD20 immunostains are reported to be less effective than examining the clinical features, cytomorphology, and blood counts.95 BREAST MARKERS
The use of cytology in breast cancer management primarily deals with the aspiration of solid organ tumors or suspicious lymph nodes to document metastatic disease, which is important for staging and prognosis but also provides pathologic material for the analysis of important prognostic and predictive tests that can help to determine the appropriate therapy. Estrogen Receptor
Estrogen receptor (ER) antibodies have been suggested as a tool to identify metastatic breast carcinoma in
effusions from patients without solid tissue metastases (Fig. 21-12, D). Although reactive mesothelial cells are ER negative, ER is not a sensitive or specific marker by itself. Gynecologic carcinomas of the vulva, vagina, cervix, endometrium, ovary, and fallopian tube are often positive for ER in a patchy fashion (see Fig. 21-7, E).96 Although ER is a helpful marker, the pathologist must exercise caution when the differential diagnosis includes pulmonary adenocarcinoma, because a subset of these tumors may be associated with significant nuclear expression with both 6F11 and 1D5 ER clones (Fig. 21-13).97 Gross Cystic Fluid Protein 15 (BRST-2)
The monoclonal antibody to BRST-2 is also known as gross cystic fluid protein 15 (GCDFP-15) and has been described in breast cyst fluid and in the plasma of invasive mammary carcinoma. In IHC analyses, it has been shown to have a sensitivity and specificity up to 70% to 90% and 95%, respectively, in primary and metastatic mammary carcinomas.98-101 However, GCDFP-15 can be positive in tumors that arise from other organs, such as prostate and salivary and sweat glands and in central (bronchial) lung carcinomas.102 GCDFP-15
A
B
C
D
Figure 21-12 A to D, Adenocarcinoma of breast primary with immunostaining (original magnification ×400). A, The cell block from this fine needle aspirate shows numerous clusters of cells with nuclear pleomorphism and prominent nucleoli. Immunostains revealed that the tumor cells were positive for mammaglobin (B), focally positive for gross cystic disease fluid protein 15 (C), and strongly positive for estrogen receptor (D).
Applications of Immunocytology
A
B
C
D
847
Figure 21-13 A to D, Computed tomography–guided fine needle aspiration shows adenocarcinoma of the lung with estrogen receptor positivity (original magnification ×400). The aspirate smear and cell block specimen show papillary clusters of cells with cytologic atypia in this solitary lung mass (A and B). The tumor cells were strongly positive for thyroid transcription factor 1 (C) and showed weak, patchy staining for estrogen receptor (D). Excision confirmed the diagnosis of a primary lung adenocarcinoma. This case illustrates unexpected staining results that can occur in some tumors, so correlation with the clinical and radiologic features becomes very important.
is now recognized in cytology samples as a relatively specific marker for breast carcinomas, both primary and metastatic. Mammaglobin
Mammaglobin is a gene-sequence fragment that has been shown to be expressed in 50% to 90% of primary breast carcinomas and 62% of metastatic breast carcinomas.101-104 It is also demonstrated in other tumors such as endometrial adenocarcinomas, salivary gland carcinomas, and endocervical carcinomas in situ.103 A
study that examined the potential use of this marker in pleural effusions found that 87% of metastatic breast carcinomas expressed mammaglobin, in comparison to only 47% of tumors that stained positive for GCDFP15; however, the specificity of GCDFP-15 was slightly higher than that of mammaglobin (96% vs. 88%).101 Therefore in clinical practice, apart from a good clinical history, use of both mammaglobin and GCDFP-15 is recommended to increase the sensitivity and specificity of the panel to make an accurate diagnosis of adenocarcinoma of breast origin (see Fig. 21-12).
848
Immunocytology
E-Cadherin and p120 Catenin
The E-cadherin complex is composed of the transmembrane E-cadherin protein and the alpha, beta, gamma, and p120 catenins that anchor the E-cadherin protein to the cytoplasmic actin filaments. The catenins are normally located at the junction of the cytoplasm and internal aspect of the cell membrane, where they link with the actin cytoskeleton. They show a variety of cellular abnormalities that may result in the absence of E-cadherin and cytoplasmic redistribution of the catenins of the E-cadherin–catenin complex in lobular neoplasia.105,106 These alterations result in a loss of immunodetectable E-cadherin in lobular neoplasia and in a diffuse cytoplasmic p120 immunostaining pattern. In contrast, ductal neoplasia retains the membranous immunostaining pattern of p120 and E-cadherin, reflecting the normal construction of the E-cadherin complex. Diffuse signet-ring carcinomas of stomach and rectum may also show p120 cytoplasmic immunostaining, because they also lack E-cadherin.106 OVARIAN MARKERS
Although many ovarian masses are not aspirated, occasional ovarian cyst fluid specimens and more numerous samples of peritoneal or ascites fluid specimens are evident in older women, found by the pathologist looking for metastatic ovarian carcinoma. In these fluids, ICC can be helpful. Wilms Tumor Gene Product
The Wilms tumor gene product WT1 has been established as a good marker for mesothelial cells but is also a highly sensitive and specific marker of serous carcinomas of ovarian surface epithelial origin, both ovarian and extraovarian.107-109 Mucinous and micropapillary breast carcinomas may express WT1 and may thus confound the distinction between breast carcinoma and metastatic ovarian serous carcinoma.110 In addition, as mentioned previously, WT1 is a marker of mesothelial cells and therefore will not distinguish a mesothelial proliferation from a metastatic ovarian carcinoma in a body cavity fluid specimen. PAX8
PAX8 is a member of the PAX gene family that has an importance in the development of certain systems in the body, including the kidneys, thyroid, and müllerian system. PAX8 is positive in 72.4% of müllerian tumors but was absent in all pancreatobiliary, GI, and breast carcinomas.111 Thus it is a relatively sensitive and specific marker for the confirmation of a tumor of müllerian or ovarian origin and appears to be more sensitive and specific than WT1 in the confirmation of nonmucinous ovarian carcinomas.112 PAX8 is valuable when considering the possibility of breast or ovarian carcinoma, given that it tends to be positive in ovarian tumors but negative in breast tumors, and thus it can be advantageous in the metastatic setting or when a patient has a history of both tumors. Conversely, NY-BR-1 immunostaining tends to show the reverse
pattern, with positivity in breast carcinoma and negative staining in ovarian tumors.111 Some studies have also shown expression in tumors of thyroid, thymic, or renal origin, thus additional markers should be used when considering these possibilities, such as TTF-1, renal cell carcinoma (RCC), and WT1.112 Hepatocyte Nuclear Factor 1β
Hepatocyte nuclear factor 1β (HNF-1β) is a transcription factor that directly binds to DNA and is shown to be unregulated in ovarian clear cell carcinomas. Mesothelial cells and non–clear cell adenocarcinomas are shown to be negative. Strong nuclear staining is shown in ovarian clear cell carcinomas and tends to be negative in other ovarian tumors that can show clear cells, such as endometrioid and serous carcinomas.113,114 However, a subset of extraovarian tumors with clear cell changes, including renal cell carcinomas and hepatocellular carcinomas, have also been shown to demonstrate positivity for HNF-1β, so it may not be helpful in distinguishing ovarian clear cell carcinomas from other extraovarian clear cell tumors.114 GYNECOLOGIC CYTOLOGY MARKERS
ICC also has a role in gynecologic cytology, including use in documenting metastatic or recurrent disease in aspiration cytology or exfoliative cytology specimens. However, the role of ICC appears to be growing in the area of primary cervical cancer screening, in which investigators are applying immunostains to Pap test specimens. Although colposcopic biopsy is the gold standard for abnormal squamous cells seen on screening, the diagnostic accuracy of detection of human papillomavirus (HPV) infection has been increasing with successful adjuvant methods such as CDKN2A (formerly p16INK4a) immunostaining and HPV DNA testing on cytologic specimens. MARKERS OF DYSPLASIA CDKN2A
Overexpression of CDKN2A has been strongly linked to high-risk HPV infection and is expressed in dysplastic squamous cells. Over 80 scientific publications demonstrate virtually 100% sensitivity for cervical intraepithelial neoplasia 3 (CIN3) combined with very high specificity for nonmalignant disease by using CINtec p16 assay, with significant improvements in interobserver reproducibility and diagnostic accuracy. In addition, CDKN2A also consistently discriminates in situ and invasive cervical adenocarcinomas from benign endocervical cells. Sensitivity and specificity of CDKN2A expression in adenocarcinoma in situ and invasive adenocarcinoma are 94.5% and 100%, respectively.115-123 CDKN2A has been successfully demonstrated on SurePath slides (Becton, Dickinson and Company, Franklin Lakes, NJ) and on ThinPrep slides as well as in cell blocks.118-120 Studies show that CDKN2A expression in simultaneously sampled tissue sections and liquid-based Pap tests carry similar sensitivity and
Applications of Immunocytology
A
849
B
C D Figure 21-14 A to D, Use of p16 immunostaining in cervicovaginal cytology. The Pap test shows cells with elevated nuclear/cytoplasmic ratios and nuclear irregularities suggestive of a high-grade squamous intraepithelial lesion (A) and atypical endocervical cells (C). Strong nuclear and cytoplasmic staining of p16 was seen in preparations from the residual material in the Pap test vials (B, inset, and D).
specificity.121 With CDKN2A immunostaining, the sensitivity and specificity of detecting a low-grade squamous intraepithelial lesion (LSIL) are 74%; for a high-grade squamous intraepithelial lesion (HSIL), sensitivity and specificity are 97%. A score of more than 10 cells showing predominantly nuclear staining but also cytoplasmic staining is considered positive.124 One study addressed the identification of HSIL by using nuclear score in patients with minor cytologic abnormalities (atypical squamous cells of undetermined significance [ASCUS] and/or those of the LSIL group). It was found that the overall sensitivity and specificity of CDKN2Apositive cells with a nuclear score greater than 2 for identification of HSIL in ASCUS and LSIL Pap tests combined was 96% and 83%, respectively.124 These data suggest that the use of CDKN2A as a biomarker combined with nuclear scoring of positive cells in cervical cytology to triage ASCUS and LSIL cases allows identification of HSIL with good sensitivity and specificity.122-124 A caveat with CDKN2A is that some nonspecific staining may occur in metaplastic cells, atrophic specimens,
and endometrial cells, and the antibody may also crossreact with Trichomonas vaginalis and other organisms in liquid-based cytology (LBC) specimens.125,126 Therefore it is critical to compare immunostaining with cytomorphology. Figure 21-14 illustrates CDKN2A immunostaining in abnormal Pap tests. In a recent study, CDKN2A assessment in urine cytology samples showed a sensitivity of 66.7% and a specificity of 82.8% in the diagnosis of low-grade urothelial carcinoma.127 In addition, CDKN2A has been a useful stain in the identification of HPV-related squamous cell carcinomas of other origins, including oropharyngeal, head and neck, and anorectal carcinomas (Fig. 21-15).128,129 ProEx C
The ProEx C ICC assay (TriPath Oncology, Burlington, NC) utilizes a cocktail of monoclonal antibodies directed against proteins associated with aberrant S-phase cellcycle induction (topoisomerase II-α, minichromosome maintenance protein 2). A few studies have addressed the role of ProEx C in liquid-based cytology, and one
850
Immunocytology
A
B
C
D
Figure 21-15 A to D, Metastatic squamous cell carcinoma of head and neck origin (original magnification ×400). This fine needle aspirate of an enlarged cervical lymph node showed clusters of squamous cells with cytological atypia (A, hematoxylin and eosin stain). Immunostains confirmed the squamous nature of the tumor cells with positivity for p63 (B) and cytokeratin 5/6 (C). In addition, the tumor cells were positive for p16 (D), which is compatible with an origin from the patient’s oropharyngeal squamous cell carcinoma.
study showed a 100% positivity for HSIL with ProEx C. It is also interesting to note that 25% of LSILs were positive. This study suggests that the assay should be integrated into the clinical cytology laboratory to increase the positive predictive value of LBC for biopsyproven HSIL.130 One of the difficulties with ProExC and p16 is that as many as 84% of cytology-negative ThinPrep specimens can show false-positive staining as a result of staining of normal-appearing cells.131 Thus further investigations are required to look at the performance of these immunostains in Pap tests. PROLIFERATION MARKERS MIB-1 (Ki-67)
Ki-67 (MIB-1) is a monoclonal antigen that interacts with human nuclear antigen Ki-67, which is present in proliferating cells. Ki-67 can be helpful in the grading of some neoplasms seen in cytologic specimens, particularly neuroendocrine tumors, mesenchymal neoplasms, and lymphoid neoplasms. Given the limited number of cells available in some specimens, Ki-67 is usually used
instead of a mitotic count when a proliferation index is needed for grading of neoplasms in cytologic specimens. Few studies have compared the expression of CDKN2A and Ki-67 in cervical intraepithelial lesions.132 One study showed positive scores for Ki-67 and CDKN2A of 68.4% and 100%, respectively, in LSIL and 94.7% and 100%, respectively, in HSIL. Positive predictive values of these three biomarkers for HPV were 82.4% and 91.4%, respectively. The studies concluded that MIB-1 (Ki-67) and CDKN2A are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease.132 INFECTIOUS DISEASE MARKERS
Immunostains are also utilized in cytopathology for the diagnosis and subtyping of certain infectious etiologies, including bacteria such as Bartonella and Helicobacter, fungi such as Aspergillus, parasites such as Toxoplasma, and viruses that include varicella zoster, herpes simplex virus (HSV), EBV, and cytomegalovirus (CMV).7 One of the most widely used antibodies is the one for EBVLMP, given the association between EBV and certain
Theranostic Applications of Immunocytology
A
851
B
Figure 21-16 A and B, Bronchoalveolar lavage specimen with herpes simplex virus (HSV) infection confirmed with immunostains. This specimen from a leukemia patient had cells with nuclear enlargement, multinucleation, and marginated chromatin (arrows), suggestive of infection by HSV (A, hematoxylin and eosin stain, original magnification ×400). An immunostain for HSV1/2 was positive in these cells, confirming the diagnosis of HSV infection (B, HSV1/2 immunostain, original magnification ×400). This illustrates the utility of immunostains in the cytologic diagnosis of infection.
lesions seen in cytology specimens, including benign processes (infectious mononucleosis) and malignant tumors (posttransplant lymphoproliferative disorders, diffuse large B-cell lymphoma of the elderly, and others). Immunostains for herpes viruses are also used in the diagnosis of infection (e.g., HSV1/2 in HSV infections) and in the diagnosis of certain tumors (human herpesvirus 8 in Kaposi sarcoma). One example of a bronchoalveolar lavage (BAL) specimen in a leukemia patient, which showed cells worrisome for cellular changes associated with an HSV infection, was positive for the HSV1/2 stain and helped to confirm the diagnosis (Fig. 21-16). Antibodies are continuing to emerge for confirmation of other infectious organisms and are helpful, particularly in scenarios of unsuspected infection without microbial cultures and in helping to make particular diagnoses with viral associations. In cases of suspected infection, it is still helpful to send for microbial cultures to look at antibiotic sensitivities and to help subclassify the infection when immunostains are not available.
Theranostic Applications of Immunocytology ICC is becoming increasingly important for the evaluation of predictive markers that can help select patients who may respond to particular targeted therapies. Some of these include Gleevec (Novartis, Basel, Switzerland) for GI stromal tumors (GISTs), Herceptin (Genentech, South San Francisco, CA) for HER2-positive breast cancers, and rituximab for CD20-positive lymphomas.
CD117 Mutations of the KIT gene are characteristically seen in GISTs and are shown as increased protein expression on
surgical specimens demonstrated by IHC. CD117 (c-Kit) protein expression has been examined in cytology specimens, particularly on cell block material, and is helpful in subclassifying epithelioid and spindle cell tumors as GISTs.133 In additional to playing a diagnostic role, CD117 helps identify patients who may benefit from treatment with anti-CD117 agents, such as imatinib mesylate (Gleevec). In addition to GISTs, CD117 is positive in mast cells and melanocytes and has also been reported in a subset of renal cell carcinomas, seminomas, sarcomas, and extramedullary myeloid tumors.134,135
ERBB2 Approximately 15% to 20% of invasive breast carcinomas express ERBB2 (formerly HER2/neu) and are associated with poor prognosis. Trastuzumab (Herceptin), a humanized monoclonal antibody directed against the extracellular domain of HER2 protein, has been shown to significantly increase complete pathologic response after adjuvant chemotherapy. Therefore it is important for the laboratory to assess HER2 status on both histologic and cytologic specimens for management of these patients. ERBB2 protein expression by ICC can be performed on formalin-fixed cell blocks and evaluated in a similar fashion to surgical specimens, with equivocal cases (score 2+) being triaged for FISH studies. Studies have also shown success on ThinPrep specimens.17 Evaluation is simplified in metastatic lesions, because all tumor cells will be invasive, as opposed to the evaluation of ERBB2 in primary breast lesions, in which only the invasive tumor cells can be evaluated. Chromogenic in situ hybridization (CISH) is a recent methodology wherein the ERBB2 gene copies are detected by a silver reaction and are visualized by light microscopy. A study that evaluated CISH on ThinPrepprocessed FNA specimens found a good concordance between CISH performed on LBC specimens compared with paraffin-embedded tissue specimens.136,137
852
Immunocytology
CD20 Of the lymphomas, B-cell non-Hodgkin lymphomas are the most common type, particularly diffuse large B-cell lymphomas (30% of cases) and follicular lymphomas (25% to 30% of cases). Given that many B-cell lymphomas are positive for a B-cell marker, CD20, a chimeric monoclonal antibody was created that recognizes the human CD20 antigen (rituximab). This was the first antibody approved by the FDA for use in the treatment of lymphoma, and it was one of the first examples of monoclonal therapy success in the treatment of cancer.138 Since that time, additional anti-CD20 agents have become available, including radiolabeled anti-CD20 agents (Y-90 ibritumomab tiuxetan [Zevalin; Spectrum Pharmaceuticals, Irvine CA] and I-131 tositumomab [Bexxar; GlaxoSmithKline, Philadelphia PA]).139 Thus CD20 is an important marker to evaluate by ICC or flow cytometry in an effort to guide treatment for the patient. In some cases, a B-cell lymphoma may lose CD20 expression after treatment with an anti-CD20 agent, and in these cases, additional B-cell markers can be helpful, including CD79a and Pax-5.140
Tumors of Unknown Primary Tumors of unknown primary account for 3% to 5% of all solid organ malignancies. These patients usually come to medical attention with multiple liver, bone, brain, or lung masses; lymphadenopathy; and/or peritoneal carcinomatosis, depending on the type of tumor. Thus these tumors can be seen in aspiration or exfoliative cytology specimens, and the tumors may be of epithelial origin, such as carcinoma, or nonepithelial origin, such as lymphoma, melanoma, sarcoma, and mesothelioma. Therapeutic strategies rely on determining a possible subtype of tumor (adenocarcinoma, lymphoma, melanoma) and origin of tumor (lung, colon), which may not be possible morphologically; thus ICC plays an important role. For this reason, allocating material for immunostaining is crucial in these cases. Given that tumors may have an atypical immunophenotype, and given the lack of highly specific markers for some types of tumors, definitive classification by ICC may not be possible. In these scenarios, additional clinical and radiologic imaging findings are helpful in evaluation. Body-fluid cytology is a common specimen to receive for evaluation of a patient with a tumor of unknown primary, because the specimen is easy to acquire through minimally invasive methods, such as thoracentesis. In fluid cytology, adenocarcinomas are the most common types of tumors seen (60%), and in the pleural cavity, the majority of these are from the lung (Fig. 21-17). Other common origins of adenocarcinoma of unknown primary include pancreas, liver, GI tract, and kidney. The characteristic staining profile of CK7 and CK20 has been shown to be of great utility in surgical pathology, and these are used in cytology specimens to help identify the origin of malignant effusions.48 The majority of lung, breast, gastric, and ovarian adenocarcinomas show
a CK7-positive, CK20-negative immunophenotype. However, the pathologist should utilize other markers of mesothelial cells in the panel, given that mesothelial cells are positive for CK7 and could mimic a CK7positive, CK20-negative metastatic adenocarcinoma. TTF-1 is a highly sensitive and specific marker in the differentiation of pulmonary adenocarcinomas from nonpulmonary adenocarcinomas in effusion cytology specimens.141,142 The CK7-negative, CK20-positive immunoprofile is highly specific for an adenocarcinoma of colorectal origin, although a significant number of gastric and pancreatic adenocarcinomas also exhibit this immunoprofile.143-145 CDX2 can also help to confirm a tumor of GI origin; however, weak positivity of CDX2 has also been seen in gastric, pancreatic, biliary, and mucinous ovarian adenocarcinomas.88,89 A few other markers can be helpful in the setting of a CK7-positive adenocarcinoma, including SMAD4 (formerly DPC4), which can be helpful in suggesting a pancreatobiliary origin.146,147 Prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) have also been reported to have utility in supporting a prostatic origin; however, prostate cancer is not commonly seen in effusion cytology or as a carcinoma of unknown primary.148 An immunopanel that comprises CK7, CK20, TTF-1, and CDX2 is a reasonable panel for tumors of unknown primary in serous effusions. When poorly differentiated squamous cell carcinoma is a consideration, p63 or p40 is helpful to confirm a squamous cell carcinoma.145 CK5/6 is not as helpful, because mesothelial cells are positive for this stain. Although most site-specific markers do not determine the site of origin for a squamous cell carcinoma, immunostaining for p16 or in situ hybridization (ISH) for HPV can also be helpful in cases of metastatic squamous cell carcinoma of unknown primary, because these stains could help confirm an HPV-related squamous cell carcinoma arising from the oropharynx, anogenital region, or uterine cervix (see Fig. 21-15).128,129 In cases of peritoneal carcinomatosis of unknown origin, the most common site of origin is the müllerian tract in women, followed by the GI tract, breast, and lymph nodes. Accurate diagnostic markers are needed to accurately determine the likely sites of origin. In these scenarios, CA125, Pax-8, and CDX2 are helpful in addition to the CK7 and CK20 immunoprofile. Nonepithelial tumors can present as tumors of unknown primary and should be suspected in tumors that fail to show cytokeratin staining or in those that have cytomorphologic features that suggest a nonepithelial tumor, such as dyscohesion. These tumors include malignant melanoma, lymphoma, sarcoma, germ cell tumor, and some pediatric malignant small round blue cell tumors. The determination of origin of nonepithelial malignancies on purely cytomorphologic grounds is difficult, so ICC plays an important role in accurate identification of these tumors. Malignant melanoma is uncommon in body-fluid cytology but can be seen in aspiration cytology for a tumor of unknown primary. ICC is helpful, because the tumor cells are immunoreactive for human melanoma black 45 (HMB-45), S-l00 protein, melan-A, tyrosinase, and other melanoma markers (see Fig. 21-4). Although vimentin is positive in
Summary
A
B
C
D
853
Figure 21-17 A to D, Poorly differentiated carcinoma of unknown primary. The aspirate smear and cell block show clusters of cells with marked nuclear pleomorphism, including large nucleoli (A and B, Diff-Quik and hematoxylin and eosin stain, original magnification ×400). Immunostains revealed that the tumor cells were positive for thyroid transcription factor 1 (TTF-1) (C, TTF-1 immunostain, original magnification ×400) and negative for p40 (D, p40 immunostain, original magnification ×400), consistent with a poorly differentiated adenocarcinoma of lung origin. Immunostains help to subclassify the tumor type (adenocarcinoma vs. squamous cell carcinoma) and the origin (lung origin or other).
melanomas, it is also positive in mesotheliomas, sarcomas, and some carcinomas, such as endometrial adenocarcinomas. Cytokeratin immunoreactivity has been reported in malignant melanoma in 1% to 27% of cases, but immunostaining in these cells is usually weak and focal.149 Sarcomas account for only 3% to 6% of malignant effusions. They usually occur in the setting of a known primary tumor, and they rarely present as a tumor of unknown origin. Synovial sarcoma, epithelioid sarcoma, vascular tumors, leiomyosarcoma, endometrial stromal sarcoma, and GIST are some of the sarcomas that can present as tumors of unknown origin. In children, the most common causes of a tumor of unknown primary include the small round blue cell tumors, such as lymphoma and leukemia, followed by Wilms tumor, neuroblastoma, Ewing sarcoma, and embryonal rhabdomyosarcoma. In these scenarios, flow cytometry and ICC play an important role. Given the range of diagnostic possibilities for a tumor of unknown primary, obtaining sufficient material for ICC is one of the most important goals in order to reach an accurate diagnosis.
Summary Modern diagnostic cytopathology is a dynamic and evolving field that utilizes a variety of ancillary studies for diagnostic, prognostic, and predictive roles. Of these studies, ICC is the most widely used and plays a critical role in establishing accurate diagnoses and in providing important information to treating clinicians. In the future, as more antibodies are developed and applied to cytologic material, the challenges will include development of optimized and standardized protocols; optimization of cells available for these studies; validation of new platforms available for immunostaining, such as multiplex staining; and new digital applications, including image analysis, to enhance interpretation of the results. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Chess Q, Hajdu S: The role of immunoperoxidase staining in diagnostic cytology. Acta Cytol. 30:1–7, 1986. 2. Flens MJ, Valk PVD, Tadema TM, et al: The contribution of immunocytochemistry in diagnostic cytology, comparison and evaluation with immunohistology. Cancer. 65:2704–2711, 1990. 3. Johnston WW, Szpak CA, Thor A, et al: Techniques in immunocytochemistry, application to diagnostic pathology. Arch Pathol Lab Med. 113:641–644, 1989. 4. Lai C-R, Pan C-C, Tsay S-H: Contribution of immunocytochemistry in routine diagnostic cytology. Diagn Cytopathol. 14:221– 225, 1996. 5. Schofield J, Krausz T: Metastatic disease and lymphomas. In Gray W, editor: Serous Cavities, London, 1995, ChurchillLivingstone. 6. Nance K, Silverman JF: Immunocytochemical panel for the identification of malignant cells in serous effusions. Am J Clin Pathol. 95:867–874, 1993. 7. Eyzaguirre E, Haque AK: Application of immunohistochemistry to infections. Arch Pathol Lab Med. 132:424–431, 2008. 8. Fowler LJ, Lachar WA: Application of Immunohistochemistry to Cytology. Arch Pathol Lab Med. 132:373–383, 2008. 9. Suthipintawong C, Leong A-Y, Vinyuvat S: Immunostaining of cell preparations: A comparative evaluation of common fixatives and protocols. Diagn Cytopathol. 15:167–174, 1996. 10. Fulciniti F, Frangella C, Staiano M, et al: Air-dried smears for optimal diagnostic immunocytochemistry. Acta Cytol. 52:178– 186, 2008. 11. Abendroth CS, Dabbs DJ: Immunocytochemical staining of unstained versus previously stained cytologic preparations. Acta Cytol. 39:379–386, 1995. 12. Leong A-Y, Suthipintawong C, Vinyuvat S: Immunostaining of cytologic preparations: A review of technical problems. Appl Immunohistochem. 7:214–220, 1999. 13. Dabbs DJ, Abendroth CS, Grenko RT, et al: Immunocytochemistry on the ThinPrep processor. Diagn Cytopathol. 17:388–392, 1997. 14. Guiter GE, Gatscha RM, Zakowski MF: ThinPrep vs conventional aspirations of sarcomas: A morphological and immunocytochemical study. Diagn Cytopathol. 21:351–354, 1999. 15. Leung SW, Bedard YC: Immunocytochemical staining on ThinPrep processed smears. Mod Pathol. 9:304–306, 1996. 16. Kaplan MA, Segura AM, Wang HH, et al: Evaluation of Cytolyt and PreservCyt as preservatives for immunocytochemistry for cytokeratin in fine needle aspiration. Appl Immunohistochem. 6:23–29, 1998. 17. Pegolo E, Machin P, Riosa F, et al: Hormone receptor and human epidermal growth factor receptor 2 status evaluation on Thin Prep specimens from breast carcinoma. Cancer Cytopathol. 120:196–205, 2012. 18. Gong Y, Symmans WF, Krishnamurthy S, et al: Optimal fixation conditions for immunocytochemical analysis of estrogen receptor in cytologic specimens of breast carcinoma. Cancer. 102:34– 40, 2004. 19. Soule N, Mui K, O’Brien M, et al: A rapid automated system for creating paraffin-embedded cell blocks. Abstract 193. Cancer Cytopathol. 108(Suppl 5):444–445, 2006. 20. Kulkarni MB, Prabhudesai NM, Desai SB, et al: Scrape cell-block technique for fine needle aspiration cytology smears. Cytopathology. 11:179–184, 2000. 21. Negri G, Moretto G, Menia E, et al: Immunocytochemistry of p16INK4a in liquid-based cervicovaginal specimens with modified Papanicolaou counterstaining. Clin Pathol. 59:827–830, 2006. 22. Gupta S, Halder K, Khan VA, et al: Cell block as an adjunct to conventional Papanicolaou smear for diagnosis of cervical cancer in resource-limited settings. Cytopathology. 18:309–315, 2007. 23. Liu H, Shi J, Wilkerson M, et al: Immunohistochemical detection of p16INK4a in liquid-based cytology specimens on cell block sections. Cancer. 111:74–82, 2007. 24. Hammond MEH, Hayes DF, Dowsett M, et al: American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer. Arch Pathol Lab Med. 134:1–29, 2010.
853.e1
25. Yildiz-Aktas IZ, Dabbs DJ, Cooper KL, et al: The effect of 96-hour formalin fixation on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Am J Clin Pathol. 137:691–698, 2012. 26. Yildiz-Aktas IZ, Dabbs DJ, Bhargava R: The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol. 25:1098–1105, 2012. 27. Yaziji H, Taylor CR, Goldstein NS, et al: Consensus recommendations on estrogen receptor testing in breast cancer byimmunohistochemistry. Appl Immunohistochem Mol Morphol. 16:513– 520, 2008. 28. Bhargava R, Chivukula M, Beriwal S, et al: Estrogen and progesterone receptors in breast carcinoma: Quantitation is therapeutically important. Adv Anat Pathol. 15:304–305, 2008. 29. McKee GT, Tambouret RH, Finkelstein D: A reliable method of demonstrating HER 2/neu, estrogen receptors, and progesterone receptors on routinely processed cytologic material. Appl Immunohistochem Mol Morphol. 9:352–357, 2001. 30. Goldstein NS, Hewitt SM, Taylor CR, et al: Members of Ad-Hoc Committee on Immunohistochemistry Standardization. Recommendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol. 15:124–133, 2007. 31. Shi SR, Liu C, Taylor CR: Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: From experiments to hypothesis. J Histochem Cytochem. 55:105–109, 2007. 32. Wolff AC, Hammond EH, Schwartz JN, et al: American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. Arch Pathol Lab Med. 131:18–43, 2007. 33. Randall B, van Amerongen L: Commercial laboratory practice evaluation of air-dried/rehydrated cervico-vaginal smears vs traditionally-fixed smears. Diagn Cytopathol. 16:174–176, 1997. 34. Ng WF, Choi FB, Cheung LLH, et al: Rehydration of air-dried smears with normal saline: Application in fluid cytology. Acta Cytol. 38:56–64, 1994. 35. Chan JKC, Kung ITM: Rehydration of air-dried smears with normal saline: Application in fine-needle aspiration cytologic examination. Am J Clin Pathol. 89:30–34, 1988. 36. Shi SR, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry: Past, present, and future. J Histochem Cytochem. 45:327–343, 1997. 37. Shi SR, Key ME, Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 39:741–748, 1991. 38. Boon ME, Kok LP: Breakthrough in pathology due to antigen retrieval. Mal J Med Lab Sci. 12:1–9, 1995. 39. Sherman ME, Jimenez-Joseph D, Gangi MD, et al: Immunostaining of small cytologic specimens. Facilitation with cell transfer. Acta Cytol. 38:18–22, 1994. 40. Denda T, Kamoshida S, Kawamura J, et al: Optimal antigen retrieval for Ethanol-Fixed Cytologic Smears. Cancer Cytopathol. 120:167–176, 2012. 41. Miller RT, Swanson PE, Wick MR: Fixation and epitope retrieval in diagnostic immunohistochemistry: A concise review with practical considerations. Appl Immunohistochem Mol Morphol. 8:228–235, 2000. 42. Colasacco C, Mount S, Leiman G: Documentation of immunocytochemistry controls in the cytopathologic literature: a metaanalysis of 100 journal articles. Diagn Cytopathol. 39:245–250, 2011. 43. Sethi S, Geng L, Shidham VB, et al: Dual color multiplex TTF-1 + Napsin A and p63 + CK5 immunostaining for subcategorizing of poorly differentiated pulmonary non-small carcinomas into adenocarcinoma and squamous cell carcinoma in fine needle aspiration specimens. Cytojournal. 9:10, 2012. 44. Dabbs DJ, Wang X: Immunocytochemistry on cytologic specimens of limited quantity. Diagn Cytopathol. 18:166–169, 1998. 45. Hunt JL, van de Rijn M, Gupta PK: Immunohistochemical analysis of gel-transferred cells in cytologic preparations following smear division. Diagn Cytopathol. 18:377–380, 1998.
853.e2
Immunocytology
46. Edwards C, Oates J: OV 632 and MOC-31 in the diagnosis of mesothelioma and adenocarcinoma: An assessment of their use in formalin-fixed and paraffin wax embedded material. J Clin Pathol. 48:626–630, 1995. 47. To A, Dearnaley DP, Omerod MG, et al: Epithelial membrane antigen: Its use in the cytodiagnosis of malignancy. Am J Clin Pathol. 78:214–219, 1982. 48. Sack MJ, Roberts SA: Cytokeratins 20 and 7 in the differential diagnosis of metastatic carcinomas in cytologic specimens. Cytopathology. 16:132–136, 1997. 49. Manosca F, Schinstine M, Fetsch PA, et al: Diagnostic effects of prolonged storage on fresh effusion samples. Diagn Cytopathol. 35:6–11, 2007. 50. Gotzos V, Vogt P, Celio MR: The calcium binding protein calretinin is a selective marker for malignant pleural mesothelioma of the epithelial type. Pathol Res Pract. 192:137–147, 1996. 51. Doglioni C, Dei Tos AP, Laurino L, et al: A novel immunocytochemical marker for mesothelioma. Am J Surg Pathol. 20:1037– 1046, 1996. 52. Lugli A, Forster Y, Haas P, et al: Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples. Hum Pathol. 34:994–1000, 2003. 53. Politi E, Kandaraki C, Apostolopoulou C, et al: Immunocytochemical panel for distinguishing between carcinoma and reactive mesothelial cells in body cavity fluids. Diagn Cytopathol. 32:151–155, 2005. 54. Fetsch PA, Simsir A, Abati A: Comparison of antibodies to HBME-1 and calretinin for the detection of mesothelial cells in effusion cytology. Diagn Cytopathol. 25:158–161, 2001. 55. Ordoñez NG: Value of calretinin immunostaining in differentiating epithelial mesothelioma from lung adenocarcinoma. Mod Pathol. 11:929–933, 1998. 56. Nagel H, Hemmerlein B, Ruschenberg I, et al: The value of anticalretinin antibody in the differential diagnosis of normal and reactive mesothelia versus metastatic tumors in effusion cytology. Pathol Res Pract. 194:759–764, 1998. 57. Yaziji H, Battifora H, Barry TS, et al: Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: Identification of a three-antibody immunohistochemical panel with maximal sensitivity and specificity. Mod Pathol. 19:514–523, 2006. 58. Moll R, Dhouailly D, Sun T-T: Expression of keratin 5 as a distinctive feature of epithelial and biphasic mesotheliomas. An immunohistochemical study using monoclonal antibody. Virchows Arch B Cell Pathol. 58:129–145, 1989. 59. Blobel GA, Moll R, Franke RR, et al: The intermediate cytoskeleton of malignant mesotheliomas and its diagnostic significance. Am J Pathol. 121:235–247, 1985. 60. Clover J, Oates J, Edwards C: Anti-cytokeratin 5/6: A positive marker for mesothelioma. Histopathology. 31:140–143, 1997. 61. Ordoñez NG: Value of cytokeratin 5/6 immunostaining in distinguishing epithelial mesothelioma of the pleura from lung adenocarcinoma. Am J Surg Pathol. 22:1215–1221, 1998. 62. Walker C, Rutten F, Yuan X, et al: Wilms’ tumor suppressor gene expression in rat and human mesothelioma. Cancer Res. 54:3101–3106, 1994. 63. Scharnhorst V, van der Eb AJ, Jochemsen AG: WT1 proteins: functions in growth and differentiation. Gene. 273:141–161, 2001. 64. Nonaka D, Chiriboga L, Soslow RA: Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol. 32:1566–1571, 2008. 65. Saad RS, Lindner JL, Lin X, et al: The diagnostic utility of D2-40 for malignant mesothelioma versus pulmonary carcinoma with pleural involvement. Diagn Cytopathol. 34:801–806, 2006. 66. Bassarova AV, Nesland JM, Davidson B: D2-40 is not a specific marker for cells of mesothelial origin in serous effusions. Am J Surg Pathol. 30:878–882, 2006. 67. Afify A, Zhou H, Howell L, et al: Diagnostic utility of GLUT-1 expression in the cytologic evaluation of serous fluids. Acta Cytol. 49:621–626, 2005. 68. Kato Y, Tsuta K, Seki K, et al: Immunohistochemical detection of GLUT-1 can discriminate between reactive mesothelium and malignant mesothelioma. Mod Pathol. 20:215–220, 2007.
69. Monaco SE, Shuai Y, Bansal M, et al: The diagnostic utility of p16 FISH and GLUT-1 immunohistochemical analysis in mesothelial proliferations. Am J Clin Pathol. 135:619–627, 2011. 70. Morgan RL, De Young BR, McGaughy VR, et al: MOC-31 aids in the differentiation between adenocarcinoma and reactive mesothelial cells. Cancer. 87:390–394, 1999. 71. Ordoñez NG: Value of the MOC-31 monoclonal antibody in differentiating epithelial pleural mesothelioma from lung adenocarcinoma. Hum Pathol. 29:166–169, 1998. 72. Gonzalez-Lois C, Ballestin C, Sotelo MT, et al: Combined use of novel epithelial (MOC-31) and mesothelial (HBME-1) immunohistochemical markers for optimal first line diagnostic distinction between mesothelioma and metastatic carcinoma in pleura. Histopathology. 38:528–534, 2001. 73. Kundu UR, Krishnamurthy S: Use of the monoclonal antibody MOC-31 as an immunomarker for detecting metastatic adenocarcinoma in effusion cytology. Cancer Cytopathol. 119:272– 278, 2011. 74. Bailey ME, Brown RW, Mody DR, et al: Ber-EP4 for differentiating adenocarcinoma from reactive and neoplastic mesothelial cells in serous effusions. Comparison with carcinoembryonic antigen, B72.3 and Leu-M1. Acta Cytol. 40:1212–1216, 1996. 75. Delahaye M, van der Ham F, van der Kwast TH: Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions. Diagn Cytopathol. 17:115–120, 1997. 76. Gaffey MJ, Mills SE, Swanson PE, et al: Immunoreactivity for Ber-EP4 in adenocarcinomas, adenomatoid tumors and malignant mesotheliomas. Am J Surg Pathol. 16:3–9, 1992. 77. Latza U, Niedobitek G, Schwarting R, et al: Ber-EP4: New monoclonal antibody which distinguishes epithelia from mesothelia. J Clin Pathol. 43:213–219, 1990. 78. Singh HK, Silverman JF, Berns L, et al: Significance of epithelial membrane antigen in the work-up of problematic serous effusions. Diagn Cytopathol. 13:3–7, 1995. 79. Dejmek A, Hjerpe A: Immunohistochemical reactivity in mesothelioma and adenocarcinoma: A stepwise logistic regression analysis. APMIS. 102:255–264, 1994. 80. Ordóñez NG: The diagnostic utility of immunohistochemistry in distinguishing between mesothelioma and renal cell carcinoma: a comparative study. Hum Pathol. 35:697–710, 2004. 81. Shield PW, Callan JJ, Devine PL: Markers for metastatic adenocarcinoma in serous effusion specimens. Diagn Cytopathol. 11:1– 9, 1995. 82. Afify AM, al-Khafaji BM: Diagnostic utility of thyroid transcription factor-1 expression in adenocarcinomas presenting in serous fluids. Acta Cytol. 46:675–678, 2002. 83. Hecht JL, Pinkus JL, Weinstein LJ, et al: The value of thyroid transcription factor-1 in cytologic preparations as a marker for metastatic adenocarcinoma of lung origin. Am J Clin Pathol. 116:483–488, 2001. 84. Mukhopadhyay S, Katzenstein AL: Subclassification of nonsmall cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol. 35:15–25, 2011. 85. Dejmek A, Naucler P, Smedjeback A, et al: Napsin A (TA02) is a useful alternative to thyroid transcription factor-1 (TTF-1) for the identification of pulmonary adenocarcinoma cells in pleural effusions. Diagn Cytopathol. 35:493–497, 2007. 86. Ordóñez NG: Napsin A expression in lung and kidney neoplasia: a review and update. Adv Anat Pathol. 19:66–73, 2012. 87. Bishop JA, Teruya-Feldstein J, Westra WH, et al: p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 25:405–415, 2012. 88. Kobayashi M, Ueyama Y, Nakanishi H, et al: Immunocytochemical detection using CDX2: An aid for discerning tumor involvement in ascites cytology. Cancer. 108:114–118, 2006. 89. Kaimaktchiev V, Terracciano L, Tornillo L, et al: The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas. Mod Pathol. 17:1392–1399, 2004. 90. Levine PH, Joutovsky A, Cangiarella J, et al: CDX-2 expression in pulmonary fine-needle aspiration specimens: A useful adjunct
References for the diagnosis of metastatic colorectal adenocarcinoma. Diagn Cytopathol. 34:191–195, 2006. 91. Saad RS, Essig DL, Silverman JF, et al: Diagnostic utility of CDX2 expression in separating metastatic gastrointestinal adenocarcinoma from other metastatic adenocarcinoma in fineneedle aspiration cytology using cell blocks. Cancer. 102:168–173, 2004. 92. Jaffee IM, Rahmani M, Singhal MG, et al: Expression of the intestinal transcription factor CDX2 in carcinoid tumors is a marker of midgut origin. Arch Pathol Lab Med. 130:1522–1526, 2006. 93. Park KJ, Bramlage MP, Ellenson LH et al: Immunoprofile of the endometrium, endocervix and ovary with mucinous differentiation. Appl Immunohistochem Mol Morphol. 17:8–11, 2009. 94. Sullivan LM, Smolkin ME, Froerson HF et al: Comprehensive evaluation of CDX2 in invasive cervical adenocarcinomas: immunopositivity in the absence of overt colorectal morphology. Am J Surg Pathol. 32:1608–1612, 2008. 95. Walts AE, Marchevsky AM: Low cost-effectiveness of CD3/ CD20 immunostains for initial triage of lymphoid-rich effusions: an evidence-based review of the utility of these stains in selecting cases for full hematopathologic workup. Diagn Cytopathol. 40:565–569, 2012. 96. Recine MA, Deavers MT, Middleton LP, et al: Serous carcinoma of the ovary and peritoneum with metastases to the breast and axillary lymph nodes: A potential pitfall. Am J Surg Pathol. 28:1646–1651, 2004. 97. Dabbs DJ, Landreneau RJ, Liu Y, et al: Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg. 73:403–405, 2002. 98. Wick MR, Lillemoe TJ, Copland GT, et al: Gross cystic disease fluid protein-15 as a marker for breast cancer: Immunohistochemical analysis of 690 human neoplasms and comparison with alpha-lactalbumin. Hum Pathol. 20:281–287, 1989. 99. Lee BH, Hecht JL, Pinkus JL, et al: WT1, estrogen receptor, and progesterone receptor as markers for breast or ovarian primary sites in metastatic adenocarcinoma to body fluids. Am J Clin Pathol. 117:745–750, 2002. 100. Fiel MI, Cernaianu G, Burstein DE, et al: Value of GCDFP-15 (BRST-2) as a specific immunocytochemical marker for breast carcinoma in cytologic specimens. Acta Cytol. 40:637–641, 1996. 101. Yan Z, Gidley J, Horton D, et al: Diagnostic utility of mammaglobin and GCDFP-15 in the identification of metastatic breast carcinoma in fluid specimens. Diagn Cytopathol. 37:475–478, 2009. 102. Striebel JM, Dacic S, Yousem SA: Gross cystic disease fluid protein-(GCDFP-15): Expression in primary lung adenocarcinoma. Am J Surg Pathol. 32:426–432, 2008. 103. Bhargava R, Beriwal S, Dabbs DJ: Mammaglobin vs GCDFP-15: An immunohistologic validation survey for sensitivity and specificity. Am J Clin Pathol. 127:103–113, 2007. 104. Ciampa A, Fanger G, Khan A, et al: Mammaglobin and CRxA-01 in pleural effusion cytology: Potential utility of distinguishing metastatic breast carcinomas from other cytokeratin 7-positive/ cytokeratin 20-negative carcinomas. Cancer. 102:368–372, 2004. 105. Dabbs D, Bhargava R, Chivukula M: The utility of p120 catenin in the diagnosis of lobular Carcinoma of the breast. Am J Surg Pathol. 31:427–437, 2007. 106. Kalogeraki A, Garbagnati F, Santinami M, et al: E-cadherin expression on fine needle aspiration biopsies of breast invasive ductal carcinomas and its relationship to clinicopathologic factors. Acta Cytol. 47:363–367, 2003. 107. Hylander B, Repasky E, Shrikant P, et al: Expression of Wilms tumor gene (WT1) in epithelial ovarian cancer. Gynecol Oncol. 101:12–17, 2006. 108. Hwang H, Quenneville L, Yaziji H, et al: Wilms tumor gene product: Sensitive and contextually specific marker of serous carcinomas of ovarian surface epithelial origin. Appl Immunohistochem Mol Morphol. 12:122–126, 2004. 109. Chivukula M, Niemeier LA, Edwards R, et al: Carcinomas of Distal Fallopian Tube and Their Association with Tubal Intraepithelial Carcinoma: Do They Share a Common “Precursor” Lesion? Loss of Heterozygosity and Immunohistochemical
853.e3
Analysis Using PAX 2, WT-1, and P53 Markers. ISRN Obstet Gynecol. 858647, 2011. 110. Karabakhtsian R, Bhargava R: WT-1 expression in primary invasive breast carcinoma: Occasional expression in micropapillary and mucinous subtypes. Mod Pathol. 20(Suppl 2):38A (abstract 151), 2007. 111. Woodard AH, Yu J, Dabbs DJ, et al: NY-BR-1 and PAX8 immunoreactivity in breast, gynecologic tract, and other CK7+ carcinomas: potential use for determining site of origin. Am J Clin Pathol. 136:428–435, 2011. 112. Laury AR, Perets R, Piao H, et al: A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 35:816–826, 2011. 113. Kato N, Toukairin M, Asanuma I, et al: Immunocytochemistry for hepatocyte nuclear factor-1beta (HNF-1beta): A marker for ovarian clear cell carcinoma. Diagn Cytopathol. 35:193–197, 2007. 114. Kao YC, Lin MC, Lin WC, et al: Utility of hepatocyte nuclear factor-1β as a diagnostic marker in ovarian carcinomas with clear cells. Histopathology. 61:760–768, 2012. 115. Agoff SN, Lin P, Morihara J, et al: p16(INK4a) expression correlates with degree of cervical neoplasia: A comparison with Ki-67 expression and detection of high-risk HPV types. Mod Pathol. 16:665–673, 2003. 116. Hariri J, Øster A: The negative predictive value of p16INK4a to assess the outcome of cervical intraepithelial neoplasia 1 in the uterine cervix. Int J Gynecol Pathol. 26:223–228, 2007. 117. Schorge JO, Lea JS, Elias KJ, et al: P16 as a molecular biomarker of cervical adenocarcinoma. Am J Obstet Gynecol. 190:668–673, 2004. 118. Bibbo M, Klump WJ, DeCecco J, et al: Procedure for immunocytochemical detection of P16INK4A antigen in thin-layer, liquid-based specimens. Acta Cytol. 46:25–29, 2002. 119. Saqi A, Pasha TL, McGrath CM, et al: Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia. Diagn Cytopathol. 27:365–370, 2002. 120. Bibbo M, DeCecco J, Kovatich AJ: P16INK4A as an adjunct test in liquid-based cytology. Anal Quant Cytol Histol. 25:8–11, 2003. 121. Yoshida T, Fukuda T, Sano T, et al: Usefulness of liquid-based cytology specimens for the immunocytochemical study of p16 expression and human papillomavirus testing: A comparative study using simultaneously sampled histology materials. Cancer. 102:100–108, 2004. 122. Pientong C, Ekalaksananan T, Kongyingyoes B, et al: Immunocytochemical staining of p16INK4a protein from conventional Pap test and its association with human papillomavirus infection. Diagn Cytopathol. 31:235–242, 2004. 123. Wentzensen N, Bergeron C, Cas F, et al: Triage of women with ASCUS and LSIL cytology: Use of qualitative assessment of p16INK4a positive cells to identify patients with highgrade cervical intraepithelial neoplasia. Cancer. 111:58–66, 2007. 124. Wentzensen N, Bergeron C, Cas F, et al: Evaluation of a nuclear score for p16INK4a-stained cervical squamous cells in liquidbased cytology samples. Cancer. 105:461–467, 2005. 125. Pantanowitz L, Florence RR, Goulart RA, et al: Trichomonas vaginalis P16 immunoreactivity in cervicovaginal Pap tests: A diagnostic pitfall. Diagn Cytopathol. 33:210–213, 2005. 126. Duncan L, Jacob S, Hubbard E: Evaluation of p16INK4a as a diagnostic tool in the triage of Pap smears demonstrating atypical squamous cells of undetermined significance. Cancer. 114:34–48, 2008. 127. Alameda F, Juanpere N, Pijuan L, et al: Value of p16(INK4a) in the diagnosis of low-grade urothelial carcinoma of the urinary bladder in urinary cytology. Cancer Cytopathol. 120:276–282, 2012. 128. Begum S, Gillison ML, Ansari-Lari MA, et al: Detection of human papillomavirus in cervical lymph nodes: a highly effective strategy for localizing site of tumor origin. Clin Cancer Res. 9:6469–6475, 2003. 129. Zhang MQ, El-Mofty SK, Dávila RM: Detection of human papillomavirus-related squamous cell carcinoma cytologically and by in situ hybridization in fine-needle aspiration biopsies of
853.e4
Immunocytology
cervical metastasis: a tool for identifying the site of an occult head and neck primary. Cancer. 114:118–123, 2008. 130. Kelly D, Kincaid E, Fansler Z, et al: Detection of cervical highgrade squamous intraepithelial lesions from cytologic samples using a novel immunocytochemical assay (ProEx C). Cancer. 108:494–500, 2006. 131. Oberg TN, Kipp BR, Vrana JA, et al: Comparison of p16INK4a and ProEx C immunostaining on cervical ThinPrep cytology and biopsy specimens. Diagn Cytopathol. 38:564–572, 2010. 132. Keating JT, Cviko A, Riethdorf S, et al: Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia. Am J Surg Pathol. 25:884–891, 2001. 133. Willmore-Payne C, Layfield LJ, Holden JA: c-KIT mutation analysis for diagnosis of gastrointestinal stromal tumors in fine needle aspiration specimens. Cancer. 105:165–170, 2005. 134. Miliaras D, Karasavvidou F, Papanikolaou A, et al: KIT expression in fetal, normal adult, and neoplastic renal tissues. J Clin Pathol. 57(5):463–466, 2004. 135. Espinosa I, Lee CH, Kim MK, et al: A novel monoclonal antibody against DOG1 is a sensitive and specifi c marker for gastrointestinal stromal tumors. Am J Surg Pathol. 32(2):210–218, 2008. 136. Vocaturo A, Novelli F, Benevolo M, et al: Chromogenic in situ hybridization to detect HER 2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: A sensitive and practical method in the trastuzumab era. Oncologist. 11:878–886, 2006. 137. Bozzetti C, Nizzoli R, Guazzi A, et al: HER 2/neu amplification detected by fluorescence in situ hybridization in fine needle aspirates from primary breast cancer. Ann Oncol. 13:1398–1403, 2002. 138. Maloney DG: Anti-CD20 antibody therapy for B-cell lymphomas. N Engl J Med. 366:2008–2016, 2012. 139. Dillman RO: Radioimmunotherapy of B-cell lymphoma with radiolabelled anti-CD20 monoclonal antibodies. Clin Exp Med. 6:1–12, 2006.
140. Duman BB, Sahin B, Ergin M, et al: Loss of CD20 antigen expression after rituximab therapy of CD20 positive B cell lymphoma (diffuse large B cell extranodal marginal zone lymphoma combination): a case report and review of the literature. Med Oncol. 29:1223–1226, 2012. 141. Jang MJ, Lee DG, Chung MJ: Utility of thyroid transcription factor-1 and cytokeratin 7 and 20 immunostaining in the identification of origin in malignant effusions. Anal Quant Cytol Histol. 23:400–404, 2001. 142. Pomjanski N, Grote HJ, Doganay P, et al: Immunocytochemical identification of carcinomas of unknown primary in serous effusions. Diagn Cytopathol. 33:309–315, 2005. 143. Ascoli V, Taccogna S, Scalzo CC, et al: Utility of cytokeratin 20 in identifying the origin of metastatic carcinomas in effusions. Diagn Cytopathol. 12:303–308, 1995. 144. Pereira TC, Saad RS, Liu Y, et al: The diagnosis of malignancy in effusion cytology: A pattern recognition approach. Adv Anat Pathol. 13:174–184, 2006. 145. Wu M, Szporn AH, Zhang D, et al: Cytology applications of p63 and TTF-1 immunostaining in differential diagnosis of lung cancers. Diagn Cytopathol. 33:223–227, 2005. 146. Schutte M, Hruban RH, Hedrick L, et al: DPC4 gene in various tumor types. Cancer Res. 56:2527–2530, 1996. 147. Hahn SA, Bartsch D, Schroers A, et al: Mutations of the DPC4/ Smad4 gene in biliary tract carcinoma. Cancer Res. 58:1124– 1126, 1998. 148. Broghamer WL, Jr, Richardson ME, Faurest S, et al: Prostatic acid phosphatase immunoperoxidase staining of cytologically positive effusions associated with adenocarcinomas of the prostate and neoplasms of undetermined origin. Acta Cytol. 29:274–278, 1985. 149. Beaty MW, Fetsch P, Wilder AM, et al: Effusion cytology of malignant melanoma. A morphologic and immunocytochemical analysis including application of the MART-1 antibody. Cancer. 81:57–63, 1997.
C H A P T E R 2 2
IMMUNOHISTOLOGY OF PEDIATRIC NEOPLASMS CHERYL M. COFFIN, MARIANA M. CAJAIBA, PAMELA LYLE, HERNAN CORREA, JENNIFER O. BLACK
Overview 854 Biology of Antigens and Antibodies 854 Specific Tumors 855 Summary 876
Overview Solid neoplasms of childhood and adolescence comprise a diverse group of diagnostically challenging entities with the basic morphologic themes of round cell, spindle cell, and epithelioid tumors. Immunohistochemistry (IHC) and molecular diagnostic tests have greatly improved our ability to classify these lesions.1-3 Ancillary diagnostic techniques have become increasingly important in the diagnosis and evaluation of recurrent or metastatic disease and, in some cases, in genomic or prognostic classification. In addition, ancillary diagnostic tests must be interpreted in the context of the light microscopic findings, and specimen adequacy is a critical factor. IHC is a valuable tool, although it has significant pitfalls in specific instances.4 Tissue fixation; necrosis; focality of marker expression; artifactual changes on very small specimens, such as core needle biopsies; and other technical factors may influence the IHC results. Techniques such as flow cytometry for leukemias and lymphomas, electron microscopy (EM), and molecular testing5 may be necessary to support the diagnosis or provide prognostic information. Therefore tumor protocols for handling specimens and procuring tissues can provide important guidelines for pathologic evaluation.6-10 This chapter reviews the diagnostic evaluation, IHC findings, and genomic and prognostic aspects of pediatric and adolescent solid neoplasms including neuroblastoma (NB) and related neuroblastic tumors, rhabdomyosarcoma (RMS), Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), desmoplastic small round cell tumor (DSRCT), malignant rhabdoid tumor (MRT), Wilms tumor (WT), and osteosarcoma (OS). In 854
many cases, the combination of the clinical and radiologic presentation and the light microscopic appearance are sufficient for diagnosis. In other cases, the tumor may have a predominantly round or spindle cell pattern, which leads to a differential diagnosis that depends on the clinical, radiologic, and morphologic findings. For example, a tumor in an infant with an adrenal mass, elevated serum and urine catecholamine levels, and the microscopic finding of neuroblasts in a background of neuropil with calcification and thin fibrovascular septa can be confidently diagnosed as NB. On the other hand, a primitive RMS may require IHC and/or molecular analysis to reach a diagnosis. The decision about when to order special tests and which to select depends on the complexity of the tumor and the individual pathologist’s experience. IHC is often the first step toward refining or confirming the diagnosis. If this results in unexpected or contradictory findings, additional IHC tests may be obtained, and cytogenetic or molecular genetic testing can be considered. Use of a panel of IHC stains, rather than overreliance on a single antibody, is an important diagnostic principle.
Biology of Antigens and Antibodies Principal Antibodies Many different antibodies are used for the IHC evaluation of pediatric solid neoplasms. The more generally utilized antibodies are discussed in other chapters. This section summarizes antibodies with particular importance for the tumors covered in this chapter; these include myogenic transcriptional regulatory proteins, CD99, the FLI1 protein, the Wilms tumor 1 (WT1) protein, and the SMARCB1 (formerly hSNF5/INI1) protein. MYOGENIC REGULATORY PROTEINS
Myogenin (myf-4), MyoD1, myf-5, and mrf-4herculin/myf-6 comprise a family of myogenic transcriptional regulatory proteins involved in skeletal
Specific Tumors
muscle differentiation that are expressed earlier than structural proteins, such as desmin or actin. Myogenin and MyoD1 are expressed in RMS, even those less differentiated and that lack definitive morphologic evidence of rhabdomyoblastic differentiation, such as strap cells with cross-striations.11-13 Numerous studies have demonstrated the specificity of myogenin and MyoD1 as markers for RMS.11-17 This contrasts with musclespecific actin (MSA) and desmin, which can be found in many different neoplasms, including skeletal muscle, smooth muscle, and fibroblastic-myofibroblastic tumors. Cytoplasmic staining for MyoD1 can be observed in many tumors, so it is important to adhere strictly to the requirement for nuclear staining for interpretation of MyoD1 stains. Tumors that display skeletal muscle differentiation—such as Wilms tumor, ectomesenchymoma, and malignant peripheral nerve sheath tumor (MPNST) with divergent differentiation—are reactive for myogenic transcriptional regulatory proteins.18 Nonneoplastic skeletal muscle fiber nuclei can stain positively for myogenin.16 Different patterns of staining for myogenin in different subtypes of RMS have also been observed.19 CD99 (p30/32 mic2)
This group of antibodies detects a transmembrane glycoprotein that is the product of the pseudoautosomal CD99 gene on chromosome Xp22.32 pter and chromosome Yq11 pter20 and is unrelated to EWSR1 gene rearrangements, although the protein may contribute to oncogenesis via inhibition of neural differentiation.21 CD99—as detected with a variety of antibodies, including O13, 12E7, and HBA-71—is expressed by 85% to 95% of ES/PNETs and demonstrates strong membranous staining in this context.22-24 Absence of staining for CD99 may be an indication to perform additional studies, such as further IHC stains or molecular analysis to support the diagnosis of ES/PNET and to exclude other small round blue cell tumors. CD99 is also positive in acute lymphoblastic leukemia/lymphoma, acute myelogenous leukemia, granulocytic sarcoma, mesenchymal chondrosarcoma, synovial sarcoma, vascular tumors, and other neoplasms.25-30 Antigen retrieval techniques may result in increased expression of CD99 in a variety of tumors.29 Caution is warranted in distinguishing between ES/PNET and acute lymphoblastic leukemia/lymphoma in similar tumors because of the IHC overlap, especially in cases with unusual clinical presentations.25,31 CD99 is especially useful in the distinction between ES/PNET and NB.32 The potential for many different types of tumors to express CD99 emphasizes the importance of a panel of antibodies in differential diagnosis of small round cell tumors. FLI1
The FLI1 protein is overexpressed in ES/PNETs, which contain the EWSR1-FLI1/1 fusion gene as a result of the translocation t(11;22)(q24;q12);33 it may also be detected in ES/PNET with other gene fusions. Nuclear FLI1 immunoreactivity is found in approximately 70%
855
of ES/PNETs but is also observed in nearly 90% of lymphoblastic lymphomas and in vascular tumors.34,35 This antibody may be useful as part of a panel for evaluation of potential ES/PNETs, although the overlap with lymphoblastic lymphomas and vascular neoplasms must be kept in mind. WILMS TUMOR 1
An antibody to the carboxy-terminal (C-terminal) region of the Wilms tumor gene, WT1, is useful in recognition of DSRCT, which shows strong nuclear staining with a C-terminal WT1 antibody.36-38 Wilms tumor and RMS can also show nuclear reactivity for WT1.27,37,38 We have observed more than occasional examples of other small round cell tumors, including ES/PNETs, with reactivity for WT1, so caution must be exercised in the use of this antibody. SMARCB1
Atypical teratoid/rhabdoid tumor (AT/RT) of the kidney, soft tissues, and central nervous system (CNS) frequently demonstrates deletion and mutation of the SMARCB1 gene, with decreased or absent protein expression. An antibody to the SMARCB1 protein can be used to assess SMARCB1 loss, characterized by absence of immunoreactivity in contrast to the functional gene with positive immunoreactivity in normal tissues and other neoplasms.39-42 However, an increasing variety of tumors are found to have SMARCB1 protein loss, so the IHC stain must be interpreted in the context of the overall clinical and pathologic findings.
Specific Tumors Neuroblastoma and Neuroblastic Tumors The neuroblastic tumor family consists of NB, ganglioneuroblastoma (GNB), and ganglioneuroma (GN), which are derived from migrating neuroectodermal cells of the primitive neural crest.43 Neuroblastic tumors are the most common solid extracranial malignant neoplasm in the first 2 years of life, and most occur in the first decade. The most common site of origin is the adrenal medulla, followed by the paraspinal ganglia in the abdomen, thorax, neck, and pelvis. Neuroblastic tumors are classified histologically according to the extent of neuroblastic differentiation and the quantity of schwannian stroma. The current classification, as defined by the International Neuroblastoma Pathology Committee, divides neuroblastic tumors into three categories: neuroblastoma (NB, schwannian stroma poor); ganglioneuroblastoma (GNB, schwannian stroma rich); and ganglioneuroma (GN, schwannian stroma dominant). Each type is further subdivided according to the amount of schwannian stroma, the degree of neuroblastic differentiation, and the gross and microscopic interface between these components.10,44,45 Classic NB is a cellular, stroma-poor tumor composed of small round cells with varying degrees
856
Immunohistology of Pediatric Neoplasms
Figure 22-1 Neuroblastoma with differentiation consists of small round blue cells and occasional larger cells with more vesicular nuclei and more abundant eosinophilic cytoplasm (hematoxylin and eosin, ×200).
a sharp transition from the surrounding schwannianrich stroma created by a pushing border or a pseudocapsule. These nodules consist of varying proportions of poorly differentiated and differentiating neuroblasts with scant stroma.10,47 Ganglioneuroma (GN) represents the most mature end of the spectrum and consists of abundant schwannian stroma with sparse, randomly arranged, mature ganglion cells; a few isolated differentiating neuroblasts or immature ganglion cells may be present without nest or nodule formation. Although NB has a neuronal phenotype, it does not have a specific IHC profile. Neuroblasts are reactive for a variety of markers that characterize neuronal differentiation, including neuron-specific enolase (NSE; Fig. 22-2), CD57, CD56, protein gene product 9.5 (PGP9.5), synaptophysin (Fig. 22-3), chromogranin, and neurofilament protein (NFP).10,44,45,48-50 Some of these markers are highly sensitive but lack specificity, such as NSE, CD56, CD57, and PGP9.5. On the other hand, the more specific markers—such as synaptophysin, chromogranin, and NFP—are less sensitive. NB84 is a very sensitive IHC marker that can aid in the identification of NB, including metastatic NB. Its specificity is limited by the fact that NB84 is expressed in many cell types and in other round cell tumors, such as ES/ PNET, DSRCT, and medulloblastoma.51 Tyrosine hydroxylase (TH) is important in the biosynthetic pathway of norepinephrine, and TH IHC reactivity is sensitive but not highly specific for NB, because it is present in other neural tissues, the gastrointestinal (GI)
of neuroblastic differentiation; Homer-Wright rosette formation; delicate fibrillary neuritic cell processes known as neuropil, differentiating neuroblasts with or without recognizable ganglion cell differentiation; and scant stroma that consists of thin fibrovascular septa (Fig. 22-1).10,44 The three histologic subtypes of NB are undifferentiated, poorly differentiated, and differentiating NB. Undifferentiated NB is composed of small round cells without identifiable neuroblastic features.45,46 Large cell NB is an aggressive variant of undifferentiated NB with pleomorphic nuclei, prominent nucleoli, and cytoplasm that may have a rhabdoid appearance.46 Poorly differentiated and differentiating NB are distinguished by their neuroblastic differentiation and the presence of neuropil and Homer-Wright rosettes. GNB has two pathologic subtypes, intermixed and nodular. Intermixed GNB is a prognostically favorable stromarich tumor, in which a wide range of primitive and differentiating neuroblasts and ganglion cells form nests intermixed with the schwannian stroma in a random fashion. The schwannian stroma occupies more than 50% of the tumor, and the neuroblastic nodules are not visible grossly.10 Nodular GNB is a biologically aggressive variant characterized by one or more grossly and/ or radiologically identifiable neuroblastic nodules within a stroma-rich background. The well-demarcated, grossly visible nodules are usually red and/or hemorrhagic, but on occasion, they may be pink or tan, and they display
Figure 22-2 Neuron-specific enolase shows diffuse cytoplasmic reactivity in neuroblastoma (immunoperoxidase, ×400).
Specific Tumors
Figure 22-3 Synaptophysin shows strong cytoplasmic reactivity in neuroblastoma (immunoperoxidase, ×400).
tract, and sympathetic ganglia.52 Endocrine markers may occasionally be expressed, especially in the context of secretory diarrhea associated with vasoactive intestinal peptide production by the tumor. The schwannian stroma may be reactive for S-100 protein, and favorable-histology NB typically has more prominent S-100 protein stromal staining.49 NB lacks vimentin expression. A variety of potential prognostic IHC markers have been assessed in NB. Proliferation markers such as Ki-67 and repp86 are more highly expressed in advanced neuroblastic tumors with unfavorable prognostic features, and their absence may be associated with longer survival.53-55 The cell surface glycoprotein CD44, an adhesion/homing molecule, is expressed in favorablehistology NB, and nonexpression is associated with unfavorable histology.56 CD44 variants such as CD44v4-v7 and CDd44v6-v9 play a role in tumor progression.57 High levels of tyrosine receptor kinase A (TRK-A) expression with or without TRK-C expression are associated with favorable histology and favorable outcome, and unfavorable tumors express TRK-B and its ligand brain-derived neurotrophic factor (BDNF)43,58,59; however, these markers are not in wide clinical use. The presence of reactivity for anaplastic lymphoma kinase (ALK) protein in more than 90% of NB is a potential diagnostic pitfall in distinguishing NB from anaplastic large cell lymphoma (ALCL) and some RMS.60 However, ALK protein expression does not distinguish between NB with or without an ALK gene mutation, so the IHC stain is not useful for clinical
857
purposes.61,62 The ALK inhibitor crizotinib is effective against NB with the R1275Q ALK mutation. Many cytogenetic and molecular genetic abnormalities have been described in NB, but none has diagnostic specificity.58,59 The MYCN gene encodes a transcriptional regulator, and MYCN amplification is a reliable marker of poor outcome in NB.63 MYCN amplification can be detected by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) on formalinfixed paraffin-embedded (FFPE) tissue.64 The DNA index is a prognostic marker in patients young than 2 years who have disseminated NB; triploidy is favorable and near-diploidy is unfavorable.65 Deletion or rearrangement of chromosome 1p36-38, present in 25% to 35% of NBs, corresponds to loss of the tumor suppressor gene CHD5, a chromatin-remodeling gene active in neural tissue. The 1p36.2 deletion is associated with increased risk of relapse in patients with localized disease. Gain of chromosome 17q, found in 80% of NB, is often present as an unbalanced translocation with chromosome 1p or 11q.58,59,65,66 Gain of chromosome 17q is associated with a poor outcome, whereas gain of all of chromosome 17 is associated with a favorable prognosis.67,68 Deletion of 11q, present in 15% to 22% of NBs, is often found in high-stage tumors without MYCN amplification and with intact 1p, and it is associated with a reduced progression-free survival. Loss of 3p is often found in association with deletion of 11q and tends to occur in older patients whose tumors lack MYCN amplification or 1p deletion.66 The International Neuroblastoma Risk Group (INRG) classification system uses clinical and biologic features to stratify NB into favorable and unfavorable prognostic groups. Clinical prognostic factors are age at diagnosis (dichotomized at 18 months) and stage of disease before treatment. Biologic factors include histologic type and genetic features such as MYCN amplification status, ploidy, loss of heterozygosity of 1p and 11q, and other structural and numerical chromosomal aberrations and gene expression signatures and polymorphisms.65,69-73 The most important applications of IHC in NB are to distinguish undifferentiated NB from its mimics, detect persistent or metastatic disease, and identify NB in small samples. For undifferentiated NB, the differential diagnosis includes ES/PNET, lymphoma/leukemia, RMS, and other blastematous or undifferentiated neoplasms. Markers nonreactive in NB are thus useful in the recognition of other round cell malignant neoplasms; these include desmin, myogenic markers such as myogenin and MyoD1, vimentin, CD45, terminal deoxynucleotidyl transferase (TdT), and CD99. Reactivity for CD45 can be used to distinguish chronic inflammation from NB in small biopsies, posttreatment specimens, or other problematic cases.49 Evaluation of neuronal markers may also be useful for detection of bone marrow metastases.74 NBs are typically negative for vimentin and CD99, which permits distinction from ES/PNET.48 MRT displays reactivity for vimentin, keratins, and epithelial membrane antigen (EMA) in addition to other mesenchymal markers, which aid in the distinction from NB.
858
Immunohistology of Pediatric Neoplasms
KEY DIAGNOSTIC POINTS Neuroblastoma and Neuroblastic Tumors • Neuroblastic tumors express neuronal markers of varying sensitivity and specificity, including NSE, PGP9.5, synaptophysin, chromogranin, NB84, TH, and others. • Neuroblastic tumors typically are nonreactive for vimentin, desmin, myogenic markers such as myogenin and MyoD1, keratins, and CD99. • S-100 protein expression may be seen in the schwannian stroma of differentiating NB, GNB, and GN. • TRK-A expression in NB correlates with favorable prognosis and correlates inversely with MYCN amplification. • ALK is constitutively expressed in NB, therefore ALK IHC is not diagnostically useful.
Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a tumor of primitive skeletal muscle differentiation that arises at all ages, although some subtypes show a greater predilection for certain age groups. In general, RMS shows a bimodal age distribution; half arise in the first decade of life, and a second peak is seen in adolescence. Among children, it is the most common soft tissue sarcoma and accounts for half of all childhood sarcomas and for 5% to 10% of pediatric solid tumors.75,76 RMS is rare in adults. Although derived from skeletal muscle, RMS does not always arise within mature skeletal muscle and is seen in a wide variety of locations, ranging from visceral organs, mucosal surfaces such as the urinary bladder and vagina, to anywhere in the musculoskeletal system, even in the skin and CNS.76,77 Multiple histologic subtypes of RMS have been well defined and have biologic and prognostic significance, and a unifying classification scheme has been proposed by the Intergroup Rhabdomyosarcoma Study, subsequently adapted by the World Health Organization (WHO) for inclusion in the WHO classification of tumors.76,78-80 Major categories include embryonal RMS (ERMS), including the botryoid subtype; alveolar RMS (ARMS); pleomorphic RMS (PRMS); and spindle cell/ sclerosing RMS (SRMS). With the exception of pleomorphic RMS, which is seen almost exclusively in adults, the other types can occur at any age. ERMS encompasses a broad morphologic spectrum of tumors that collectively account for half of RMS. Of these, one third occur in children younger than 5 years of age, and ERMS comprises 20% of adult RMS.81 These tumors occur more commonly in the head and neck and in genitourinary sites. ERMS is a variably cellular small round cell neoplasm with minimal to abundant loose myxoid stroma (Fig. 22-4). The lesional cells may demonstrate mild nuclear pleomorphism with occasional nucleoli and multinucleation. A variable number of rhabdomyoblastic cells may show eccentric cytoplasmic eosinophilic globules that ultrastructurally contain elements of the skeletal muscle Z-bands (rhabdomyoblasts). The cytoplasm may be elongated and may contain cross-striations that are visible with light
Figure 22-4 Embryonal rhabdomyosarcoma consists of small round, polygonal, and elongated tumor cells with variable amounts of eosinophilic cytoplasm and hyperchromatic nuclei (hematoxylin and eosin, ×200).
microscopy; these cells have been referred to as “strap” or “tadpole” cells. The degree of differentiation ranges from tumors with abundant rhabdomyoblasts and strap cells to very undifferentiated tumors with primitive morphologic characteristics. Botryoid RMS is considered a subtype of ERMS, although it has distinctive morphologic and prognostic features. Botryoid RMS occurs with greatest frequency in children younger than 5 years and arises in submucosal sites, especially in the genitourinary tract. The tumor forms an exophytic polypoid mass that bulges from the visceral surface in discrete tumor lobules to create soft, mucoid, clustered grapelike structures covered by epithelium. Botryoid RMS is distinguished histologically from other ERMS by the presence of a condensed “cambium” layer of tumor cells immediately beneath the epithelial surface. ARMS accounts for 31% of RMS (20% of pediatric RMS) and generally is associated with an unfavorable clinical course.82 It can occur at any age but is uncommon among very young children, with highest frequency in teens and young adults. ARMS arises most commonly in the extremities but is also seen in the paraspinal, perineal, and paranasal sinus regions; in the female breast; and in a disseminated form.76,83 ARMS classically demonstrates a distinct pattern of anastomosing fibrovascular septa lined by tumor cells, with dyscohesion of tumor cells between the septa, which creates a nested or alveolar pattern with a “falling apart” of tumor cells
Specific Tumors
(Fig. 22-5). Most ARMS falls into the category of small round blue cell tumors, and individual cells are round to polygonal with scant cytoplasm, although crossstriations may be observed. The nuclei are typically round and uniform and may occasionally form wreathlike multinucleated structures. SRMS is an uncommon variant of RMS that demonstrates a predominantly spindle cell pattern and accounts for 5% to 10% of RMS. Both adults and children are affected, although with a male predilection.84-90 The paratesticular region is a favored site in children, and other common locations include periorbital and other deep soft tissues of the head and neck or somatic soft tissue of the extremities. SRMS in children has a favorable prognosis with a 95% 5-year survival rate; SRMS in adults has a 40% to 50% risk of recurrence and metastasis. SRMS is often circumscribed but not encapsulated; it displays a gritty, gray and white whorled cut surface and variable necrosis and hemorrhage. Microscopically the tumor has infiltrative borders and a fascicular or storiform spindle cell pattern. The cells have elongated nuclei with vesicular chromatin, inconspicuous nucleoli, and scant cytoplasm, although scattered rhabdomyoblasts may be seen. Nuclear atypia, hyperchromasia, and mitotic figures, including atypical forms, are common. Prominent stromal hyalinization characterizes sclerosing RMS, especially in tumors that occur in the extremities. The matrix can be so dense as to be confused with cartilage or osteoid and may account for as much as 40% to 60% of the tumor mass. Cytogenetic data is limited in SRMS, with only one t(2;13)-positive
case described and others with nonspecific genetic abnormalities.87 Pleomorphic RMS (PRMS) is an aggressive form of RMS seen almost exclusively in adults.91,92 It can be morphologically indistinguishable from high-grade undifferentiated pleomorphic sarcoma, except that PRMS displays myogenic differentiation with spindled and round cells with bizarre cellular atypia.92 Anaplasia, defined as nuclear hyperchromasia, pleomorphism (with tumor size variation of at least 3 times), and atypical mitotic figures has been described in ERMS, SRMS, and ARMS and may be associated with a worse prognosis than tumors without anaplasia.93 Genomic amplification and p53 overexpression have been associated with anaplasia, whether of the ARMS or ERMS subtype.94 Other rare morphologic findings in RMS include clear cell and lipid-rich types that ultrastructurally show increased cytoplasmic content of glycogen and lipid, respectively.95,96 A recently described morphologic variant known as epithelioid rhabdomyosarcoma resembles a poorly differentiated carcinoma or melanoma and is more prevalent in older male patients.97 Histologic characteristics include infiltrative sheetlike growth with tumor cells, showing cells with abundant cytoplasm, vesicular chromatin, prominent nucleoli, extensive necrosis, and high mitotic activity. These tumors are aggressive, and early metastasis is common. The diagnostic IHC profile of RMS (Fig. 22-6) is defined by its myogenic properties and centers on the use of antibodies for desmin (Figs. 22-7 and 22-8), muscle-specific actin (MSA; Figs. 22-9 and 22-10), myogenin (Figs. 22-11 and 22-12), and MyoD1 (Fig. 22-13).16,76,98,99 Myogenin and MyoD1 protein expression characterizes cells committed to myogenesis in their earliest phase. A nuclear staining pattern for myogenin and MyoD1 has high sensitivity and specificity for RMS (90% and 95%). MyoD1 may also be associated with nonspecific cytoplasmic staining in RMS and in other tumors, but nuclear staining in the specific setting of RMS is its most useful application. MyoD1 IHC is performed optimally on freshly cut tissue 100% 95% 93%
93%
93%
86% Positive
Figure 22-5 Alveolar rhabdomyosarcoma displays fibrovascular septa lined by small round tumor cells surrounding spaces with dispersed individual tumor cells (hematoxylin and eosin, ×200).
859
46%
0% Vimentin Myogenin
MyoD
MSA
Desmin Myoglobin
Figure 22-6 An immunohistogram of rhabdomyosarcoma. MSA, Muscle-specific actin.
860
Immunohistology of Pediatric Neoplasms
Figure 22-7 Desmin displays cytoplasmic reactivity in embryonal rhabdomyosarcoma and highlights elongated straplike cells (immunoperoxidase, ×200).
Figure 22-8 Desmin displays strong cytoplasmic reactivity in alveolar rhabdomyosarcoma (immunoperoxidase, ×400).
Figure 22-9 Muscle-specific actin displays cytoplasmic reactivity and highlights spindled and rhabdomyoblastic cells in embryonal rhabdomyosarcoma (immunoperoxidase, ×400).
Figure 22-10 Muscle-specific actin demonstrates diffuse cytoplasmic reactivity in alveolar rhabdomyosarcoma (immunoperoxidase, ×400).
Specific Tumors
Figure 22-11 Myogenin decorates nuclei and highlights the architecture of alveolar rhabdomyosarcoma (immunoperoxidase, ×400).
Figure 22-12 Myogenin stains variable proportions of nuclei in embryonal rhabdomyosarcoma (immunoperoxidase, ×400).
861
Figure 22-13 MyoD1 displays nuclear reactivity in rhabdomyosarcoma but may be difficult to interpret because of background cytoplasmic staining (immunoperoxidase, ×400).
sections, because antigen reactivity fades when unstained slides are stored at room temperature.11,16 Myogenin tends to be a cleaner stain with less background, but institutional experience varies in this respect; in general, overall performance may vary, depending on the quality and nature of tissue preservation. Staining may be negative, for instance when mercury-based fixatives are used.98 The extent of staining for MyoD1 or myogenin within a given tumor may correlate to some degree with tumor subtype. Diffuse nuclear staining is typical of ARMS, whereas nuclear staining in ERMS may be sparse and variable. Additionally, diffuse nuclear staining with myogenin appears to portend poor survival in pediatric RMS.100 SRMS can show very focal and weak staining for myogenin and desmin, although MyoD1 may stain more strongly in SRMS than these other markers.77,85 Other proteins expressed in RMS that are less specific include vimentin, myosin, actin, creatine kinase, α-actinin, and tropomyosin. Myoglobin is also a very specific marker of late skeletal muscle differentiation, but it is not sensitive and is therefore seldom used. Desmin and MSA are sensitive but not specific and can also be expressed in many benign or malignant smooth muscle, myofibroblastic, or other mesenchymal proliferations.98,101 Additionally, desmin expression may be absent in a small proportion of RMS, particularly in the most poorly differentiated tumors. Recent gene-expression analysis studies have identified genes that are differentially expressed between ARMS and ERMS, some of which may be detected with
862
Immunohistology of Pediatric Neoplasms
new IHC markers that may distinguish among RMS subtypes. These markers include AP2β and P-cadherin for ARMS (translocation positive) and epidermal growth factor receptor (EGFR) and fibrillin 2 as markers of ERMS, all of which showed high specificity (90% and 98% respectively) and lower sensitivity (60% and 64% respectively).102,103 Some prognostic correlations were noted: AP2β and P-cadherin were associated with worse outcomes, and EGFR and fibrillin 2 were associated with significantly better outcomes, matching known ERMS and ARMS subtype prognostic data. However, other studies have suggested that IHC expression of EGFR and ERBB2 may not correlate with actual mutation status or amplification of these genes.104 Additionally, insulin-like growth factors (IGFs) have been implicated as important signaling components in the pathogenesis of RMS; increased expression is seen in all types of RMS, but preferential expression of IGF2 is found in translocation-negative RMS.105 Several prognostic markers may be useful in evaluating RMS, such as Ki-67 proliferation index106 and p53 overexpression.107 These are not in wide clinical use. The genetic aberrations in RMS are varied and complex and correlate to some extent with the histologic subtype. Some ARMS demonstrates translocations that involve the FOXO1 gene (formerly FKHR) on chromosome 13 and either the PAX3 or PAX7 gene, found on chromosomes 2 and 1, respectively.108 Translocations involving PAX3 are more common, whereas PAX7 involvement is seen in a smaller subset of tumors. Not all RMS with ARMS morphology is translocation positive, and evidence suggests that the translocationnegative tumors may behave more like ERMS in terms of outcome and disease progression.109,110 Translocationpositive tumors also tend more commonly to exhibit genomic amplification.94 Amplification of MYCN, an oncogene in 2p24, may occur with either translocation, although it is not clear whether MYCN amplification has prognostic significance for RMS.111 The morphologic and IHC differential diagnosis of RMS must take into account not only other tumors with the capacity for muscle differentiation but also a wide variety of round and spindle cell tumors that simulate the morphology of RMS. Myogenin and MyoD1 expression can be seen in other neoplasms with skeletal muscle differentiation, such as Wilms tumor, pleuropulmonary blastoma, hepatoblastoma, MPNST, some germ cell tumors, and ectomesenchymoma. Another diagnostic pitfall is the presence of entrapped atrophic or regenerating nonneoplastic skeletal muscle fibers in an infiltrative process with expression of myogenin and MyoD1. Both of these myogenic markers are generally negative in other small round blue cell tumors in the differential diagnosis for RMS, including DSRCT, ES/PNET, MRT, NB, epithelioid sarcoma, and leukemia/lymphoma. Nonspecific or aberrant IHC reactivity for a variety of markers can lead to diagnostic difficulty in RMS, especially if interpreted out of the context of histologic features combined with myogenic staining. CD99, smooth muscle actin (SMA), cytokeratin (CK), neuroendocrine markers, CD34, ALK-1, CD117 (KIT), glypican-3, p63, NFP, and S-100 protein all may be
expressed in RMS.1,112-119 CD99 may show cytoplasmic, Golgi, or membranous positivity in RMS. SMA is positive in a small subset of tumors, but other smooth muscle or myofibroblastic proliferations lack nuclear staining of myogenic markers. ARMS may show concomitant CK and neuroendocrine staining, similar to small cell carcinoma, but myogenic markers easily clarify the diagnosis.115 Limited studies of ALK in RMS have shown that ALK staining correlates with amplification and/or upregulation and is more frequent in ARMS, but it is unclear whether ALK plays a role in RMS oncogenesis or whether ALK-inhibitor treatment is efficacious. Nonspecific cytoplasmic staining for placental alkaline phosphatase (PLAP) and WT1 has been described in a high proportion of RMS.120,121 Caveolin 1, expressed in muscle satellite cells, shows increased IHC expression in ERMS, whereas caveolin 3, expressed in differentiating rhabdomyoblasts and more mature muscle fibers, shows increased expression in ERMS.117 Glypican-3, a proteoglycan expressed in multiple visceral malignancies, is expressed in RMS and is negative in mature skeletal muscle and in a wide variety of other soft tissue tumors.118 Staining for p63 strongly highlights Z-band cross-striations in RMS cells,119 but the nuclear staining observed in myoepithelial cells and some carcinomas is absent. PAX genes, a family that encodes paired box transcription factors, are abnormally upregulated in many malignancies, and nuclear PAX5 expression is seen in ARMS but not in other subtypes nor in most other small round blue cell tumors.122 Additionally, weak PAX2 nuclear staining, expressed in Wilms tumor, can also be seen in RMS, especially ARMS.123 CD133 and nestin are stem cell markers that also are positive in RMS.124 KEY DIAGNOSTIC POINTS Rhabdomyosarcoma • RMS is best evaluated with a panel of myogenic markers, including myogenic transcriptional regulatory proteins myogenin and MyoD1 and also desmin. • MyoD1 and myogenin are both highly sensitive and specific, but staining can be focal or sparse in some types of RMS. • Subtypes of RMS are defined by morphologic characteristics recognizable with light microscopy, and the pattern of myogenic staining can also vary according to subtype. • Other diagnostic markers, such as CD99 and cytokeratin, can be expressed in RMS but should not be misinterpreted when the clinical, morphologic, and appropriate IHC information is considered. • Addition of myogenic markers to the diagnostic panel of any small round blue cell tumor in adults and children is important to rule out a diagnosis of RMS, which can occur in any age group and in many different contexts.
Ewing Sarcoma/Primitive Neuroectodermal Tumor Ewing sarcoma/primitive neuroectodermal tumor (ES/ PNET) is a primitive round cell sarcoma that shows
Specific Tumors
Figure 22-14 Ewing sarcoma/primitive neuroectodermal tumor displays cohesive sheets of small to medium-sized round cells with round to oval nuclei, fine chromatin, scant clear cytoplasm, and indistinct cell borders (hematoxylin and eosin, ×200).
100% 92% 86% 71% Positive
varying degrees of neuroectodermal differentiation.125,126 In the past, the diagnoses were separated based on light microscopic, electron microscopic, and IHC features of neuroectodermal differentiation; but in recent years, it has been recognized that ES/PNET is a single entity with a shared clinical course and prognosis and similar groups of molecular genetic abnormalities. ES/PNET is the second most common bone and soft tissue sarcoma in children and adolescents; peak incidence is in the second decade, although it occurs throughout life with a predilection for Caucasians and males (3 : 2 male/female).127,128 In bone, ES/PNET tends to arise in long bones, pelvis, and ribs.125 It can also occur in superficial or deep soft tissues, as a disseminated neoplasm without an obvious primary site, or in organs such as the kidney.129-131 The tumor is a tan-gray color and is frequently a necrotic, hemorrhagic mass. Occasional cases are associated with a peripheral nerve. The histologic spectrum of ES/PNET ranges from a neoplasm composed of uniform small round cells with round nuclei, fine chromatin, scant cytoplasm, and indistinct cell borders to a neoplasm with larger, more irregular cells with irregular nuclear contours, pseudorosettes, a nesting pattern, and even spindle cell morphology (Fig. 22-14). Geographic zones of necrosis are frequently observed, with preserved perivascular clusters of tumor cells. Ultrastructurally, ES/PNET shows a similar spectrum, from relatively undifferentiated mesenchymal cells to neural differentiation.132
863
49% 46%
0% CD99
Vimentin
Fli-1
Syn
NSE
Figure 22-15 An immunohistogram of Ewing sarcoma/primitive neuroectodermal tumor. NSE, Neuron-specific enolase; Syn, synaptophysin.
The IHC profile of ES/PNET is illustrated in Figure 22-15, compiled from several series.32,133-135 The typical IHC profile includes reactivity for vimentin and CD99 (O13, HBA-71, mic-2) and variable reactivity for NSE, CD57, synaptophysin, and CK. Rare cases stain positively for desmin and glial fibrillary acidic protein (GFAP), but ES/PNET does not typically stain for leukocyte common antigen (LCA) or actins.20,49,134,136,137 Expression of neural markers does not correlate with outcome.133 CD99 typically displays a membranous staining pattern (Fig. 22-16) and was initially thought to be a highly specific marker for ES/PNET. However, it is now recognized that the specificity is limited, although its sensitivity ranges from 84% to nearly 100% in ES/PNET.23,24,30,32,138 Recently, the membrane protein caveolin 1 (CAV1) has been described as a sensitive marker (96% positivity) for Ewing family tumors139,140 and was expressed in exceptionally rare CD99-negative genetically confirmed cases.139 Further study of CAV1 is required to determine its specificity for ES/PNET. NSE is sensitive but not specific (Fig. 22-17). A translocation involving the EWSR1 gene on chromosome 22q12 and other partners, most frequently the FLI1 gene on chromosome 11q24, is typical for ES/ PNET, and several different subtypes of EWSR1/FLI1 fusion transcripts are possible.141-146 Other fusion partners for EWSR1 include ERG, ETV1 (formerly E1AF), FEV, and other genes.147-149 These genetic abnormalities are detectable by conventional cytogenetics, FISH, and reverse transcription PCR (RT-PCR).136,148,149,150-154 Exceptional cases show variant translocations that involve the FUS gene rather than EWSR1.155,156 Overexpression of the FLI1 protein in a nuclear pattern is observed in 71% to 100% of ES/PNETs (Fig. 22-18)34,35,157 and may be detected in ES/PNETs with gene fusions other than the EWSR1/FLI1 transcript. Unfortunately, a number of other mimics of ES/PNET can also exhibit FLI1 reactivity by IHC, including lymphoblastic
864
Immunohistology of Pediatric Neoplasms
Figure 22-16 CD99 reactivity in Ewing sarcoma/primitive neuroectodermal tumor typically shows a strong membranous staining pattern (immunoperoxidase, ×400).
Figure 22-17 Neuron-specific enolase is frequently positive in Ewing sarcoma/primitive neuroectodermal tumor but can be seen in many other tumors (immunoperoxidase, ×400).
Figure 22-18 FLI1 protein expression with nuclear reactivity is found in Ewing sarcoma/primitive neuroectodermal tumor but can also be observed in other small round blue cell tumors, particularly lymphoblastic lymphoma (immunoperoxidase, ×400).
lymphoma in nearly 90% of cases, myeloid neoplasms, DSRCT, malignant melanoma, Merkel cell carcinoma, synovial sarcoma, some carcinomas, and vascular proliferations and neoplasms such as benign hemangioma, angiosarcoma, epithelioid hemangioendothelioma, glomus tumor, and Kaposi sarcoma. We have also seen cases of RMS, particularly the alveolar subtype, with nuclear FLI1 protein reactivity. When the morphology is characteristic, the diagnosis of ES/PNET can be made on the basis of light microscopy and IHC reactivity for CD99 and FLI1 protein with absence of lymphoblastic, epithelial, and myogenic markers.158 Others have advocated for more routine use of ancillary molecular studies for unequivocal identification of ES/PNET,148,149,159 but current evidence indicates that genetic confirmation is not required, unless unusual morphologic variants of ES/PNET are encountered such as epithelioid, spindle cell, or desmin-positive tumors.136,158,160 Recently, a group of Ewing-like sarcomas has been identified with a translocation that involves chromosomes 19 and 4 with a CIC and DUX4 gene fusion, and these rare tumors have pathologic features nearly identical to Ewing sarcomas with EWSR1 gene rearrangements.161 This emphasizes that even with the advances of the past several decades, the understanding of this group of tumors continues to evolve. Prognostic and biologic IHC markers for ES/PNET include p53 and p16. Overexpression of p53 by IHC has been associated with a poor outcome,162,163 whereas loss of p16 may be associated with more aggressive
Specific Tumors
clinical behavior.164 Although IHC staining for CD117 can be strong and diffuse in some cases of ES/PNET, its therapeutic and genetic significance is unclear.165 A recent study showed that increased numbers of CD68positive tumor-infiltrating macrophages were associated with poorer overall survival.166 Neural differentiation or neural marker expression by light microscopy, IHC, or ultrastructure is not prognostically significant.134,167-169 Some studies have suggested that a type 1 EWSR1/FLI1 fusion transcript may signify an improved survival among patients with localized disease,170 however, improved current treatment protocols appear to have eliminated this prognostic advantage.171 In addition, a large multivariate analysis showed a female survival benefit limited to Caucasian patients.127 The differential diagnosis of ES/PNET encompasses a broad range of round cell neoplasms with varying phenotypes, usually distinguishable by a combination of clinicopathologic and IHC features. Other CD99-positive tumors that mimic ES/PNET include RMS, DSRCT, glial tumors, neuroendocrine tumors, some carcinomas, lymphoblastic lymphoma, other primitive hematolymphoid neoplasms, Wilms tumor, uterine sarcomas, clear cell sarcoma of the kidney, teratoma, synovial sarcoma, osteosarcoma, and mesenchymal chondrosarcoma.23,24,30,36,157,159,172 Fortunately, the blastemal elements of Wilms tumor are not CD99 positive. A significant diagnostic challenge is distinction of lymphoblastic lymphoma from ES/PNET, because both can be reactive for CD99 in a membranous cytoplasmic pattern, and lymphoblastic lymphoma may not be reactive for CD45.23,173,174 A combination of IHC reactivity for TdT, CD43, CD34, CD10, and CD79a and generearrangement studies can distinguish lymphoblastic lymphoma from ES/PNET.56,173,174 Vimentin is reactive in a high proportion of ES/PNET but in less than 25% of lymphoblastic lymphoma, and it is absent in NB.
Desmoplastic Small Round Cell Tumor Desmoplastic small round cell tumor (DSRCT) is an aggressive, polyphenotypic, malignant tumor that occurs
865
predominantly in children and young adults (mean 22 years)175 with a striking male predominance and an age range that spans the first through eighth decades.176-182 A large epidemiologic study showed that DSRCT was significantly more common in the African-American population compared with whites (incidence rate ratio 3.0).128 DSRCT typically presents as widespread tumor nodules in the abdominal cavity, but occasionally it occurs in the thoracic cavity, paratesticular region, head and neck, CNS, extremities, kidney, and other solid organs.183-190 Grossly, the tumor forms a solid multinodular mass with peritoneal studding and a firm graywhite cut surface that usually shows foci of hemorrhage and necrosis.38,172,175 Histologically, DSRCT is characterized by variably sized, sharply delineated nests of relatively uniform small round cells encased in a prominent desmoplastic stroma (Fig. 22-19).175 The larger nests commonly show central necrosis, and cystic degeneration can also be seen. Occasionally, the neoplastic cells show other architectural arrangements that include trabeculae, rosette formation, foci of epithelial differentiation, and cords of single cells reminiscent of lobular breast carcinoma. The tumor cells are typically small and uniform with hyperchromatic nuclei with inconspicuous nucleoli, scant cytoplasm, and indistinct cell borders, but larger atypical cells are sometimes present focally and rarely comprise the major cell type in a given tumor. A component of neoplastic cells with intracytoplasmic rhabdoid eosinophilic inclusions composed of aggregates of intermediate filaments is commonly found,191 as are frequent mitoses and individual cell necrosis. The typical desmoplastic stroma is composed of fibroblasts or myofibroblasts in a collagenous matrix. By IHC, DSRCT exhibits a distinct polyphenotypic profile characterized by concurrent expression of epithelial, mesenchymal, and neural markers. The IHC profile compiled from eight published series is shown in Figure 22-20.27,36-38,172,175,177,192 DSRCTs are dependably immunoreactive for vimentin (Fig. 22-21) and epithelial markers that include cytokeratins (AE1, AE3, CAM5.2; Fig. 22-22) and EMA (Fig. 22-23). In one study, MOC-31 and BerEP4 were positive in 90% and
KEY DIAGNOSTIC POINTS Ewing Sarcoma/Primitive Neuroectodermal Tumor • ES/PNET shows a morphologic spectrum that ranges from a primitive small round cell tumor to a predominantly round cell neoplasm with a lobular architecture and pseudorosette formation. • The characteristic IHC profile includes reactivity for vimentin, CD99 (usually membranous), and FLI1 (nuclear) with variable IHC reactivity for neural markers. • ES/PNET has a variety of cytogenetic abnormalities that involve the EWSR1 gene, and the most common is a translocation that involves chromosomes 11 and 22 with an EWSR1/FLI1 gene fusion. • Nuclear expression of FLI1 protein is typical for an ES/PNET with a translocation that involves the EWSR1 gene on chromosome 22 and the FLI1 gene on chromosome 11, but it is not entirely specific. • IHC can be used to distinguish ES/PNET from RMS and other small blue cell tumor mimics, with the recognition that neither CD99 nor FLI1 protein are completely specific for ES/PNET in this context and that a diagnostic panel of IHC stains is necessary. • Strong CD99 staining in synovial sarcoma and in primitive hematolymphoid neoplasms, especially lymphoblastic lymphoma, is a potentially serious diagnostic trap that can be avoided by using a panel of IHC studies and molecular analyses. • In problematic cases, cytogenetic and molecular genetic tests are useful in the differential diagnosis.
866
Immunohistology of Pediatric Neoplasms
Figure 22-19 Desmoplastic small round cell tumor displays round and polygonal tumor cells arranged in geographic nests separated by a fibrous stroma (hematoxylin and eosin, ×100).
Figure 22-21 Vimentin displays diffuse cytoplasmic reactivity in desmoplastic small round cell tumor (immunoperoxidase, ×400).
71% of cases, respectively.172 Desmin shows cytoplasmic positivity (Fig. 22-24) and may display a distinctive dotlike perinuclear expression pattern that corresponds to aggregates of intracytoplasmic intermediate filaments. The neoplastic cells are consistently negative for myogenin and MyoD1 expression,27,175 and immunoreactivity for MSA and SMA is rare.27,37,38,175,192 NSE and CD57 (Leu-7) expression are frequent but nonspecific.172 Synaptophysin172,192 and S-100 protein are expressed in fewer than 25% of cases.175,177 100%
98% 93% 88%
88%
89%
Positive
79%
29%
0% Vimentin Keratin
EMA
Desmin
WT1
NSE
CD99
Figure 22-20 An immunohistogram of desmoplastic small round cell tumor. EMA, Epithelial membrane antigen; NSE, neuronspecific enolase.
Figure 22-22 Cytokeratin demonstrates cytoplasmic reactivity in desmoplastic small round cell tumor (immunoperoxidase, ×200).
Specific Tumors
Figure 22-23 Epithelial membrane antigen demonstrates cytoplasmic and membranous reactivity in desmoplastic small round cell tumor (immunoperoxidase, ×400).
Figure 22-24 Desmin shows diffuse strong cytoplasmic reactivity in desmoplastic small round cell tumor (immunoperoxidase, ×400).
867
CD99 and NB84 are sensitive but nonspecific markers routinely utilized in the small round blue cell differential diagnoses of ES/PNET and NB, respectively. Of importance, significant immunophenotypic overlap exists between DSRCT and these entities. Approximately 30% of DSRCT cases demonstrate positivity for CD99. In one large series,175 the pattern of reactivity was cytoplasmic in all nine positive cases (HBA71 and O13 clones); however, two smaller series36,172 showed a membranous pattern. Similarly, NB84 is expressed in 30% to 50% of DSRCT, although the immunoreactivity pattern is usually focal and is present in fewer than 50% of tumor cells.172,193 DSRCT is associated with a unique chromosomal translocation, t(11;22)(p13;q12); it involves the EWSR1 and WT1 genes, leading to a fusion protein and the consequent nuclear expression of WT1, which is demonstrated by antibodies to the C-terminus (Fig. 22-25).36,192 However, at least one DSRCT that occurred in soft tissue was negative for the WT1 C-terminal and rather showed nuclear staining with the N-terminal antibody.194 This case was confirmed as having two variant fusion transcripts by RT-PCR and sequencing.194 We have also observed WT1 reactivity in other childhood tumors and have encountered technical challenges with the IHC stain, which may limit its utility. A number of other positive markers have been described in DSRCT but are not particularly helpful in the diagnostic workup; these include CA125
Figure 22-25 Wilms tumor 1, using an antibody to the C-terminus, shows diffuse nuclear reactivity in desmoplastic small round cell tumor (immunoperoxidase, ×400).
868
Immunohistology of Pediatric Neoplasms
(42%),176 PLAP (81%), calretinin (19%), ERBB2 (formerly HER2/neu; 39%), c-Kit (14%),37 p63 (10%), and CD15 (70%).192 As previously mentioned, DSRCT harbors a unique and specific chromosomal translocation, t(11;22) (p13;q12), which involves the N-terminal domain of the EWSR1 gene on chromosome 22 (22q12) and the C-terminal domain of the Wilms tumor suppressor gene, WT1, on chromosome 11p13. The signature aberration results in an in-frame fusion between a potent transcriptional activation domain from EWSR1 and a zinc-finger DNA-binding domain from WT1. The chimeric gene is abundantly transcribed, and via alternative splicing, it ultimately results in two EWSR1-WT1 isoform proteins with different oncogenic properties.195,196 Two spindle cell tumors with an EWSR1-WT1 transcript and a favorable clinical course have been reported, raising the question of a variant of DSRCT or leiomyosarcoma or a unique entity among mesenchymal tumors.197 At present, no predictive or prognostic markers for DSRCT have been identified, so it must be distinguished from other tumors that demonstrate small round cell histology, including ES/PNET, MRT, RMS, blastemapredominant WT, NB, synovial sarcoma with round cell features, lymphoma, and poorly differentiated carcinoma. A panel of IHC studies is essential given the partial immunophenotypic overlap with the aforementioned tumors. Coexpression of epithelial, mesenchymal, and neural markers is key to the diagnosis. The presence of nuclear WT1 can differentiate DSRCT from MRT and exceptional cases of desmin-positive ES/PNET.136,137 RMS is distinguished by the presence of staining for myogenin and MyoD1. Blastemapredominant WT and synovial sarcoma with round cell features could pose challenges in the differential diagnosis if epithelial components are not identified, but it can be definitively distinguished by molecular genetic studies. In such diagnostically challenging cases or in unusual clinical settings, molecular confirmation of the EWSR1-WT1 gene rearrangement supports the diagnosis of DSRCT193,198-203 and can be detected by tumor karyotype, dual probe FISH, or RT-PCR.136,144,152-154,158,204
Although not specific for the EWSR1-WT1 fusion, FISH with a commercially available EWSR1(22q12) dualcolor break-apart probe has been successfully used as a diagnostic adjunct to show chromosomal rearrangement of the EWSR1 gene.204-206
Malignant Rhabdoid Tumor Malignant rhabdoid tumor (MRT) is a highly aggressive neoplasm of infancy and childhood with a tendency for widespread metastases.207-210 Originally described in the kidney209,211,212 and CNS,213,214 the clinicopathologic spectrum is now known to include other organs, extrarenal soft tissue, and skin and also has a congenital disseminated form.208,215-218 Familial cases with involvement of the CNS and other sites have been reported.219-221 Although most tumors occur in children, rare examples of true MRT have been observed in adults. No gender predilection has been noted. These primitive tumors are polyphenotypic, and their name is derived from the rhabdoid appearance of the tumor cells. The histopathologic appearance of MRT is that of a densely cellular neoplasm composed of sheets or cords of cells with large, vesicular, round or oval nuclei; prominent central eosinophilic nucleoli; and abundant eccentric eosinophilic cytoplasm (Fig. 22-26). Histologic variability is typical, and some cases have smaller numbers of characteristic rhabdoid cells or display a primitive round cell pattern, cellular dyscohesion, a myxoid background, increased collagen between sheets,
KEY DIAGNOSTIC POINTS Desmoplastic Small Round Cell Tumor • Coexpression of vimentin, cytokeratin, EMA, desmin (often perinuclear and dotlike), and nuclear WT1 support the diagnosis of DSRCT. • Molecular or cytogenetic confirmation of the EWSR1-WT1 gene fusion supports the diagnosis of DSRCT. • Absence of staining for the myogenic regulatory proteins, myogenin and MyoD1, helps to distinguish DSRCT from RMS; both tumors can be immunoreactive for desmin, myoglobin, and actin. • Presence of WT1 staining and the EWSR1-WT1 gene fusion distinguish DSRCT from MRT, synovial sarcoma, and blastema-predominant Wilms tumor, other polyphenotypic malignant neoplasms that can show similar histology.
Figure 22-26 Malignant rhabdoid tumor consists of sheets of round cells with vesicular nuclei, prominent nucleoli, and variable amounts of eosinophilic cytoplasm with occasional large cytoplasmic eosinophilic globules (hematoxylin and eosin, ×200).
Specific Tumors
869
100% 93% 80% 73% 66%
66%
Positive
61%
37% 20%
Figure 22-27 An immunohistogram of malignant rhabdoid tumor. EMA, Epithelial membrane antigen; MSA, muscle-specific actin; NSE, neuron-specific enolase; Syn, synaptophysin.
0%
or cords of tumor cells and scattered nonneoplastic osteoclast like giant cells. Occasional cases have focal epithelial areas, and mitoses are frequent. Electron microscopy reveals cytoplasmic whorls of intermediate filaments, which correspond to the eosinophilic cytoplasmic globules in classic rhabdoid cells. IHC is an invaluable adjunct technique because of the potential for many different neoplasms to display a rhabdoid appearance. Figure 22-27 shows an immunohistogram of the most frequent markers in MRT, compiled from four series.208,213,215,217 MRT is typically a polyphenotypic neoplasm with coexpression of vimentin (Fig. 22-28); at least one epithelial marker, such as cytokeratin (Fig. 22-29) or EMA (Fig. 22-30); neural or neuroectodermal markers such as S-100 protein, GFAP, NSE, and synaptophysin (Fig. 22-31); and mesenchymal markers such as MSA, desmin, CD99, and SMA (Fig. 22-32).42,208,213,215,217 However, myogenin, myoglobin, human melanoma black 45 (HMB-45), chromogranin, and CD34 are typically absent,217 and claudin-6 expression is nonspecific.222 The cytoplasmic filaments are composed of vimentin and CK8, and MRT has been found to have mutations of the human cytokeratin gene.223 Stem cell–associated markers are also identified.224,225 Dysadherin, a cancer-associated cell membrane glycoprotein expressed in epithelioid sarcoma, is not found in MRT, a fact that may be useful in differential diagnosis.226 Regardless of location, approximately 75% of MRTs harbor deletions or mutations of the SMARCB1 gene at chromosome 22q11.2.42,215,227-229 The spectrum of cytogenetic abnormalities includes monosomy 22, deletion of 22q11, translocations that involve chromosome 22q11.2, and mutations and homozygous deletions of the SMARCB1 gene.219,230-232 Germline SMARCB1 mutations occur in both familial and sporadic cases. A subgroup of MRTs have intact SMARCB1 gene regions with paradoxical loss of protein expression, likely related to epigenetic factors, and a variety of other chromosomal abnormalities.229,233 Retention of SMARCB1 protein expression has also been reported with a nonsense mutation and inactivation of SMARCA4
Vimentin Cytokeratin
EMA
MSA
Desmin
CD99
NSE
Syn
(formerly BRG1), which is another SWF/SNF chromatin-remodeling complex member.234 FISH is useful for detection of deletions.235 IHC analyses for the SMARCB1 protein in MRT of the CNS, kidney, and extrarenal soft tissue have shown that absence of protein expression by IHC (Fig. 22-33) correlates well with deletion and mutation of the SMARCB1 gene.39-41 Pathologic-prognostic features beyond the diagnosis of MRT have not been identified. The polyphenotypic IHC profile of MRT presents challenges in differential diagnosis, particularly with
Figure 22-28 Vimentin displays cytoplasmic reactivity in malignant rhabdoid tumor (immunoperoxidase, ×400).
870
Immunohistology of Pediatric Neoplasms
Figure 22-29 CAM5.2 cytokeratin shows variable cytoplasmic reactivity in malignant rhabdoid tumor (immunoperoxidase, ×400).
Figure 22-30 Epithelial membrane antigen shows focal strong cytoplasmic reactivity in malignant rhabdoid tumor (immunoperoxidase, ×400).
Figure 22-31 Weak synaptophysin reactivity is present in the cytoplasm of malignant rhabdoid tumor (immunoperoxidase, ×400).
Figure 22-32 Muscle-specific actin demonstrates focal cytoplasmic reactivity in malignant rhabdoid tumor (immunoperoxidase, ×400).
Specific Tumors
871
clinicopathologic (age, site), IHC (CD34 reactivity), and genetic features of epithelioid sarcoma, such as a lower frequency of SMARCB1 gene alterations, help to distinguish it from MRT.240 Loss of SMARCB1 protein expression in sarcomas with a translocation that involves the EWSR1 gene at 22q11.2, such as extraskeletal myxoid chondrosarcoma, is another pitfall.241 The lack of expression of SMARCB1 protein in MRT contrasts with other histologic mimics, such as RMS, ES, and other round cell tumors with eosinophilic cytoplasm. Analysis of specific markers of skeletal muscle differentiation, such as myogenin and MyoD1, is very helpful in the differential diagnosis. In questionable cases, cytogenetic and molecular genetic analysis can be used as a diagnostic adjunct.
Wilms Tumor
Figure 22-33 Absence of nuclear staining for SMARCB1 protein in tumor cells correlates with deletion and mutation of the SMARCB1 gene, formerly hSNF5/INI1, in malignant rhabdoid tumor (immunoperoxidase, ×400).
DSRCT, RMS, choroid plexus carcinoma, epithelioid sarcoma, synovial sarcoma with rhabdoid or round cell features, and rhabdomyomatous Wilms tumor. In particular, the absence of SMARCB1 protein in epithelioid sarcoma, in a subset of CNS PNETs without a rhabdoid phenotype, in schwannomatosis, and in renal medullary carcinoma suggests the existence of a wider family of SMARCB1-deficient tumors.40,236-239 Other KEY DIAGNOSTIC POINTS Malignant Rhabdoid Tumor • MRT is a highly aggressive, polyphenotypic, malignant neoplasm with a tendency for widespread metastases and a disseminated presentation. • The proportion of rhabdoid cells within an MRT varies, and the histopathologic spectrum mimics ES/PNET, medulloblastoma, RMS, DSRCT, epithelioid sarcoma, and other primitive malignant neoplasms. • MRT characteristically shows diffuse reactivity for vimentin, focal reactivity for at least one epithelial marker, and variable expression of mesenchymal and neuroectodermal markers. • Absence of SMARCB1 protein expression is useful for recognition of MRT and its distinction from other neoplasms, which lack SMARCB1 deletions or mutations and show SMARCB1 protein immunoreactivity. • Molecular genetics analysis to identify abnormalities of chromosome 22q11 provides additional support for the diagnosis of MRT.
Wilms tumor (WT), or nephroblastoma, is the most common renal neoplasm in children. No sex predilection has been noted, and 98% of all cases occur in patients younger than 10 years. WT is derived from nephrogenic blastemal cells and thus can exhibit a wide range of histologic appearances that replicate the developing kidney and display divergent differentiation. Most tumors are unicentric, but 5% to 10% are bilateral or multicentric. Approximately 10% to 15% of WTs are syndromic, most frequently associated with different germline mutations within the Wilms tumor gene, WT1. Defined syndromes associated with WT1 mutations and an increased risk for WT development are WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation), Denys-Drash, and Frasier syndromes.242 In contrast, the genetic basis of sporadic tumors appears to be heterogeneous. Up to one third of these tumors have been shown to harbor somatic mutations on the WT1, AMER1 (formerly WTX), and/or CTNNB1 (β-catenin) genes,243,244 and activation of the Wnt pathway is thought to play an important role in their pathogenesis.245,246 Although WT is not characterized by recurrent molecular abnormalities, a few molecular aberrations have been associated with poor outcome in favorablehistology tumors, including loss of heterozygosity (LOH) for chromosomes 1p, 16q, and 11p15 (associated with WT1 mutations); whereas TP53 mutations, MYCN gain, and loss of chromosomes 4q, 11q, and 14q have been demonstrated in association with anaplasia.247-251 LOH for 1p and 16q may affect therapeutic stratification in ongoing COG phase III studies for favorable histology tumors, therefore tissue harvesting for molecular studies should be included in the handling of WTs. The classic histologic pattern of WT consists of triphasic elements that include blastemal, epithelial, and stromal components (Fig. 22-34). Proportions of these three elements vary, and biphasic or monophasic lesions may be encountered. Blastemal cells are small, round, or oval and are densely packed in diffuse, nodular, or serpentine patterns. The epithelial component ranges from primitive tubular forms with a rosettelike appearance to more obvious tubular or papillary structures.
872
Immunohistology of Pediatric Neoplasms
A
B
Figure 22-34 Distinctive morphologic features in Wilms tumors. A, Triphasic pattern with blastemal epithelial and stromal components (hematoxylin and eosin [H&E], ×200). B, Anaplasia (H&E, ×400).
Heterologous epithelial elements include mucinous and squamous epithelium. Stromal differentiation includes mesenchymal components such as nondescript fibrous tissue, undifferentiated mesenchyme, smooth muscle, skeletal muscle, adipose tissue, cartilage, bone, and also neural tissue, including ganglion cells, nerve, and neuroglia.252 Pathologic evaluation for WT has been standardized with both the Children’s Oncology Group (COG) and the International Society of Paediatric Oncology (SIOP) protocols in North America and
A
Europe, respectively.6,253 The histologic pattern most likely to cause diagnostic difficulty is the blastemal pattern; monomorphous sheets of primitive blue cells can appear dyscohesive and may simulate other small round cell neoplasms (Fig. 22-35). The differential diagnosis includes lymphoma, ES/PNET, NB, DSRCT, synovial sarcoma, and RMS. WTs do not exhibit a specific immunophenotype, and the most important clue to the correct diagnosis is the clinical history of a renal mass in a young child and
B
Figure 22-35 Wilms tumor. A, Blastemal component with monomorphous sheets of primitive cells that can mimic other small round blue cell neoplasms (hematoxylin and eosin, ×200). B, Wilms tumor 1 (WT1) shows nuclear reactivity in blastemal and primitive epithelial areas (immunoperoxidase, ×400).
Specific Tumors
the presence of a mixture of blastemal, epithelial, and mesenchymal components. As mentioned previously, blastemal-predominant tumors may be difficult to distinguish from other uncommon small round blue cell tumors that arise in the kidney; however, because of overlapping immunophenotypical features among these tumors, molecular and/or cytogenetic studies should be incorporated into their workup.254 The blastemal component is typically reactive for vimentin and nestin and usually shows desmin reactivity.255,256 Other muscle markers such as myogenin and MyoD1 are absent from blastemal foci but can be positive in areas of skeletal muscle differentiation within the stromal component. Nuclear staining for WT1 is positive in blastemal areas, in foci of early epithelial differentiation (Fig. 22-36),257 and in undifferentiated stromal areas.258 Pax-2, a transcription factor involved in normal renal development, shows strong nuclear staining predominantly within the epithelial and blastemal components and less frequently within stromal areas.123 Although nonspecific, WT1 and Pax-2 expression within the blastemal component can aid in the differential diagnosis with other small round blue cell neoplasms; however, the utility of Pax-2 in differentiating WT from other primary renal tumors has not been explored. Blastema is also focally positive for NSE, CD56, and cytokeratin but not for CD99, and the absence of CD99 reactivity may also help to distinguish blastemal-predominant WT from ES/PNET. More mature areas of epithelial differentiation, such as tubules, are typically reactive for cytokeratin (Fig. 22-37), CD56, and CD57.257 The stromal component consistently expresses Bcl-2 and lacks CD34 expression.258 Neural differentiation within a WT is associated with reactivity for NSE, chromogranin, and synaptophysin, but staining for GFAP and S-100 protein is variable.252 Nuclear anaplasia occurs in approximately
Figure 22-36 Desmin shows strong cytoplasmic reactivity in a blastemal area of Wilms tumor (immunoperoxidase, ×400).
873
Figure 22-37 Cytokeratin highlights an area of tubular epithelial differentiation in Wilms tumor (immunoperoxidase, ×400).
5% of WTs and is characterized by extreme nuclear atypia with multipolar polyploid mitotic figures, marked nuclear enlargement (at least three times larger than the adjacent nonanaplastic nuclei), and hyperchromasia (Fig. 22-38). Anaplastic WT may also express p53 (Fig. 22-39). Diffuse anaplasia is an unfavorable histologicprognostic indicator. Prognostically significant IHC markers have not been sufficiently explored as to their utility. In addition to WT1 and Pax-2, IHC expression of several markers
Figure 22-38 Myogenin shows focal nuclear reactivity in an area of skeletal muscle differentiation in Wilms tumor (immunoperoxidase, ×400).
874
Immunohistology of Pediatric Neoplasms
WT.259,260 Other transcription factors involved in normal renal development have also been shown to be aberrantly expressed in WT by IHC, such as CITED1 and SALL4.224,261
Osteosarcoma
Figure 22-39 Nuclear reactivity for p53 is present in anaplastic Wilms tumor and is associated with p53 mutation (immunoperoxidase, ×400).
involved in normal renal development has been demonstrated in WTs, but their diagnostic utility remains unexplored. Nuclear accumulation of β-catenin has been observed in up to 67% of WTs, restricted mostly to the stromal component and less frequently to the blastema, with lack of nuclear staining in the epithelial component; nuclear accumulation of β-catenin can be seen regardless of the presence of CTNNB1 mutations, suggesting alternative ways of Wnt activation in
KEY DIAGNOSTIC POINTS Wilms Tumor • Wilms tumor can usually be diagnosed in a combination of classic histologic findings combined with clinical information about the age and site of the tumor. • Blastemal-predominant WT, especially in small biopsy specimens, can mimic other small blue cell tumors. A pitfall in the diagnosis of WT versus other kidney tumors includes confusion with primary renal ES/PNET, synovial sarcoma, leukemia/lymphoma, NB, DSRCT, and ARMS. • Potentially useful markers in blastemal-predominant WT include WT1 and Pax-2. Muscle markers, such as myogenin and MyoD1, are not expressed within the blastemal component and may serve as a useful differential diagnostic tool in desmin-positive, blastemal-predominant WTs. CD99 expression is also a useful distinguishing feature of ES/PNET in contrast to blastemal-predominant WT.
Osteosarcoma (OS) is the most common primary bone malignancy, and the classic type is a high-grade neoplasm in which tumor cells characteristically produce osteoid.262,263 Although OS is primarily a malignancy of the young, with a peak in the second decade of life and 60% occurrence in patients younger than 25 years, approximately 30% occur in individuals older than 40 years. Males are more often affected than females, especially in the younger age range. The most frequent sites are the long bones of the appendicular skeleton, with a tumor origin in the metaphysis or diaphysis. The mass is large, fleshy, or hard and contains variable amounts of calcification, bone, and cartilage. Microscopically, OS is composed of anaplastic pleomorphic cells with a morphologic spectrum that includes spindle, round, ovoid, epithelioid, plasmacytoid, clear, and multinucleated cells. Usually more than one cell type is present in individual OS. Osteoid is required for the diagnosis, and variable bone formation, cartilage, and fibrous tissue may also be encountered. Histologic subtypes of conventional OS include osteoblastic, chondroblastic, and fibroblastic patterns. Ultrastructural studies have also demonstrated multiple cell types that include osteoblastic, osteoclastic, chondroblastic, and fibroblastic features. OS has no specific immunophenotype, and it is immunohistochemically heterogenous with expression of osteocalcin, osteonectin, cytokeratin, EMA, desmin, SMA, type IV collagen, S-100 protein, factor XIII, p63, ezrin, caveolin-1, podoplanin (D2-40), nestin, and CD99 in varying proportions.149,264-268 IHC is useful for the differential diagnostic distinction of osteosarcomatous mimics such as sarcomatoid carcinoma and other sarcomas, including synovial sarcoma, lymphoma, and malignant melanoma. The presence of reactivity for CD99 is a potential diagnostic pitfall, especially if the OS has a predominantly small round cell pattern, although nuclear Fli-1 reactivity may help to distinguish ES from small cell OS,269 and small cell OS typically forms osteoid.149 Cyclooxygenase 2 (COX-2) and galectin-1 expression in chondroblastic osteosarcoma may be useful in the distinction from osteoblastoma.270,271 MDM2 and CDK4 are expressed in low-grade and dedifferentiated OS and may aid in distinction from benign mimics.272-274 Complex cytogenetic abnormalities are often detected in OS and are nonspecific.263 The most significant pathologic-prognostic indicator in OS is the extent of necrosis after chemotherapy.275-277 A variety of IHC markers with potential prognostic or biologic significance have been investigated. Although some controversy surrounds the genetic basis and significance of cytoplasmic versus nuclear patterns of immunoreactivity for ERBB2,278,279 it now appears that IHC expression of ERBB2 portends a poor
Specific Tumors
875
TABLE 22-1 Key Pathologic, Immunohistochemical, and Genetic Features of Selected Pediatric Neoplasms Diagnosis
Histopathology
Immunophenotype
Genetic Aberrations
Neuroblastoma
SRCs in sheets or nests with variable mitoses, karyorrhexis, neuropil, rosettes, fibrovascular septa, and schwannian stroma
NSE, synaptophysin, chromogranin, neurofilament, CD57, CD56, PGP9.5
MYCN amplification; deletion of chromosomes 1p or 11q
Rhabdomyosarcoma
Alveolar: Alveolar or solid architecture; SRCs with variable rhabdomyoblastic differentiation, giant rhabdomyoblastic cells, clear cells Embryonal: primitive spindled, stellate, round, and polygonal cells in variably myxoid background; Variable rhabdomyoblasts, strap cells, multinucleated cells, and myotube forms
Alveolar: Myogenin, MyoD1, desmin, MSA Embryonal: Myogenin, MyoD1, MSA, desmin.
t(2;13)(q35;q14) with PAX3/FOXO1 fusion, t(1;13)(p36;q14) with PAX7/FOXO1 fusion, loss of heterozygosity at 11p15.5, and other nonspecific abnormalities
Ewing sarcoma/ primitive neuroectodermal tumor
SRCs in sheets with round nuclei, fine chromatin, scanty cytoplasm; variable rosettes, prominent nucleoli, spindle cells, necrosis
Vimentin, CD99, synaptophysin, FLI1
t(11;22)(q24;q12) with EWSR1/FLI1 fusion t(21;22)(q22;q12) with EWSR1/ERG fusion t(7;22)(p22;q12) with EWSR1/ETV1 fusion t(17;22)(q12;q12) with EWSR1/ETVF fusion t(2;22)(q33;q12) with EWSR1/FEV fusion Other translocations
Desmoplastic small round cell tumor
SRCs in nests with prominent stromal desmoplasia, central necrosis, variable cystic degeneration and epithelial differentiation
Polyphenotypic: cytokeratins, EMA, vimentin, desmin, WT-1 (C-terminus), CD99
t(11;22)(p13;q12) with EWSR1/WT1 fusion
Malignant rhabdoid tumor
Polygonal cells or SRCs in sheets or trabeculae with large, vesicular nuclei, prominent central nucleoli, abundant eccentric cytoplasm, frequent mitoses and globoid, hyaline, eosinophilic cytoplasmic inclusions
Polyphenotypic: vimentin, cytokeratin, EMA Variable CD99, synaptophysin, S-100 protein, MSA Absence of SMARCB1
Deletion or mutation of SMARCB1 on chromosome 22q11
Wilms tumor
Triphasic blastemal, epithelial, and stromal components; blastemal cells small, round, or oval in nodules or serpentine patterns; epithelial component is primitive rosettelike to tubular or papillary; mesenchymal component is fibrous, myoid, adipose, chondroid, osseous, or neural
Blastemal: vimentin, desmin Epithelial: cytokeratin Mesenchymal: variable according to differentiation pattern
Various abnormalities, including mutations of WT1 at 11p13 and WT2 at 11p15; numerical and structural karyotypic abnormalities of chromosomes 1, 11, 13, 14, 16, 17, 19, and 22
Osteosarcoma
Anaplastic pleomorphic cells with morphologic spectrum, osteoid, bone formation
Heterogeneous, without a specific immunophenotype
Nonspecific numeric and structural abnormalities
EMA, Epithelial membrane antigen; MSA, muscle-specific actin; NSE, neuron-specific enolase; SRCs, small round cells; PGP9.5, protein gene product 9.5.
876
Immunohistology of Pediatric Neoplasms
prognosis and increased risk of lung metastases.280,281 Other potential poor prognostic markers include ephrin,282 ezrin,283,284 cytochrome P450 (CYP) 3A4/5,285 WT1,286 chemokine receptor CXC R4,236 vascular endothelial growth factor (VEGF),236 yin-yang 1 (YY1),287 drug-related genes IMPDH2 and FTL,288 p-glycoprotein,280,289,290 p53,280,291,292 and proliferative markers such as Ki-67 and proliferating cell nuclear agent (PCNA).29,291,292 Survivin expression may have prognostic relevance.293,294
KEY DIAGNOSTIC POINTS Osteosarcoma • OS is an immunohistochemically heterogeneous neoplasm that lacks a characteristic IHC profile. • The presence of osteoid is the key distinguishing feature of OS. • Expression of cytokeratin, EMA, CD99, and S-100 protein are potential diagnostic pitfalls in the distinction of OS from metastatic or sarcomatoid carcinoma, ES/PNET, synovial sarcoma, malignant melanoma, and other malignancies.
Summary In conclusion, the availability of more specific and more sensitive IHC markers has enabled a more accurate categorization of pediatric tumors, especially round cell tumors and spindle cell tumors (Table 22-1). As therapeutic protocols are refined, precise diagnoses are required. IHC is only one of the tools available to the pathologist to categorize solid tumors of childhood and adolescence, with proven utility of cytogenetic and molecular analysis, flow cytometry, and electron microscopy for selected cases. The ready availability, ease of use, and cost-effectiveness of IHC make it an important diagnostic tool in pediatric surgical pathology. Knowledge of reactivity patterns, sensitivity, specificity, and potential diagnostic pitfalls is essential.
Acknowledgements Devin Jacobsen, BA, and Kristi Kelley provided support for manuscript preparation. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Coindre JM: Immunohistochemistry in the diagnosis of soft tissue tumours. Histopathology. 43:1–16, 2012. 2. Bridge JA, Cushman-Vokoun AM: Molecular diagnostics of soft tissue tumors. Arch Pathol Lab Med. 135(5):588–601, 2011. 3. Coffin CM, Alaggio R, Dehner LP: Some general considerations about the clinicopathologic aspects of soft tissue tumors in children and adolescents. Pediatr Dev Pathol. 15(1 Suppl):11–25, 2012. 4. Leong AS: Pitfalls in diagnostic immunohistology. Adv Anat Pathol. 11(2):86–93, 2004. 5. de Alava E: Molecular pathology in sarcomas. Clin Transl Oncol. 9(3):130–144, 2007. 6. Qualman SJ, Bowen J, Amin MB, et al: Protocol for the examination of specimens from patients with Wilms tumor (nephroblastoma) or other renal tumors of childhood. Arch Pathol Lab Med. 127(10):1280–1289, 2003. 7. Qualman SJ, Bowen J, Parham DM, et al: Protocol for the examination of specimens from patients (children and young adults) with rhabdomyosarcoma. Arch Pathol Lab Med. 127(10):1290– 1297, 2003. 8. Carpentieri DF, Qualman SJ, Bowen J, et al: Protocol for the examination of specimens from pediatric and adult patients with osseous and extraosseous Ewing sarcoma family of tumors, including peripheral primitive neuroectodermal tumor and ewing sarcoma. Arch Pathol Lab Med. 129(7):866–873, 2005. 9. Coffin CM, Dehner LP: Pathologic evaluation of pediatric soft tissue tumors. Am J Clin Pathol. 109(4 Suppl 1):S38–S52, 1998. 10. Shimada H, Ambros IM, Dehner LP, et al: Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee. Cancer. 86(2):349–363, 1999. 11. Wang NP, Marx J, McNutt MA, et al: Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood. Am J Pathol. 147(6):1799–1810, 1995. 12. Dias P, Parham DM, Shapiro DN, et al: Myogenic regulatory protein (myoD1) expression in childhood solid tumors: diagnostic utility in rhabdomyosarcoma. Am J Pathol. 137(6):1283– 1291, 1990. 13. Cui S, Hano H, Harada T, et al: Evaluation of new monoclonal anti-myoD1 and anti-myogenin antibodies for the diagnosis of rhabdomyosarcoma. Pathol Int. 49(1):62–68, 1999. 14. Engel ME, Mouton SC, Emms M: Paediatric rhabdomyosarcoma: MyoD1 demonstration in routinely processed tissue sections using wet heat pretreatment (pressure cooking) for antigen retrieval. J Clin Pathol. 50(1):37–39, 1997. 15. Kumar S, Perlman E, Harris CA, et al: Myogenin is a specific marker for rhabdomyosarcoma: an immunohistochemical study in paraffin-embedded tissues. Mod Pathol. 13(9):988–993, 2000. 16. Cessna MH, Zhou H, Perkins SL, et al: Are myogenin and myoD1 expression specific for rhabdomyosarcoma? A study of 150 cases, with emphasis on spindle cell mimics. Am J Surg Pathol. 25(9):1150–1157, 2001. 17. Sebire NJ, Malone M: Myogenin and myoD1 expression in paediatric rhabdomyosarcomas. J Clin Pathol. 56(6):412–416, 2003. 18. Dias P, Parham DM, Shapiro DN, et al: Monoclonal antibodies to the myogenic regulatory protein myoD1: epitope mapping and diagnostic utility. Cancer Res. 52(23):6431–6439, 1992. 19. Dias P, Chen B, Dilday B, et al: Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass. Am J Pathol. 156(2):399–408, 2000. 20. Stevenson AJ, Chatten J, Bertoni F, et al: (p30/32MIC2) neuroectodermal/Ewing’s sarcoma antigen as an immunohistochemical marker: Review of more than 600 tumors and the literature experience. Appl Immunohistochem. 2:231–240, 1994. 21. Rocchi A, Manara MC, Sciandra M, et al: CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. J Clin Invest. 120(3):668–680, 2010. 22. Perlman EJ, Dickman PS, Askin FB, et al: Ewing’s sarcoma– routine diagnostic utilization of MIC2 analysis: a Pediatric Oncology Group/Children’s Cancer Group Intergroup Study. Hum Pathol. 25(3):304–307, 1994.
876.e1
23. Weidner N, Tjoe J: Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing’s sarcoma and peripheral neuroepithelioma. Am J Surg Pathol. 18(5):486–494, 1994. 24. Ambros IM, Ambros PF, Strehl S, et al: MIC2 is a specific marker for Ewing’s sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of Ewing’s sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer. 67(7):1886–1893, 1991. 25. Ramani P, Rampling D, Link M: Immunocytochemical study of 12E7 in small round-cell tumours of childhood: an assessment of its sensitivity and specificity. Histopathology. 23(6):557–561, 1993. 26. Riopel M, Dickman PS, Link MP, et al: MIC2 analysis in pediatric lymphomas and leukemias. Hum Pathol. 25(4):396–399, 1994. 27. Zhang PJ, Goldblum JR, Pawel BR, et al: Immunophenotype of desmoplastic small round cell tumors as detected in cases with EWS-WT1 gene fusion product. Mod Pathol. 16(3):229–235, 2003. 28. Granter SR, Renshaw AA, Fletcher CD, et al: CD99 reactivity in mesenchymal chondrosarcoma. Hum Pathol. 27(12):1273– 1276, 1996. 29. Folpe AL, Schmidt RA, Chapman D, et al: Poorly differentiated synovial sarcoma: immunohistochemical distinction from primitive neuroectodermal tumors and high-grade malignant peripheral nerve sheath tumors. Am J Surg Pathol. 22(6):673–682, 1998. 30. Fellinger EJ, Garin-Chesa P, Triche TJ, et al: Immunohistochemical analysis of Ewing’s sarcoma cell surface antigen p30/32MIC2. Am J Pathol. 139(2):317–325, 1991. 31. Ozdemirli M, Fanburg-Smith JC, Hartmann DP, et al: Differentiating lymphoblastic lymphoma and Ewing’s sarcoma: lymphocyte markers and gene rearrangement. Mod Pathol. 14(11):1175–1182, 2001. 32. Pappo AS, Douglass EC, Meyer WH, et al: Use of HBA 71 and anti-beta 2-microglobulin to distinguish peripheral neuroepithelioma from neuroblastoma. Hum Pathol. 24(8):880–885, 1993. 33. Nilsson G, Wang M, Wejde J, et al: Detection of EWS/FLI-1 by immunostaining. An adjunctive tool in diagnosis of Ewing’s sarcoma and primitive neuroectodermal tumour on cytological samples and paraffin-embedded archival material. Sarcoma. 3(1):25–32, 1999. 34. Folpe AL, Hill CE, Parham DM, et al: Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on CD99-positive mimics of Ewing’s sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol. 24(12):1657–1662, 2000. 35. Rossi S, Orvieto E, Furlanetto A, et al: Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody. Mod Pathol. 17(5):547–552, 2004. 36. Hill DA, Pfeifer JD, Marley EF, et al: WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing sarcoma/primitive neuroectodermal tumor. An immunohistochemical and molecular diagnostic study. Am J Clin Pathol. 114(3):345–353, 2000. 37. Barnoud R, Sabourin JC, Pasquier D, et al: Immunohistochemical expression of WT1 by desmoplastic small round cell tumor: a comparative study with other small round cell tumors. Am J Surg Pathol. 24(6):830–836, 2000. 38. Lae ME, Roche PC, Jin L, et al: Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol. 26(7):823–835, 2002. 39. Judkins AR, Mauger J, Ht A, et al: Immunohistochemical analysis of hSNF5/INI1 in pediatric CNS neoplasms. Am J Surg Pathol. 28(5):644–650, 2004. 40. Sigauke E, Rakheja D, Maddox DL, et al: Absence of expression of SMARCB1/INI1 in malignant rhabdoid tumors of the central nervous system, kidneys and soft tissue: an immunohistochemical study with implications for diagnosis. Mod Pathol. 19(5):717– 725, 2006.
876.e2
Immunohistology of Pediatric Neoplasms
41. Hoot AC, Russo P, Judkins AR, et al: Immunohistochemical analysis of hSNF5/INI1 distinguishes renal and extra-renal malignant rhabdoid tumors from other pediatric soft tissue tumors. Am J Surg Pathol. 28(11):1485–1491, 2004. 42. Hollmann TJ, Hornick JL: INI1-deficient tumors: diagnostic features and molecular genetics. Am J Surg Pathol. 35(10):e47–e63, 2011. 43. Schwab M, Shimada H, Joshi V, et al: Neuroblastic tumors of adrenal gland and sympathetic nervous system. In Kleihues P, Cavenee WK, editors: World Health Organization Classification of Tumors Tumors of the Nervous System, Lyon, 2000, IARC Press, pp 153–161. 44. Shimada H, Ambros IM, Dehner LP, et al: The International Neuroblastoma Pathology Classification (the Shimada system). Cancer. 86(2):364–372, 1999. 45. Joshi VV: Peripheral neuroblastic tumors: pathologic classification based on recommendations of International Neuroblastoma Pathology Committee (modification of Shimada classification). Pediatr Dev Pathol. 3(2):184–199, 2000. 46. Tornoczky T, Kalman E, Kajtar PG, et al: Large cell neuroblastoma: a distinct phenotype of neuroblastoma with aggressive clinical behavior. Cancer. 100(2):390–397, 2004. 47. Peuchmaur M, d’Amore ES, Joshi VV, et al: Revision of the International Neuroblastoma Pathology Classification: confirmation of favorable and unfavorable prognostic subsets in ganglioneuroblastoma, nodular. Cancer. 98(10):2274–2281, 2003. 48. Miettinen M, Chatten J, Paetau A, et al: Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors. Am J Surg Pathol. 22(3):327–332, 1998. 49. Parham DM: Neuroectodermal and neuroendocrine tumors principally seen in children. Am J Clin Pathol. 115(Suppl):S113– S128, 2001. 50. Wick MR: Immunohistology of neuroendocrine and neuroectodermal tumors. Semin Diagn Pathol. 17(3):194–203, 2000. 51. Bomken SN, Redfern K, Wood KM, et al: Limitations in the ability of NB84 to detect metastatic neuroblastoma cells in bone marrow. J Clin Pathol. 59(9):927–929, 2006. 52. Dunkley PR, Bobrovskaya L, Graham ME, et al: Tyrosine hydroxylase phosphorylation: regulation and consequences. J Neurochem. 91(5):1025–1043, 2004. 53. Mejia C, Navarro S, Pellin A, et al: Prognostic significance of cell proliferation in human neuroblastoma: comparison with other prognostic factors. Oncol Rep. 10(1):243–247, 2003. 54. Krams M, Hero B, Berthold F, et al: Proliferation marker KI-S5 discriminates between favorable and adverse prognosis in advanced stages of neuroblastoma with and without MYCN amplification. Cancer. 94(3):854–861, 2002. 55. Krams M, Heidebrecht HJ, Hero B, et al: Repp86 expression and outcome in patients with neuroblastoma. J Clin Oncol. 21(9):1810–1818, 2003. 56. Munchar MJ, Sharifah NA, Jamal R, et al: CD44s expression correlated with the International Neuroblastoma Pathology Classification (Shimada system) for neuroblastic tumours. Pathology. 35(2):125–129, 2003. 57. Misra S, Heldin P, Hascall VC, et al: Hyaluronan-CD44 interactions as potential targets for cancer therapy. FEBS J. 278(9):1429– 1443, 2011. 58. Brodeur GM, Maris JM, Yamashiro DJ, et al: Biology and genetics of human neuroblastomas. J Pediatr Hematol Oncol. 19(2):93– 101, 1997. 59. Brodeur GM, Nakagawara A, Yamashiro DJ, et al: Expression of TrkA, TrkB and TrkC in human neuroblastomas. J Neurooncol. 31(1-2):49–55, 1997. 60. Lamant L, Pulford K, Bischof D, et al: Expression of the ALK tyrosine kinase gene in neuroblastoma. Am J Pathol. 156(5):1711– 1721, 2000. 61. George RE, Sanda T, Hanna M, et al: Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature. 455(7215):975–978, 2008. 62. Mosse YP, Laudenslager M, Longo L, et al: Identification of ALK as a major familial neuroblastoma predisposition gene. Nature. 455(7215):930–935, 2008. 63. Brodeur GM: Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 3(3):203–216, 2003.
64. Westermark UK, Wilhelm M, Frenzel A, et al: The MYCN oncogene and differentiation in neuroblastoma. Semin Cancer Biol. 21(4):256–266, 2011. 65. Maris JM, Hogarty MD, Bagatell R, et al: Neuroblastoma. Lancet. 369(9579):2106–2120, 2007. 66. Van Roy N, De Preter K, Hoebeeck J, et al: The emerging molecular pathogenesis of neuroblastoma: implications for improved risk assessment and targeted therapy. Genome Med. 1(7):74, 2009. 67. Bown N, Cotterill S, Lastowska M, et al: Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma. N Engl J Med. 340(25):1954–1961, 1999. 68. Vandesompele J, Baudis M, De Preter K, et al: Unequivocal delineation of clinicogenetic subgroups and development of a new model for improved outcome prediction in neuroblastoma. J Clin Oncol. 23(10):2280–2299, 2005. 69. George RE, London WB, Cohn SL, et al: Hyperdiploidy plus nonamplified MYCN confers a favorable prognosis in children 12 to 18 months old with disseminated neuroblastoma: a Pediatric Oncology Group study. J Clin Oncol. 23(27):6466–6473, 2005. 70. Spitz R, Betts DR, Simon T, et al: Favorable outcome of triploid neuroblastomas: a contribution to the special oncogenesis of neuroblastoma. Cancer Genet Cytogenet. 167(1):51–56, 2006. 71. Spitz R, Hero B, Ernestus K, et al: FISH analyses for alterations in chromosomes 1, 2, 3, and 11 define high-risk groups in neuroblastoma. Med Pediatr Oncol. 41(1):30–35, 2003. 72. Haber M, Smith J, Bordow SB, et al: Association of high-level MRP1 expression with poor clinical outcome in a large prospective study of primary neuroblastoma. J Clin Oncol. 24(10):1546– 1553, 2006. 73. Asgharzadeh S, Pique-Regi R, Sposto R, et al: Prognostic significance of gene expression profiles of metastatic neuroblastomas lacking MYCN gene amplification. J Natl Cancer Inst. 98(17):1193–1203, 2006. 74. Moss TJ, Reynolds CP, Sather HN, et al: Prognostic value of immunocytologic detection of bone marrow metastases in neuroblastoma. N Engl J Med. 324(4):219–226, 1991. 75. Asmar L, Gehan EA, Newton WA, et al: Agreement among and within groups of pathologists in the classification of rhabdomyosarcoma and related childhood sarcomas. Report of an international study of four pathology classifications. Cancer. 74(9):2579–2588, 1994. 76. Parham DM, Alaggio R, Coffin CM: Myogenic tumors in children and adolescents. Pediatr Dev Pathol. 15(1 Suppl):211–238, 2012. 77. Parham DM, Ellison DA: Rhabdomyosarcomas in adults and children: an update. Arch Pathol Lab Med. 130(10):1454–1465, 2006. 78. Newton WA, Jr, Gehan EA, Webber BL, et al: Classification of rhabdomyosarcomas and related sarcomas. Pathologic aspects and proposal for a new classification–an Intergroup Rhabdomyosarcoma Study. Cancer. 76(6):1073–1085, 1995. 79. Qualman SJ, Coffin CM, Newton WA, et al: Intergroup Rhabdomyosarcoma Study: update for pathologists. Pediatr Dev Pathol. 1(6):550–561, 1998. 80. Coffin CM: The new International Rhabdomyosarcoma Classification, its progenitors, and considerations beyond morphology. Adv Anat Pathol. 4:1–16, 1997. 81. Hawkins WG, Hoos A, Antonescu CR, et al: Clinicopathologic analysis of patients with adult rhabdomyosarcoma. Cancer. 91(4):794–803, 2001. 82. Parham DM, Barr FG: Alveolar rhabdomyosarcoma. In Fletcher CDM, Unni KK, Mertens F, editors: World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Soft Tissue and Bone, Lyon, 2002, IARC Press, pp 150–152. 83. Williamson D, Missiaglia E, de Reynies A, et al: Fusion genenegative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J Clin Oncol. 28(13):2151–2158, 2010. 84. Mentzel T, Katenkamp D: Sclerosing, pseudovascular rhabdomyosarcoma in adults. Clinicopathological and immunohistochemical analysis of three cases. Virchows Arch. 436(4):305–311, 2000.
References 85. Folpe AL, McKenney JK, Bridge JA, et al: Sclerosing rhabdomyosarcoma in adults: report of four cases of a hyalinizing, matrix-rich variant of rhabdomyosarcoma that may be confused with osteosarcoma, chondrosarcoma, or angiosarcoma. Am J Surg Pathol. 26(9):1175–1183, 2002. 86. Croes R, Debiec-Rychter M, Cokelaere K, et al: Adult sclerosing rhabdomyosarcoma: cytogenetic link with embryonal rhabdomyosarcoma. Virchows Arch. 446(1):64–67, 2005. 87. Chiles MC, Parham DM, Qualman SJ, et al: Sclerosing rhabdomyosarcomas in children and adolescents: a clinicopathologic review of 13 cases from the Intergroup Rhabdomyosarcoma Study Group and Children’s Oncology Group. Pediatr Dev Pathol. 7(6):583–594, 2004. 88. Vadgama B, Sebire NJ, Malone M, et al: Sclerosing rhabdomyosarcoma in childhood: case report and review of the literature. Pediatr Dev Pathol. 7(4):391–396, 2004. 89. Zambrano E, Perez-Atayde AR, Ahrens W, et al: Pediatric sclerosing rhabdomyosarcoma. Int J Surg Pathol. 14(3):193–199, 2006. 90. Wang J, Tu X, Sheng W: Sclerosing rhabdomyosarcoma: a clinicopathologic and immunohistochemical study of five cases. Am J Clin Pathol. 129(3):410–415, 2008. 91. Furlong MA, Fanburg-Smith JC: Pleomorphic rhabdomyosarcoma in children: four cases in the pediatric age group. Ann Diagn Pathol. 5(4):199–206, 2001. 92. Furlong MA, Mentzel T, Fanburg-Smith JC: Pleomorphic rhabdomyosarcoma in adults: a clinicopathologic study of 38 cases with emphasis on morphologic variants and recent skeletal muscle-specific markers. Mod Pathol. 14(6):595–603, 2001. 93. Qualman S, Lynch J, Bridge J, et al: Prevalence and clinical impact of anaplasia in childhood rhabdomyosarcoma : a report from the Soft Tissue Sarcoma Committee of the Children’s Oncology Group. Cancer. 113(11):3242–3247, 2008. 94. Bridge JA, Liu J, Qualman SJ, et al: Genomic gains and losses are similar in genetic and histologic subsets of rhabdomyosarcoma, whereas amplification predominates in embryonal with anaplasia and alveolar subtypes. Genes Chromosomes Cancer. 33(3):310–321, 2002. 95. Zuppan CW, Mierau GW, Weeks DA: Lipid-rich rhabdomyosarcoma–a potential source of diagnostic confusion. Ultrastruct Pathol. 15(4-5):353–359, 1991. 96. Boman F, Champigneulle J, Schmitt C, et al: Clear cell rhabdomyosarcoma. Pediatr Pathol Lab Med. 16(6):951–959, 1996. 97. Jo VY, Marino-Enriquez A, Fletcher CD: Epithelioid rhabdomyosarcoma: clinicopathologic analysis of 16 cases of a morphologically distinct variant of rhabdomyosarcoma. Am J Surg Pathol. 35(10):1523–1530, 2011. 98. Morotti RA, Nicol KK, Parham DM, et al: An immunohistochemical algorithm to facilitate diagnosis and subtyping of rhabdomyosarcoma: the Children’s Oncology Group experience. Am J Surg Pathol. 30(8):962–968, 2006. 99. Coffin CM, Rulon J, Smith L, et al: Pathologic features of rhabdomyosarcoma before and after treatment: a clinicopathologic and immunohistochemical analysis. Mod Pathol. 10(12):1175– 1187, 1997. 100. Heerema-McKenney A, Wijnaendts LC, Pulliam JF, et al: Diffuse myogenin expression by immunohistochemistry is an independent marker of poor survival in pediatric rhabdomyosarcoma: a tissue microarray study of 71 primary tumors including correlation with molecular phenotype. Am J Surg Pathol. 32(10):1513– 1522, 2008. 101. Truong LD, Rangdaeng S, Cagle P, et al: The diagnostic utility of desmin. A study of 584 cases and review of the literature. Am J Clin Pathol. 93(3):305–314, 1990. 102. Wachtel M, Runge T, Leuschner I, et al: Subtype and prognostic classification of rhabdomyosarcoma by immunohistochemistry. J Clin Oncol. 24(5):816–822, 2006. 103. Grass B, Wachtel M, Behnke S, et al: Immunohistochemical detection of EGFR, fibrillin-2, P-cadherin and AP2beta as biomarkers for rhabdomyosarcoma diagnostics. Histopathology. 54(7):873–879, 2009. 104. Ganti R, Skapek SX, Zhang J, et al: Expression and genomic status of EGFR and ErbB-2 in alveolar and embryonal rhabdomyosarcoma. Mod Pathol. 19(9):1213–1220, 2006.
876.e3
105. Makawita S, Ho M, Durbin AD, et al: Expression of insulin-like growth factor pathway proteins in rhabdomyosarcoma: IGF-2 expression is associated with translocation-negative tumors. Pediatr Dev Pathol. 12(2):127–135, 2009. 106. San Miguel-Fraile P, Carrillo-Gijon R, Rodriguez-Peralto JL, et al: Prognostic significance of DNA ploidy and proliferative index (MIB-1 index) in childhood rhabdomyosarcoma. Am J Clin Pathol. 121(3):358–365, 2004. 107. Takahashi Y, Oda Y, Kawaguchi K, et al: Altered expression and molecular abnormalities of cell-cycle-regulatory proteins in rhabdomyosarcoma. Mod Pathol. 17(6):660–669, 2004. 108. Barr FG: Molecular genetics and pathogenesis of rhabdomyosarcoma. J Pediatr Hematol Oncol. 19(6):483–491, 1997. 109. Barr FG, Qualman SJ, Macris MH, et al: Genetic heterogeneity in the alveolar rhabdomyosarcoma subset without typical gene fusions. Cancer Res. 62(16):4704–4710, 2002. 110. Sorensen PH, Lynch JC, Qualman SJ, et al: PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group. J Clin Oncol. 20(11):2672–2679, 2002. 111. Barr FG, Duan F, Smith LM, et al: Genomic and clinical analyses of 2p24 and 12q13-q14 amplification in alveolar rhabdomyosarcoma: a report from the Children’s Oncology Group. Genes Chromosomes Cancer. 48(8):661–672, 2009. 112. Miettinen M, Rapola J: Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcoma-like tumors. Expression of cytokeratin and the 68-kD neurofilament protein. Am J Surg Pathol. 13(2):120–132, 1989. 113. Diniz G, Aktas S, Ortac R, et al: Kit expression in spindle cell rhabdomyosarcoma can possibly create a different approach for its tumorigenesis and therapy. Pathol Res Pract. 202(9):671–677, 2006. 114. Stock N, Chibon F, Binh MB, et al: Adult-type rhabdomyosarcoma: analysis of 57 cases with clinicopathologic description, identification of 3 morphologic patterns and prognosis. Am J Surg Pathol. 33(12):1850–1859, 2009. 115. Bahrami A, Gown AM, Baird GS, et al: Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol. 21(7):795–806, 2008. 116. Corao DA, Biegel JA, Coffin CM, et al: ALK expression in rhabdomyosarcomas: correlation with histologic subtype and fusion status. Pediatr Dev Pathol. 12(4):275–283, 2009. 117. Rossi S, Stoppani E, Puri PL, et al: Differentiation of human rhabdomyosarcoma RD cells is regulated by reciprocal, functional interactions between myostatin, p38 and extracellular regulated kinase signalling pathways. Eur J Cancer. 47(7):1095– 1105, 2011. 118. Thway K, Selfe J, Missiaglia E, et al: Glypican-3 is expressed in rhabdomyosarcomas but not adult spindle cell and pleomorphic sarcomas. J Clin Pathol. 64(7):587–591, 2011. 119. Martin SE, Temm CJ, Goheen MP, et al: Cytoplasmic p63 immunohistochemistry is a useful marker for muscle differentiation: an immunohistochemical and immunoelectron microscopic study. Mod Pathol. 24(10):1320–1326, 2011. 120. Goldsmith JD, Pawel B, Goldblum JR, et al: Detection and diagnostic utilization of placental alkaline phosphatase in muscular tissue and tumors with myogenic differentiation. Am J Surg Pathol. 26(12):1627–1633, 2002. 121. Carpentieri DF, Nichols K, Chou PM, et al: The expression of WT1 in the differentiation of rhabdomyosarcoma from other pediatric small round blue cell tumors. Mod Pathol. 15(10):1080– 1086, 2002. 122. Sullivan LM, Atkins KA, LeGallo RD: PAX immunoreactivity identifies alveolar rhabdomyosarcoma. Am J Surg Pathol. 33(5):775–780, 2009. 123. Davis JL, Matsumura L, Weeks DA, et al: PAX2 expression in Wilms tumors and other childhood neoplasms. Am J Surg Pathol. 35(8):1186–1194, 2011. 124. Sana J, Zambo I, Skoda J, et al: CD133 expression and identification of CD133/nestin positive cells in rhabdomyosarcomas and rhabdomyosarcoma cell lines. Anal Cell Pathol (Amst). 34(6):303–318, 2011. 125. Ushigome S, Machinami R, Sorensen PH: Ewing sarcoma/ primitive neuroectodermal tumour (PNET). In Fletcher CD,
876.e4
Immunohistology of Pediatric Neoplasms
Unni KK, Mertens F, editors: Pathology and Genetics of Tumors of Soft Tissue and Bone World Health Organization Classification of Tumours, Lyon, 2002, IARC Press, pp 298–300. 126. Khoury JD: Ewing sarcoma family of tumors. Adv Anat Pathol. 12(4):212–220, 2005. 127. Jawad MU, Cheung MC, Min ES, et al: Ewing sarcoma demonstrates racial disparities in incidence-related and sex-related differences in outcome: an analysis of 1631 cases from the SEER database, 1973-2005. Cancer. 115(15):3526–3536, 2009. 128. Worch J, Cyrus J, Goldsby R, et al: Racial differences in the incidence of mesenchymal tumors associated with EWSR1 translocation. Cancer Epidemiol Biomarkers Prev. 20(3):449–453, 2011. 129. de Alava E, Gerald WL: Molecular biology of the Ewing’s sarcoma/primitive neuroectodermal tumor family. J Clin Oncol. 18(1):204–213, 2000. 130. Hasegawa SL, Davison JM, Rutten A, et al: Primary cutaneous Ewing’s sarcoma: immunophenotypic and molecular cytogenetic evaluation of five cases. Am J Surg Pathol. 22(3):310–318, 1998. 131. Marley EF, Liapis H, Humphrey PA, et al: Primitive neuroectodermal tumor of the kidney–another enigma: a pathologic, immunohistochemical, and molecular diagnostic study. Am J Surg Pathol. 21(3):354–359, 1997. 132. Llombart-Bosch A, Lacombe MJ, Peydro-Olaya A, et al: Malignant peripheral neuroectodermal tumours of bone other than Askin’s neoplasm: characterization of 14 new cases with immunohistochemistry and electron microscopy. Virchows Arch A Pathol Anat Histopathol. 412(5):421–430, 1988. 133. Shanfeld RL, Edelman J, Willis JE, et al: Immunohistochemical analysis of neural markers in peripheral primitive neuroectodermal tumors (pPNET) without light microscopic evidence of neural differentiation. Appl Immunohistochem. 5:78–86, 1997. 134. Fellinger EJ, Garin-Chesa P, Glasser DB, et al: Comparison of cell surface antigen HBA71 (p30/32MIC2), neuron-specific enolase, and vimentin in the immunohistochemical analysis of Ewing’s sarcoma of bone. Am J Surg Pathol. 16(8):746–755, 1992. 135. Gu M, Antonescu CR, Guiter G, et al: Cytokeratin immunoreactivity in Ewing’s sarcoma: prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol. 24(3):410– 416, 2000. 136. Folpe AL, Goldblum JR, Rubin BP, et al: Morphologic and immunophenotypic diversity in Ewing family tumors: a study of 66 genetically confirmed cases. Am J Surg Pathol. 29(8):1025– 1033, 2005. 137. Barisella M, Collini P, Orsenigo M, et al: Unusual myogenic and melanocytic differentiation of soft tissue pPNETs: an immunohistochemical and molecular study of 3 cases. Am J Surg Pathol. 34(7):1002–1006, 2010. 138. Hamilton G, Fellinger EJ, Schratter I, et al: Characterization of a human endocrine tissue and tumor-associated Ewing’s sarcoma antigen. Cancer Res. 48(21):6127–6131, 1988. 139. Llombart-Bosch A, Machado I, Navarro S, et al: Histological heterogeneity of Ewing’s sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support. Virchows Arch. 455(5):397–411, 2009. 140. Tirado OM, Mateo-Lozano S, Villar J, et al: Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing’s sarcoma cells. Cancer Res. 66(20):9937–9947, 2006. 141. Kumar S, Pack S, Kumar D, et al: Detection of EWS-FLI-1 fusion in Ewing’s sarcoma/peripheral primitive neuroectodermal tumor by fluorescence in situ hybridization using formalin-fixed paraffin-embedded tissue. Hum Pathol. 30(3):324–330, 1999. 142. Fritsch MK, Bridge JA, Schuster AE, et al: Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue. Pediatr Dev Pathol. 6(1):43–53, 2003. 143. Dagher R, Pham TA, Sorbara L, et al: Molecular confirmation of Ewing sarcoma. J Pediatr Hematol Oncol. 23(4):221–224, 2001. 144. Sandberg AA, Bridge JA: Updates on cytogenetics and molecular genetics of bone and soft tissue tumors: Ewing sarcoma and peripheral primitive neuroectodermal tumors. Cancer Genet Cytogenet. 123(1):1–26, 2000.
145. Delattre O, Zucman J, Melot T, et al: The Ewing family of tumors–a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 331(5):294–299, 1994. 146. Wang L, Bhargava R, Zheng T, et al: Undifferentiated small round cell sarcomas with rare EWS gene fusions: identification of a novel EWS-SP3 fusion and of additional cases with the EWS-ETV1 and EWS-FEV fusions. J Mol Diagn. 9(4):498–509, 2007. 147. Sankar S, Lessnick SL: Promiscuous partnerships in Ewing’s sarcoma. Cancer Genet. 204(7):351–365, 2011. 148. Gamberi G, Cocchi S, Benini S, et al: Molecular diagnosis in Ewing family tumors: the Rizzoli experience–222 consecutive cases in four years. J Mol Diagn. 13(3):313–324, 2011. 149. Machado I, Noguera R, Pellin A, et al: Molecular diagnosis of Ewing sarcoma family of tumors: a comparative analysis of 560 cases with FISH and RT-PCR. Diagn Mol Pathol. 18(4):189–199, 2009. 150. Kushner BH, LaQuaglia MP, Cheung NK, et al: Clinically critical impact of molecular genetic studies in pediatric solid tumors. Med Pediatr Oncol. 33(6):530–535, 1999. 151. McManus AP, Gusterson BA, Pinkerton CR, et al: The molecular pathology of small round-cell tumours—relevance to diagnosis, prognosis, and classification. J Pathol. 178(2):116–121, 1996. 152. Antonescu CR: The role of genetic testing in soft tissue sarcoma. Histopathology. 48(1):13–21, 2006. 153. Lazar A, Abruzzo LV, Pollock RE, et al: Molecular diagnosis of sarcomas: chromosomal translocations in sarcomas. Arch Pathol Lab Med. 130(8):1199–1207, 2006. 154. Lewis TB, Coffin CM, Bernard PS: Differentiating Ewing’s sarcoma from other round blue cell tumors using a RT-PCR translocation panel on formalin-fixed paraffin-embedded tissues. Mod Pathol. 20(3):397–404, 2007. 155. Ng TL, O’Sullivan MJ, Pallen CJ, et al: Ewing sarcoma with novel translocation t(2;16) producing an in-frame fusion of FUS and FEV. J Mol Diagn. 9(4):459–463, 2007. 156. Shing DC, McMullan DJ, Roberts P, et al: FUS/ERG gene fusions in Ewing’s tumors. Cancer Res. 63(15):4568–4576, 2003. 157. Olsen SH, Thomas DG, Lucas DR: Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol. 19(5):659– 668, 2006. 158. Folpe AL, Goldblum JR, Rubin BP, et al: When can Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) be diagnosed without genetic confirmation? A study of 62 proven cases. Mod Pathol. 17(Suppl 1):13A, 2004. 159. Scotlandi K, Serra M, Manara MC, et al: Immunostaining of the p30/32MIC2 antigen and molecular detection of EWS rearrangements for the diagnosis of Ewing’s sarcoma and peripheral neuroectodermal tumor. Hum Pathol. 27(4):408–416, 1996. 160. Jambhekar NA, Bagwan IN, Ghule P, et al: Comparative analysis of routine histology, immunohistochemistry, reverse transcriptase polymerase chain reaction, and fluorescence in situ hybridization in diagnosis of Ewing family of tumors. Arch Pathol Lab Med. 130(12):1813–1818, 2006. 161. Kawamura-Saito M, Yamazaki Y, Kaneko K, et al: Fusion between CIC and DUX4 up-regulates PEA3 family genes in Ewing-like sarcomas with t(4;19)(q35;q13) translocation. Hum Mol Genet. 15(13):2125–2137, 2006. 162. Amir G, Issakov J, Meller I, et al: Expression of p53 gene product and cell proliferation marker Ki-67 in Ewing’s sarcoma: correlation with clinical outcome. Hum Pathol. 33(2):170–174, 2002. 163. de Alava E, Antonescu CR, Panizo A, et al: Prognostic impact of P53 status in Ewing sarcoma. Cancer. 89(4):783–792, 2000. 164. Maitra A, Roberts H, Weinberg AG, et al: Aberrant expression of tumor suppressor proteins in the Ewing family of tumors. Arch Pathol Lab Med. 125(9):1207–1212, 2001. 165. Smithey BE, Pappo AS, Hill DA: C-kit expression in pediatric solid tumors: a comparative immunohistochemical study. Am J Surg Pathol. 26(4):486–492, 2002. 166. Fujiwara T, Fukushi J, Yamamoto S, et al: Macrophage infiltration predicts a poor prognosis for human ewing sarcoma. Am J Pathol. 179(3):1157–1170, 2011. 167. Daugaard S, Kamby C, Sunde LM, et al: Ewing’s sarcoma. A retrospective study of histological and immunohistochemical
References factors and their relation to prognosis. Virchows Arch A Pathol Anat Histopathol. 414(3):243–251, 1989. 168. Parham DM, Hijazi Y, Steinberg SM, et al: Neuroectodermal differentiation in Ewing’s sarcoma family of tumors does not predict tumor behavior. Hum Pathol. 30(8):911–918, 1999. 169. Pinto A, Grant LH, Hayes FA, et al: Immunohistochemical expression of neuron-specific enolase and Leu 7 in Ewing’s sarcoma of bone. Cancer. 64(6):1266–1273, 1989. 170. de Alava E, Kawai A, Healey JH, et al: EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing’s sarcoma. J Clin Oncol. 16(4):1248–1255, 1998. 171. van Doorninck JA, Ji L, Schaub B, et al: Current treatment protocols have eliminated the prognostic advantage of type 1 fusions in Ewing sarcoma: a report from the Children’s Oncology Group. J Clin Oncol. 28(12):1989–1994, 2010. 172. Ordonez NG: Desmoplastic small round cell tumor: II: an ultrastructural and immunohistochemical study with emphasis on new immunohistochemical markers. Am J Surg Pathol. 22(11):1314–1327, 1998. 173. Lucas DR, Bentley G, Dan ME, et al: Ewing sarcoma vs lymphoblastic lymphoma. A comparative immunohistochemical study. Am J Clin Pathol. 115(1):11–17, 2001. 174. Lin O, Filippa DA, Teruya-Feldstein J: Immunohistochemical evaluation of FLI-1 in acute lymphoblastic lymphoma (ALL): a potential diagnostic pitfall. Appl Immunohistochem Mol Morphol. 17(5):409–412, 2009. 175. Gerald WL, Ladanyi M, de Alava E, et al: Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22) (p13;q12): desmoplastic small round-cell tumor and its variants. J Clin Oncol. 16(9):3028–3036, 1998. 176. Ordonez NG, Zirkin R, Bloom RE: Malignant small-cell epithelial tumor of the peritoneum coexpressing mesenchymal-type intermediate filaments. Am J Surg Pathol. 13(5):413–421, 1989. 177. Gerald WL, Miller HK, Battifora H, et al: Intra-abdominal desmoplastic small round-cell tumor. Report of 19 cases of a distinctive type of high-grade polyphenotypic malignancy affecting young individuals. Am J Surg Pathol. 15(6):499–513, 1991. 178. Gonzalez-Crussi F, Crawford SE, Sun CC: Intraabdominal desmoplastic small-cell tumors with divergent differentiation. Observations on three cases of childhood. Am J Surg Pathol. 14(7):633–642, 1990. 179. Livaditi E, Mavridis G, Soutis M, et al: Diffuse intraabdominal desmoplastic small round cell tumor: a ten-year experience. Eur J Pediatr Surg. 16(6):423–427, 2006. 180. Saab R, Khoury JD, Krasin M, et al: Desmoplastic small round cell tumor in childhood: the St. Jude Children’s Research Hospital experience. Pediatr Blood Cancer. 49(3):274–279, 2007. 181. Wolf AN, Ladanyi M, Paull G, et al: The expanding clinical spectrum of desmoplastic small round-cell tumor: a report of two cases with molecular confirmation. Hum Pathol. 30(4):430– 435, 1999. 182. Heikkila AJ, Prebtani AP: Desmoplastic small round cell tumour in a 74 year old man: an uncommon cause of ascites (case report). Diagn Pathol. 6:55, 2011. 183. Kawano N, Inayama Y, Nagashima Y, et al: Desmoplastic small round-cell tumor of the paratesticular region: report of an adult case with demonstration of EWS and WT1 gene fusion using paraffin-embedded tissue. Mod Pathol. 12(7):729–734, 1999. 184. Parkash V, Gerald WL, Parma A, et al: Desmoplastic small round cell tumor of the pleura. Am J Surg Pathol. 19(6):659–665, 1995. 185. Tison V, Cerasoli S, Morigi F, et al: Intracranial desmoplastic small-cell tumor. Report of a case. Am J Surg Pathol. 20(1):112– 117, 1996. 186. Backer A, Mount SL, Zarka MA, et al: Desmoplastic small round cell tumour of unknown primary origin with lymph node and lung metastases: histological, cytological, ultrastructural, cytogenetic and molecular findings. Virchows Arch. 432(2):135–141, 1998. 187. Collardeau-Frachon S, Ranchere-Vince D, Delattre O, et al: Primary desmoplastic small round cell tumor of the kidney: a case report in a 14-year-old girl with molecular confirmation. Pediatr Dev Pathol. 10(4):320–324, 2007. 188. Wang LL, Perlman EJ, Vujanic GM, et al: Desmoplastic small round cell tumor of the kidney in childhood. Am J Surg Pathol. 31(4):576–584, 2007.
876.e5
189. Qureshi SS, Shrikhande S, Ramadwar M, et al: Desmoplastic small round cell tumor of the pancreas: An unusual primary site for an uncommon tumor. J Indian Assoc Pediatr Surg. 16(2):66– 68, 2011. 190. Neder L, Scheithauer BW, Turel KE, et al: Desmoplastic small round cell tumor of the central nervous system: report of two cases and review of the literature. Virchows Arch. 454(4):431– 439, 2009. 191. Peydro-Olaya A, Llombart-Bosch A, Carda-Batalla C, et al: Electron microscopy and other ancillary techniques in the diagnosis of small round cell tumors. Semin Diagn Pathol. 20(1):25–45, 2003. 192. Ordonez NG, el-Naggar AK, Ro JY, et al: Intra-abdominal desmoplastic small cell tumor: a light microscopic, immunocytochemical, ultrastructural, and flow cytometric study. Hum Pathol. 24(8):850–865, 1993. 193. Barnoud R, Delattre O, Peoc’h M, et al: Desmoplastic small round cell tumor: RT-PCR analysis and immunohistochemical detection of the Wilm’s tumor gene WT1. Pathol Res Pract. 194(10):693–700, 1998. 194. Murphy AJ, Bishop K, Pereira C, et al: A new molecular variant of desmoplastic small round cell tumor: significance of WT1 immunostaining in this entity. Hum Pathol. 39(12):1763–1770, 2008. 195. Kim J, Lee K, Pelletier J: The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally distinct. Oncogene. 16(8):1021–1030, 1998. 196. Gerald WL, Haber DA: The EWS-WT1 gene fusion in desmoplastic small round cell tumor. Semin Cancer Biol. 15(3):197– 205, 2005. 197. Alaggio R, Rosolen A, Sartori F, et al: Spindle cell tumor with EWS-WT1 transcript and a favorable clinical course: a variant of DSCT, a variant of leiomyosarcoma, or a new entity? Report of 2 pediatric cases. Am J Surg Pathol. 31(3):454–459, 2007. 198. Antonescu CR, Gerald WL, Magid MS, et al: Molecular variants of the EWS-WT1 gene fusion in desmoplastic small round cell tumor. Diagn Mol Pathol. 7(1):24–28, 1998. 199. de Alava E, Ladanyi M, Rosai J, et al: Detection of chimeric transcripts in desmoplastic small round cell tumor and related developmental tumors by reverse transcriptase polymerase chain reaction. A specific diagnostic assay. Am J Pathol. 147(6):1584–1591, 1995. 200. Gerald WL, Rosai J, Ladanyi M: Characterization of the genomic breakpoint and chimeric transcripts in the EWS-WT1 gene fusion of desmoplastic small round cell tumor. Proc Natl Acad Sci U S A. 92(4):1028–1032, 1995. 201. Ladanyi M, Gerald W: Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res. 54(11):2837– 2840, 1994. 202. Ladanyi M: The emerging molecular genetics of sarcoma translocations. Diagn Mol Pathol. 4(3):162–173, 1995. 203. Leuschner I, Radig K, Harms D: Desmoplastic small round cell tumor. Semin Diagn Pathol. 13(3):204–212, 1996. 204. Zhang J, Dalton J, Fuller C: Epithelial marker-negative desmoplastic small round cell tumor with atypical morphology: definitive classification by fluorescence in situ hybridization. Arch Pathol Lab Med. 131(4):646–649, 2007. 205. Waugh MS, Dash RC, Turner KC, et al: Desmoplastic small round cell tumor: using FISH as an ancillary technique to support cytologic diagnosis in an unusual case. Diagn Cytopathol. 35(8):516–520, 2007. 206. Yamaguchi U, Hasegawa T, Morimoto Y, et al: A practical approach to the clinical diagnosis of Ewing’s sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue. J Clin Pathol. 58(10):1051–1056, 2005. 207. Schofield D: Extrarenal rhabdoid tumour. In Fletcher CDM, Unni KK, Merten F, editors: Pathology and Genetics of Tumors of Soft Tissue and Bone World Health Organization Classification of Tumours, Lyon, 2002, IARC Press, pp 219–220. 208. Kodet R, Newton WA, Jr, Sachs N, et al: Rhabdoid tumors of soft tissues: a clinicopathologic study of 26 cases enrolled on the
876.e6
Immunohistology of Pediatric Neoplasms
Intergroup Rhabdomyosarcoma Study. Hum Pathol. 22(7):674– 684, 1991. 209. Tsuneyoshi M, Daimaru Y, Hashimoto H, et al: Malignant soft tissue neoplasms with the histologic features of renal rhabdoid tumors: an ultrastructural and immunohistochemical study. Hum Pathol. 16(12):1235–1242, 1985. 210. Morgenstern DA, Gibson S, Brown T, et al: Clinical and pathological features of paediatric malignant rhabdoid tumours. Pediatr Blood Cancer. 54(1):29–34, 2010. 211. Beckwith JB, Palmer NF: Histopathology and prognosis of Wilms tumors: results from the First National Wilms’ Tumor Study. Cancer. 41(5):1937–1948, 1978. 212. Tsokos M, Kouraklis G, Chandra RS, et al: Malignant rhabdoid tumor of the kidney and soft tissues. Evidence for a diverse morphological and immunocytochemical phenotype. Arch Pathol Lab Med. 113(2):115–120, 1989. 213. Rorke LB, Packer RJ, Biegel JA: Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood: definition of an entity. J Neurosurg. 85(1):56–65, 1996. 214. Burger PC, Yu IT, Tihan T, et al: Atypical teratoid/rhabdoid tumor of the central nervous system: a highly malignant tumor of infancy and childhood frequently mistaken for medulloblastoma: a Pediatric Oncology Group study. Am J Surg Pathol. 22(9):1083–1092, 1998. 215. White FV, Dehner LP, Belchis DA, et al: Congenital disseminated malignant rhabdoid tumor: a distinct clinicopathologic entity demonstrating abnormalities of chromosome 22q11. Am J Surg Pathol. 23(3):249–256, 1999. 216. Parham DM, Weeks DA, Beckwith JB: The clinicopathologic spectrum of putative extrarenal rhabdoid tumors. An analysis of 42 cases studied with immunohistochemistry or electron microscopy. Am J Surg Pathol. 18(10):1010–1029, 1994. 217. Fanburg-Smith JC, Hengge M, Hengge UR, et al: Extrarenal rhabdoid tumors of soft tissue: a clinicopathologic and immunohistochemical study of 18 cases. Ann Diagn Pathol. 2(6):351– 362, 1998. 218. Alaggio R, Boldrini R, Di Venosa B, et al: Pediatric extra-renal rhabdoid tumors with unusual morphology: a diagnostic pitfall for small biopsies. Pathol Res Pract. 205(7):451–457, 2009. 219. Bordeaut F, Lequin D, Brugieres L, et al: Frequent hSNF5/INI1 germline mutations in patients with rhabdoid tumor. Clin Cancer Res. 17:31–38, 2011. 220. Gessi M, Giangaspero F, Pietsch T: Atypical teratoid/rhabdoid tumors and choroid plexus tumors: when genetics “surprise” pathology. Brain Pathol. 13(3):409–414, 2003. 221. Eaton KW, Tooke LS, Wainwright LM, et al: Spectrum of SMARCB1/INI1 mutations in familial and sporadic rhabdoid tumors. Pediatr Blood Cancer. 56(1):7–15, 2011. 222. Sullivan LM, Yankovich T, Le P, et al: Claudin-6 is a nonspecific marker for malignant rhabdoid and other pediatric tumors. Am J Surg Pathol. 36(1):73–80, 2012. 223. Shiratsuchi H, Saito T, Sakamoto A, et al: Mutation analysis of human cytokeratin 8 gene in malignant rhabdoid tumor: a possible association with intracytoplasmic inclusion body formation. Mod Pathol. 15(2):146–153, 2002. 224. Deisch J, Raisanen J, Rakheja D: Immunohistochemical expression of embryonic stem cell markers in malignant rhabdoid tumors. Pediatr Dev Pathol. 14(5):353–359, 2011. 225. Venneti S, Le P, Martinez D, et al: Malignant rhabdoid tumors express stem cell factors, which relate to the expression of EZH2 and Id proteins. Am J Surg Pathol. 35(10):1463–1472, 2011. 226. Izumi T, Oda Y, Hasegawa T, et al: Prognostic significance of dysadherin expression in epithelioid sarcoma and its diagnostic utility in distinguishing epithelioid sarcoma from malignant rhabdoid tumor. Mod Pathol. 19(6):820–831, 2006. 227. Biegel JA, Burk CD, Parmiter AH, et al: Molecular analysis of a partial deletion of 22q in a central nervous system rhabdoid tumor. Genes Chromosomes Cancer. 5(2):104–108, 1992. 228. Schofield DE, Beckwith JB, Sklar J: Loss of heterozygosity at chromosome regions 22q11-12 and 11p15.5 in renal rhabdoid tumors. Genes Chromosomes Cancer. 15(1):10–17, 1996. 229. Biegel JA: Molecular genetics of atypical teratoid/rhabdoid tumor. Neurosurg Focus. 20(1):E11, 2006. 230. Uno K, Takita J, Yokomori K, et al: Aberrations of the hSNF5/ INI1 gene are restricted to malignant rhabdoid tumors or
atypical teratoid/rhabdoid tumors in pediatric solid tumors. Genes Chromosomes Cancer. 34(1):33–41, 2002. 231. Sevenet N, Lellouch-Tubiana A, Schofield D, et al: Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotypephenotype correlations. Hum Mol Genet. 8(13):2359–2368, 1999. 232. Sevenet N, Sheridan E, Amram D, et al: Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers. Am J Hum Genet. 65(5):1342–1348, 1999. 233. Tsai CY, Wong TT, Lee YH, et al: Intact INI1 gene region with paradoxical loss of protein expression in AT/RT: implications for a possible novel mechanism associated with absence of INI1 protein immunoreactivity. Am J Surg Pathol. 36(1):128–133, 2012. 234. Hasselblatt M, Gesk S, Oyen F, et al: Nonsense mutation and inactivation of SMARCA4 (BRG1) in an atypical teratoid/ rhabdoid tumor showing retained SMARCB1 (INI1) expression. Am J Surg Pathol. 35(6):933–935, 2011. 235. Bruch LA, Hill DA, Cai DX, et al: A role for fluorescence in situ hybridization detection of chromosome 22q dosage in distinguishing atypical teratoid/rhabdoid tumors from medulloblastoma/central primitive neuroectodermal tumors. Hum Pathol. 32(2):156–162, 2001. 236. Oda Y, Yamamoto H, Tamiya S, et al: CXCR4 and VEGF expression in the primary site and the metastatic site of human osteosarcoma: analysis within a group of patients, all of whom developed lung metastasis. Mod Pathol. 19(5):738–745, 2006. 237. Bourdeaut F, Freneaux P, Thuille B, et al: hSNF5/INI1-deficient tumours and rhabdoid tumours are convergent but not fully overlapping entities. J Pathol. 211(3):323–330, 2007. 238. Haberler C, Laggner U, Slavc I, et al: Immunohistochemical analysis of INI1 protein in malignant pediatric CNS tumors: Lack of INI1 in atypical teratoid/rhabdoid tumors and in a fraction of primitive neuroectodermal tumors without rhabdoid phenotype. Am J Surg Pathol. 30(11):1462–1468, 2006. 239. Oda Y, Tsuneyoshi M: Extrarenal rhabdoid tumors of soft tissue: clinicopathological and molecular genetic review and distinction from other soft-tissue sarcomas with rhabdoid features. Pathol Int. 56(6):287–295, 2006. 240. Kohashi K, Izumi T, Oda Y, et al: Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor. Hum Pathol. 40(3):349–355, 2009. 241. Kohashi K, Oda Y, Yamamoto H, et al: SMARCB1/INI1 protein expression in round cell soft tissue sarcomas associated with chromosomal translocations involving EWS: a special reference to SMARCB1/INI1 negative variant extraskeletal myxoid chondrosarcoma. Am J Surg Pathol. 32(8):1168–1174, 2008. 242. Dome JS, Huff V: Wilms Tumor Overview. In Pagon RA, Bird TD, Dolan CR, et al, editors: GeneReviews, Seattle, 1993, University of Washington, Seattle. 243. Ruteshouser EC, Robinson SM, Huff V: Wilms tumor genetics: mutations in WT1, WTX, and CTNNB1 account for only about one-third of tumors. Genes Chromosomes Cancer. 47(6):461– 470, 2008. 244. Li CM, Kim CE, Margolin AA, et al: CTNNB1 mutations and overexpression of Wnt/beta-catenin target genes in WT1-mutant Wilms’ tumors. Am J Pathol. 165(6):1943–1953, 2004. 245. Fukuzawa R, Heathcott RW, Sano M, et al: Myogenesis in Wilms’ tumors is associated with mutations of the WT1 gene and activation of Bcl-2 and the Wnt signaling pathway. Pediatr Dev Pathol. 7(2):125–137, 2004. 246. Corbin M, de Reynies A, Rickman DS, et al: WNT/beta-catenin pathway activation in Wilms tumors: a unifying mechanism with multiple entries? Genes Chromosomes Cancer. 48(9):816–827, 2009. 247. Stock C, Ambros IM, Lion T, et al: Genetic changes of two Wilms tumors with anaplasia and a review of the literature suggesting a marker profile for therapy resistance. Cancer Genet Cytogenet. 135(2):128–138, 2002. 248. Grundy PE, Breslow NE, Li S, et al: Loss of heterozygosity for chromosomes 1p and 16q is an adverse prognostic factor in favorable-histology Wilms tumor: a report from the National Wilms Tumor Study Group. J Clin Oncol. 23(29):7312–7321, 2005.
References 249. Perlman EJ, Grundy PE, Anderson JR, et al: WT1 mutation and 11p15 loss of heterozygosity predict relapse in very low-risk Wilms tumors treated with surgery alone: a Children’s Oncology Group study. J Clin Oncol. 29(6):698–703, 2011. 250. Williams RD, Al-Saadi R, Natrajan R, et al: Molecular profiling reveals frequent gain of MYCN and anaplasia-specific loss of 4q and 14q in Wilms tumor. Genes Chromosomes Cancer. 50(12):982–995, 2011. 251. Wittmann S, Zirn B, Alkassar M, et al: Loss of 11q and 16q in Wilms tumors is associated with anaplasia, tumor recurrence, and poor prognosis. Genes Chromosomes Cancer. 46(2):163– 170, 2007. 252. Hussong JW, Perkins SL, Huff V, et al: Familial Wilms’ tumor with neural elements: characterization by histology, immunohistochemistry, and genetic analysis. Pediatr Dev Pathol. 3(6):561– 567, 2000. 253. Vujanic GM, Sandstedt B, Harms D, et al: Revised International Society of Paediatric Oncology (SIOP) working classification of renal tumors of childhood. Med Pediatr Oncol. 38(2):79–82, 2002. 254. Ellison DA, Parham DM, Bridge J, et al: Immunohistochemistry of primary malignant neuroepithelial tumors of the kidney: a potential source of confusion? A study of 30 cases from the National Wilms Tumor Study Pathology Center. Hum Pathol. 38(2):205–211, 2007. 255. Folpe AL, Patterson K, Gown AM: Antibodies to desmin identify the blastemal component of nephroblastoma. Mod Pathol. 10(9):895–900, 1997. 256. Murphy AJ, Viero S, Ho M, et al: Diagnostic utility of nestin expression in pediatric tumors in the region of the kidney. Appl Immunohistochem Mol Morphol. 17(6):517–523, 2009. 257. Muir TE, Cheville JC, Lager DJ: Metanephric adenoma, nephrogenic rests, and Wilms’ tumor: a histologic and immunophenotypic comparison. Am J Surg Pathol. 25(10):1290–1296, 2001. 258. Shao L, Hill DA, Perlman EJ: Expression of WT-1, Bcl-2, and CD34 by primary renal spindle cell tumors in children. Pediatr Dev Pathol. 7(6):577–582, 2004. 259. Koesters R, Niggli F, von Knebel Doeberitz M, et al: Nuclear accumulation of beta-catenin protein in Wilms’ tumours. J Pathol. 199(1):68–76, 2003. 260. Fukuzawa R, Anaka MR, Weeks RJ, et al: Canonical WNT signalling determines lineage specificity in Wilms tumour. Oncogene. 28(8):1063–1075, 2009. 261. Lovvorn HN, Westrup J, Opperman S, et al: CITED1 expression in Wilms’ tumor and embryonic kidney. Neoplasia. 9(7):589– 600, 2007. 262. Klein MJ, Siegal GP: Osteosarcoma: anatomic and histologic variants. Am J Clin Pathol. 125(4):555–581, 2006. 263. Raymond AK: Conventional Osteosarcoma. In Fletcher CDM, Unni KK, Merten F, editors: Pathology and Genetics: Tumours of Soft Tissue and Bone, Lyon, 2002, IARC Press, pp 264–270. 264. Okada K, Hasegawa T, Yokoyama R, et al: Osteosarcoma with cytokeratin expression: a clinicopathological study of six cases with an emphasis on differential diagnosis from metastatic cancer. J Clin Pathol. 56(10):742–746, 2003. 265. Hasegawa T, Hirose T, Kudo E, et al: Immunophenotypic heterogeneity in osteosarcomas. Hum Pathol. 22(6):583–590, 1991. 266. de la Roza G: p63 expression in giant cell-containing lesions of bone and soft tissue. Arch Pathol Lab Med. 135(6):776–779, 2011. 267. Ariizumi T, Ogose A, Kawashima H, et al: Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors. Pathol Int. 60(3):193–202, 2010. 268. Veselska R, Hermanova M, Loja T, et al: Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines. BMC Cancer. 8:300, 2008. 269. Lee AF, Hayes MM, Lebrun D, et al: FLI-1 distinguishes Ewing sarcoma from small cell osteosarcoma and mesenchymal chondrosarcoma. Appl Immunohistochem Mol Morphol. 19(3):233– 238, 2011. 270. Hosono A, Yamaguchi U, Makimoto A, et al: Utility of immunohistochemical analysis for cyclo-oxygenase 2 in the differential diagnosis of osteoblastoma and osteosarcoma. J Clin Pathol. 60(4):410–414, 2007.
876.e7
271. Gomez-Brouchet A, Mourcin F, Gourraud PA, et al: Galectin-1 is a powerful marker to distinguish chondroblastic osteosarcoma and conventional chondrosarcoma. Hum Pathol. 41(9):1220– 1230, 2010. 272. Yoshida A, Ushiku T, Motoi T, et al: Immunohistochemical analysis of MDM2 and CDK4 distinguishes low-grade osteosarcoma from benign mimics. Mod Pathol. 23(9):1279–1288, 2010. 273. Yoshida A, Ushiku T, Motoi T, et al: MDM2 and CDK4 immunohistochemical coexpression in high-grade osteosarcoma: correlation with a dedifferentiated subtype. Am J Surg Pathol. 36(3):423–431, 2012. 274. Dujardin F, Binh MB, Bouvier C, et al: MDM2 and CDK4 immunohistochemistry is a valuable tool in the differential diagnosis of low-grade osteosarcomas and other primary f ibro-osseous lesions of the bone. Mod Pathol. 24(5):624–637, 2011. 275. Bacci G, Bertoni F, Longhi A, et al: Neoadjuvant chemotherapy for high-grade central osteosarcoma of the extremity. Histologic response to preoperative chemotherapy correlates with histologic subtype of the tumor. Cancer. 97(12):3068–3075, 2003. 276. Coffin CM, Lowichik A, Zhou H: Treatment effects in pediatric soft tissue and bone tumors: practical considerations for the pathologist. Am J Clin Pathol. 123(1):75–90, 2005. 277. Hauben EI, Weeden S, Pringle J, et al: Does the histological subtype of high-grade central osteosarcoma influence the response to treatment with chemotherapy and does it affect overall survival? A study on 570 patients of two consecutive trials of the European Osteosarcoma Intergroup. Eur J Cancer. 38(9):1218–1225, 2002. 278. Kilpatrick SE, Geisinger KR, King TS, et al: Clinicopathologic analysis of HER-2/neu immunoexpression among various histologic subtypes and grades of osteosarcoma. Mod Pathol. 14(12):1277–1283, 2001. 279. Tsai JY, Aviv H, Benevenia J, et al: HER-2/neu and p53 in osteosarcoma: an immunohistochemical and fluorescence in situ hybridization analysis. Cancer Invest. 22(1):16–24, 2004. 280. Ferrari S, Bertoni F, Zanella L, et al: Evaluation of P-glycoprotein, HER-2/ErbB-2, p53, and Bcl-2 in primary tumor and metachronous lung metastases in patients with high-grade osteosarcoma. Cancer. 100(9):1936–1942, 2004. 281. Zhou H, Randall RL, Brothman AR, et al: Her-2/neu expression in osteosarcoma increases risk of lung metastasis and can be associated with gene amplification. J Pediatr Hematol Oncol. 25(1):27–32, 2003. 282. Varelias A, Koblar SA, Cowled PA, et al: Human osteosarcoma expresses specific ephrin profiles: implications for tumorigenicity and prognosis. Cancer. 95(4):862–869, 2002. 283. Kim MS, Song WS, Cho WH, et al: Ezrin expression predicts survival in stage IIB osteosarcomas. Clin Orthop Relat Res. 459:229–236, 2007. 284. Carneiro A, Bendahl PO, Akerman M, et al: Ezrin expression predicts local recurrence and development of metastases in soft tissue sarcomas. J Clin Pathol. 64(8):689–694, 2011. 285. Dhaini HR, Thomas DG, Giordano TJ, et al: Cytochrome P450 CYP3A4/5 expression as a biomarker of outcome in osteosarcoma. J Clin Oncol. 21(13):2481–2485, 2003. 286. Srivastava A, Fuchs B, Zhang K, et al: High WT1 expression is associated with very poor survival of patients with osteogenic sarcoma metastasis. Clin Cancer Res. 12(14 Pt 1):4237–4243, 2006. 287. de Nigris F, Zanella L, Cacciatore F, et al: YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma. BMC Cancer. 11:472, 2011. 288. Fellenberg J, Bernd L, Delling G, et al: Prognostic significance of drug-regulated genes in high-grade osteosarcoma. Mod Pathol. 20(10):1085–1094, 2007. 289. Serra M, Scotlandi K, Reverter-Branchat G, et al: Value of P-glycoprotein and clinicopathologic factors as the basis for new treatment strategies in high-grade osteosarcoma of the extremities. J Clin Oncol. 21(3):536–542, 2003. 290. Pakos EE, Ioannidis JP: The association of P-glycoprotein with response to chemotherapy and clinical outcome in patients with osteosarcoma. A meta-analysis. Cancer. 98(3):581–589, 2003.
876.e8
Immunohistology of Pediatric Neoplasms
291. Nakashima H, Nishida Y, Sugiura H, et al: Telomerase, p53 and PCNA activity in osteosarcoma. Eur J Surg Oncol. 29(7):564– 567, 2003. 292. Junior AT, de Abreu Alves F, Pinto CA, et al: Clinicopathological and immunohistochemical analysis of twenty-five head and neck osteosarcomas. Oral Oncol. 39(5):521–530, 2003.
293. Trieb K, Lehner R, Stulnig T, et al: Survivin expression in human osteosarcoma is a marker for survival. Eur J Surg Oncol. 29(4):379–382, 2003. 294. Wang W, Luo H, Wang A: Expression of survivin and correlation with PCNA in osteosarcoma. J Surg Oncol. 93(7):578–584, 2006.
C H A P T E R 2 3
IMAGING AND QUANTITATIVE IMMUNOHISTOCHEMISTRY LIRON PANTANOWITZ, DAVID L. RIMM
Overview 877 Imaging Systems 877 Software Algorithms 878 Strengths and Limitations 881 Clinical Applications 882 Summary 884
Overview Pathologists frequently use immunohistochemistry (IHC) to evaluate antigen location and expression patterns in tissue for diagnostic and/or prognostic purposes. This process often also requires quantification of these immunostains to assist in therapeutic drug selection. Traditional scoring systems for IHC have relied on manual subjective interpretation and semiquantification of immunostains by pathologists examining microscopic glass slides. With the advent of digital images, multispectral imaging, and immunofluorescent microscopy, automated image analysis tools have been developed that can be used by pathologists for automated scoring of immunostains.1-5 Image analysis is defined as the extraction of meaningful information from digital images by using imageprocessing tools. Computers are necessary to analyze large datasets and extract quantitative information. Most image-analysis tools operate by measuring the number of pixels showing staining for one or more antigens and then quantifying colocalization of these stains. They automate and quantify groups of pixels or a region of interest (ROI) with greater consistency and accuracy than light microscopy, because they eliminate interobserver and intraobserver variations that occur with human interpretation. They also permit new staining protocols, such as multiplexed antibody studies, to be used that were previously impossible to accurately quantify by using analog-driven approaches. Digital imaging also makes it easier to use immunofluorescence as a primary diagnostic tool. Quantitative immunofluorescence (QIF) has been used extensively
in research labs and recently has also seen limited usage in the clinical setting. Now that entire glass slides can be scanned with whole-slide digital scanners, digitized slides are increasingly used to perform image analysis and apply machine-learning techniques. Improvements in computation power and development of sophisticated image-analysis algorithms continue to offer pathologists more innovative tools to perform computer-aided image analysis (CAIA), which has been most widely used for estrogen receptor (ER), progesterone receptor (PR), and Her2 assessments in breast cancer. However, in spite of decades of improvement, CAIA is still predominantly used as a research tool. It has been applied in a limited way in clinical practice, including quantification of breast carcinoma biomarkers, some use in proliferation markers, microvessel density (MVD) analysis, and other less commonly used applications. This chapter provides an overview of image-analysis theory and technology and focuses on common examples used in current pathology practice.
Imaging Systems A digital image is a numeric representation of an image captured with a device, such as a digital camera. Digital cameras can be attached (coupled with a C-mount adaptor) to light microscopes, or they may be more specialized for advanced imaging (e.g., fluorescence or multispectral imaging). Digital images are made up of thousands of small rectangular pixels (PIcture ELements). Each pixel contains binary data that stores values such as brightness and color. Digital images can range from static images (stills or “snapshots”) to wholeslide images of digitized slides, sometimes called “virtual” images. Several vendors now offer whole-slide imaging (WSI) scanners with built-in robotics, optical microscopes, and digital cameras capable of automatically scanning glass slides at high speeds (1 to 4 minutes) to produce highresolution whole-slide images.5 As a result, image analysis of tissue need not be limited to still images of selected fields of view (FOV) but can now be performed on the entire tissue section present on a digitized slide.6 Because WSI allows the entire slide to be analyzed, field selection can also be automated. 877
878
Imaging and Quantitative Immunohistochemistry
12000
DAB concentration score
10000 8000 6000 4000 y 1491.7In(x) 8048.3 R2 0.6211
2000 0 0
2000
4000
6000
8000 10000 Nuclear AQUA score
12000
14000
16000
Figure 23-1 Measurement by effective concentration of estrogen receptor by diaminobenzidene (DAB) on the Y-axis versus quantitative immunofluorescence on the X-axis. To compare the measurement methods, estrogen receptor was measured by using the SP1 antibody on a cohort of approximately 240 patients from tissue collected between 2002 and 2007 and was displayed on a tissue microarray. AQUA, automated quantitative analysis.
The digital imaging process involves four steps: 1) image acquisition, or capture; 2) archiving, or the saving, retrieval, and compression of digital files; 3) editing, postcapture manipulation that includes annotation; and 4) sharing the image for viewing, reporting, displaying, or printing. Unfortunately, no standards have been set regarding these various imaging steps in the field of pathology.7 For CAIA to be reliable, the imageacquisition step must be standardized. Static image acquisition may vary, however, because digital cameras are subject to drift over time. Therefore regular calibration of digital cameras will be needed to adjust for several variables, such as light source and so on. This calibration is especially critical for standardization of quantitative analysis and QIF. The use of WSI provides a method to standardize image acquisition for image analysis. However, unlike glass slides, whole-slide scanners are presently unforgiving when there are artifacts such as tissue folds, bubbles under the coverslip, or poor staining of material.8 Pathologists must be aware that such variation may impact the outcome of image analysis. Image compression does not seem to significantly compromise the accuracy of image analysis.9,10 Features (structures) in an image can be classified, or segmented, according to their shape (morphology), spatial arrangement (topology), texture (smoothness, roughness, or coarseness of the image), intensity (brightness), and color. The term multispectral microscopic imaging refers to the capture of spectrally resolved data (i.e., wavelengths) from each pixel in an image by using bright-field and/ or fluorescence modalities. Although it is feasible to capture a multispectral image of a whole slide, this is not done routinely, because only a few dozen fields of information are presently required to create a statistically meaningful sample of a tissue slide. Spectral imaging systems allow pathology slides stained with multiple antibodies to be analyzed, and most spectral
imaging systems are able to resolve at least three or more chromogens. Analysis of multiple chromogenic stains begins by unmixing, or spectrally separating, the individual stains, which should take into account the counterstain.11 This requires specialized imaging hardware and software to automate both the image-acquisition process and the resolution of spectral (color) information across a broad range of visible and infrared bands. Determining the spectral patterns (signatures) and intensity of each individual stain in the image can then help analyze cells and/ or tissue. Although it has been used in experimental settings, the inherent variability of hematoxylin has limited its utility in clinical settings. Furthermore, the physics of light absorption limits the number of multiplex channels for chromogens, even with spectral unmixing. QIF allows more flexibility in multiplexing and can routinely accommodate five or more channels. Although not in common usage, investigators have claimed to be able to multiplex up to 40 channels using QIF by bleaching, restaining, and reimaging. Fluorescence also has a broader dynamic range than chromogenic staining. Although this is widely accepted, limited data are available to compare approaches by using the same patient population. Figure 23-1 shows an example of more than 200 cases of breast cancer read by using the Aperio pixel counter–based CAIA system for bright-field versus Automated Quantitative Analysis (AQUA)–based fluorescence. The limited dynamic range of the diaminobenzidene (DAB)-stained population is evidence of system saturation at the high end of the scale.
Software Algorithms Image analysis tools are used to analyze digital images and slides to provide accurate quantitative data about the amount and intensity of individual stains. Such
Software Algorithms
analyses involve multiple computations based on mathematic and statistical algorithms. Several image-analysis tools are currently available that perform these tasks. This includes open-source applications (ImageJ, ImmunoRatio, ImmunoMembrane) and commercial products, such as AQUA technology (Genoptix), Genetic Imagery Exploitation (Genie) from Aperio, Definiens TissueStudio, INform from Caliper/Perkin Elmer (formerly CRI), Olympus Cell Imaging Software, TissuemorphDP cell-analysis software module from Visiopharm, HistoQuant from 3DHistech, HistoQuest from TissueGnostics, and others. Many researchers have also developed custom software applications and algorithms. Image analysis is a multistep process that involves feature extraction, feature selection, and classification steps (Figs. 23-2 and 23-3). Feature extraction transforms large sets of data, such as topology, into a reduced representation set of features, including graphs that represent structural and spatial information. Feature selection using heuristic algorithms helps determine which features are relevant at a given resolution, but features present within a dataset that may not be generalizable often limit this approach. The end result is the compilation of a set of features that can be used for image classification and/or quantification. Manual, semiautomatic, and automatic selection can be used to determine features or ROIs for measurement. Segmentation algorithms can be based on intensity, texture, and/or colors. Algorithms have been developed to determine positive pixel counts—looking for positive, negative, and neutral areas—to quantify the amount and intensity of a specific stain present in a digital image. Some software packages include region segmentation algorithms (i.e., to identify ROIs) designed to classify
879
regions to be analyzed based on a user training paradigm, in which an experienced end user trains the segmentation algorithm by showing the software a few example regions of different disease classes.12 Thereafter the algorithm is able to classify the remaining image and additional images that might be needed for analysis. These systems have largely been used in research settings, because generalization to the varied conditions of staining in clinical labs around the world has confounded even the best classifiers. Very few systems have been tested by using multicenter prospective experimental approaches. The highest level of evidence published for any classifier is the work by Beck and colleagues13 using a Definiens-based system called C-PATH. Once algorithms have been developed, or manual region selection has been applied to the image, further algorithms can be used for classification or quantification. The algorithm can be applied specifically for evaluating nuclear staining, such as for ER and PR, or membrane staining, such as for Her2.14 Membrane segmentation may be tricky in IHC tissue images, because the cellular membranes are visible only in the stained tracts of the cell, whereas the unstained tracts are not visible.15 Cytoplasm can be detected by using specific cytoplasmic stains, or it can be detected by using computational methods that exploit the fact that cytoplasmic areas are between nuclear and membrane areas. For carcinomas, algorithms must differentiate epithelial parenchyma from desmoplastic stroma, so that only the stain-expression levels in the epithelial regions are quantified. To evaluate IHC in which the immunostains generate pixels with different colors, unmixing (separating colors) is often required. This result can be achieved by color deconvolution, separating the image into different
Step 1: Background-corrected tissue image
Step 2: Morphology-based classification of objects (epithelial vs. stromal cells)
Step 3: Automatic ROI identification
Step 4: (bring in color metric information): cell count, measure protein expression
Figure 23-2 Example of the multistep process involved in image analysis. Computer-aided image-analysis systems rely on algorithms such as background extraction, classification, identification of the region of interest subject to further image analysis, and quantification of results (i.e., diagnostic score). ROI, region of interest. Reproduced with permission from Ventana Medical Systems, Oro Valley, AZ.
880
Imaging and Quantitative Immunohistochemistry
A
B Figure 23-3 Examples of image quantification results for nuclear (A) and membranous (B) immunohistochemical stains. Reproduced with permission from Ventana Medical Systems, Oro Valley, AZ.
channels that correspond to the actual colors of the stains used. This approach permits a pathologist to accurately measure the area or intensity of each stain separately, even when the stains are superimposed at the same location. These algorithms usually include control parameters, such as intensity settings the user can tailor to meet their specific needs. An alternative approach is segmentation or ROI definition by molecular colocalization. This approach is challenging in chromogen-based systems, because even
with unmixing, the number of chromogens that can be resolved is limited. Fluorescence-based systems often capitalize on this approach. For example, the AQUA technology of other QIF-based software can use colocalization with cytokeratin to define an ROI in epithelial neoplasms to avoid issues of inaccuracy associated with automated feature identification. This technology further uses molecular methods to define subcellular compartments, and then it quantifies the amount of protein expressed within the compartment
Strengths and Limitations
A
B
C
D target intensity in compartment pixels compartment pixel area
881
AQUA score
Figure 23-4 Schematic of the automated quantitative analysis (AQUA) method for assessment of quantitative protein expression. AQUA begins with creation of a “tumor mask,” shown magnified in panel B, based on a dilation and filling of a cytokeratin stain shown in panel A. This binary gating allows distinction between epithelial tumors and stroma or empty space. The image is enhanced by dilating, filling holes, and removing small objects. First, 4′,6-diamidino-2-phenylindole (DAPI) is used to tag the nuclei, and then the DAPI image and cytokeratin image are combined in a clustering algorithm to define compartments based on the intensity of each marker in each pixel. These compartments are combined to give a composite or total compartment image, shown in C. An image of the target-specific marker, estrogen receptor (ER), is taken, and the sum of the intensity of the markers in all compartment pixels is divided into the subcellular compartment area on a pixel-by-pixel basis. An AQUA score (intensity/area) is generated for the target within the subcellular compartments as shown by the equation below the figures. The resultant AQUA score is standardized by using cell line controls with known content of the protein of interest.
by colocalization. Colocalization with 4′,6-diamidino2-phenylindole (DAPI) staining can be used to define nuclei or colocalization with CD31 to define endothelial cells. Another example is the construction of a ratio between the nuclear and cytoplasmic levels of a protein that revealed relationships to outcome that met biologic hypotheses but were not revealed by overall measures of expression.16,17 By inclusion of a series of cell line controls, the quantitative result achieved by AQUA is comparable to an enzyme-linked immunosorbent assay (ELISA) within a subcellular compartment.18,19 This approach has been
tested on multiple machines with multiple users on different days and has been shown to have an average coefficient of variation of less than 5%.20 The key principles of this technology and how it works21 when applied to ER are illustrated in Figure 23-4.
Strengths and Limitations Scoring of an IHC stain involves a preanalytic phase that includes tissue preparation—as in fixation, processing, and staining—and an analytic phase and
882
Imaging and Quantitative Immunohistochemistry
postanalytic phase for the quantification and reporting of results. Significant considerations must be addressed related to each of the aforementioned steps. Preanalytic variation is probably the most problematic. Delayed time before fixation of tissue, or cold ischemic time, is a significant problem. A range of work has shown that assessment of critical biomarkers may be altered by prolonged time to fixation. Nkoy and colleagues22 in Elisabeth Hammond’s group showed a higher percentage of ER-negative cases in surgeries done on Friday or Saturday, illustrating how delay to fixation that occurs over the weekend can have dramatic effects on patient care. Subsequently, a number of studies have been done to suggest that ER and other markers show degradation with delayed fixation.23-25 This problem is particularly troublesome in applications where users wish to assess phosphorylation using phosphate-specific antibodies.23,26 In attempts to address this problem, guidelines have set the target time to fixation in formalin to less than 1 hour.27 Preanalytic variables also include tissue processing and immunostaining. This process has been dramatically improved over the years as a result of the introduction of automated staining devices. However, the preparation of tissue, also referred to as antigen retrieval, represents a daunting variable. Although standard methods with standard buffers are often used and desirable, some antigens require protease-mediated antigen retrieval, which is much more challenging to standardize. The failure of epidermal growth factor receptor (EGFR) as a biomarker for EGFR antibody therapeutics was likely due to this issue or to the issue of antibody validation.28 Antibody selection or validation of selected antibodies can also be a preanalytic variable. Studies that use different antibodies for both ER29 and Her230 have shown different results that may affect treatment. Antibody validation is also critical in the research setting, where new antibodies may not recognize the protein stated on the label.31 Uncontrolled variables in the analytic/postanalytic phase are also a key limitation of IHC staining. As interpretation of immunostaining has shifted from qualitative (positive or negative) to semiquantitative (0, 1, 2, 3)32 to automated semiquantitative or truly quantitative methods, new challenges arise. Traditional “by eye” microscopy by pathologists is subject to variable interpretation and also to the imprecision of the human eye. A number of studies have revealed the weaknesses of traditional subjective scoring. For example, one study showed that the discrepancy between ERBB2 IHC and fluorescence in situ hybridization (FISH) was most often due to manual interpretation and not to reagent limitations.33 Manual scoring is susceptible to interobserver and intraobserver variability.34 The use of scales (0, 1+, 2+, 3+ staining) and H scores acknowledges the inherent imprecision and subjectivity involved. Human variability in scoring is particularly notable with borderline and weakly stained cases. In addition, human scoring is associated with added subjectivity and fatigue. In fact, when attempts are made to apply semiquantitative tools, it can result in compression of the scale; this has been seen in measurement of ER in breast cancer, in
which a continuous distribution of scores appears bimodal when scored by eye.35,36 In addition, the evaluation of immunostaining may be influenced by the heterogeneity of epitope biologic expression that exists between tissue sections and blocks of a tumor.37 Early studies showed that CAIA was no better than visual analysis.38 However, with advanced computer technology and image-analysis algorithms, newer published data has subsequently shown that CAIA is comparable, or in small studies, superior to manual methods.39-44 Whereas broad adoption awaits more comprehensive trials and higher levels of evidence, automated image-analysis methods appear to offer more objective, precise, and reproducible quantification on a continuous scale than manual scoring. Limited studies have compared different CAIA technologies to demonstrate the agreement between these systems.45 Problems that may be encountered with CAIA include discrepancies associated with low-level staining, artifacts such as dust particles, interfering nonspecific staining in selected areas, and erroneous low scores generated by small amounts of stained tissue. Furthermore, not all aspects of performing image analysis have been adequately addressed in the literature. These include the number of images (ROIs), appropriate tissue areas (highest labeling areas, or “hot spots”), and level of tissue magnification (×20 or ×40) to be used for analysis. Also, whereas WSI has been used to conduct quantitative image analysis,46 limited data are available to indicate that analyzing an entirely scanned slide, instead of several FOVs from a single slide, overcomes the problem of tumor heterogeneity and sampling issues.47 Whereas some CAIA systems can automate selecting the ROIs to be analyzed, one study has been published suggesting that pathologists are perhaps better at selecting the appropriate areas of a slide to be analyzed.48 Image-analysis tools are not yet in broad clinical use. In clinical practice, they should be used by trained pathologists who have an understanding of the algorithm, the input parameters that may need to be adjusted, and potential pitfalls that may occur when running the algorithm (e.g., counting lymphocytes with nonspecific staining in regions of breast carcinoma designated for analysis). Unfortunately, very few prospective trials of automated analysis methods have been done, and none have been performed in accordance with recent guidelines for evaluation of levels of evidence for biomarkers.49 Given recent Food and Drug Administration (FDA) statements, it is likely that more comprehensive trials will be required before acceptance is widespread and reimbursement for CAIA is enhanced.
Clinical Applications Image analysis has been successfully applied to quantify IHC stains in specific tissues. These include the scoring of breast biomarkers, determination of the proliferation index in certain tumors, and the evaluation of MVD. Image analysis is increasingly being used for many research purposes, including the analysis of tissue microarrays.50,51 The FDA has cleared only a limited number
Clinical Applications
883
TABLE 23-1 List of FDA-Approved Digital Imaging Immunohistochemical Applications Vendor Product PATHIAM (Ventana)
ScanScope XT (Aperio)
Clearance Date
Tissue
Immunostain
Reagent
Application
2010/10
Breast
P53/Ki-67
Dako
Image analysis
2009/02
Breast
ERBB2
Dako
Image analysis
2007/02
Breast
ERBB2
Dako
Image analysis
2009/08
Breast
ERBB2
Dako
Image analysis
2008/10
Breast
PR
Dako
Reading on monitor
2008/08
Breast
ER/PR
Dako
Image analysis
2007/12
Breast
ERBB2
Dako
Reading on monitor
2007/10
Breast
ERBB2
Dako
Image analysis
2996/09
Breast
p53
Ventana
Image analysis
2006/04
Breast
Li-67
Ventana
Image analysis
2005/08
Breast
ERBB2
Ventana
Image analysis
2005/05
Breast
ER/PR
Ventana
Image analysis
2004/03
Breast
ER/PR
Dako
Image analysis
2004/01
Breast
ERBB2
Dako
Image analysis
ACIS (Clarient/Chroma Vision)
2004/02
Breast
ER/PR
Dako
Image analysis
2003/12
Breast
ERBB2
Dako
Image analysis
QCA
2003/12
Breast
ER
Dako
Image analysis
VIAS (TriPath Imaging and Ventana)
ARIOL (Applied Imaging)
Modified from Lange H: Digital pathology: a regulatory overview. Lab Medicine 2011;42:588-591. ER, Estrogen receptor; FDA, Food and Drug Administration; PR, progesterone receptor; QCA, quantum-dot cellular automata.
of digital imaging systems (Table 23-1) for the evaluation of IHC staining reactions.52
Breast Biomarkers Determination of ER and PR expression status in breast carcinoma is of prognostic importance and is a strong predictor of patient response to hormonal therapy. IHC performed on formalin-fixed tissue sections is the most commonly used assay, having replaced biochemicalbased methods. Currently, a pathologist assesses hormone receptor status with a cutoff threshold of positive tumor cells commonly used to predict responsiveness to adjuvant hormonal therapy. In 2010, a panel of members from the American Society of Clinical Oncologists (ASCO) and the College of American Pathologists (CAP) met to examine this assay, and they changed the standard to 1% of cells with “any immunoreactivity.”27 This threshold has been automated and is part of some of the FDA-approved systems. It is similarly important to identify those breast carcinomas with human epidermal receptor protein 2 (ERBB2) alterations, because such tumors may respond to targeted therapy with trastuzumab. ERBB2 alterations at the DNA (amplification) and protein (overexpression) level usually occur in concert. FISH or IHC can be used to assess for these alterations. Automated image analysis for all of these breast biomarkers have been shown to have the potential for increased accuracy
of their quantification, standardization, and throughput and even for the identification of potentially new prognostic subgroups, which may not have been evident following initial manual analysis alone.53,54 These tools help diminish the “equivocal” category in ERBB2 assessment. Guidelines published by the ASCO/CAP panel for ERBB2 testing in breast cancer55 indicate that image analysis can serve as an effective tool for achieving consistent interpretation. The guidelines also indicate that a pathologist is required to confirm image-analysis results and that image-analysis equipment, including optical microscopes, must be calibrated and subjected to regular maintenance. Similar recommendations for image-analysis systems to enhance reproducibility of scoring were published following the Canadian national consensus meeting on ERBB2 testing in breast cancer.56 ERBB2 expression has been shown to be helpful in managing gastroesophageal adenocarcinoma. However, the ERBB2 IHC scoring system for gastroesophageal adenocarcinomas differs from that of breast carcinoma. Automated image analysis, validated for scoring of ERBB2 immunostaining in breast cancer, cannot be reliably used in the interpretation of ERBB2 expression in gastroesophageal adenocarcinomas.57 Whereas CAIA has been cleared by the FDA for both ER and ERBB2 testing, the level of acceptance is still low. Large cooperative group trials are still required to compare CAIA with conventional “by eye” analysis.
884
Imaging and Quantitative Immunohistochemistry
Proliferation Index Cell proliferation is a major determinant of the biologic behavior of several neoplasms, including brain tumors and breast carcinoma. The proliferation index is also important in grading certain non-Hodgkin lymphomas and neuroendocrine tumors. The MIB-1 antibody permits the IHC detection of the nuclear Ki-67 antigen in tissue sections, therefore this Ki-67 labeling index serves as a good proliferation marker.58 However, the utility of the proliferative index is hampered by interobserver variability. Studies have shown that the application of quantitative image analysis algorithms gives a more accurate estimate of the proliferation rate.59,60 One study involving pancreatic neuroendocrine tumors showed that although it was practical to perform only a single-field hot-spot analysis to determine the proliferative activity, the results varied when using 10 consecutive fields for image analysis.61 The measurement of Ki-67 is a good target for automation efforts and has been assessed by a range of automated methods. However, to date, no widely accepted standard has been applied for automated measurement of Ki-67. Further work to standardize CAIA, including prospectively designed trials, may be required.
Microvessel Density Assessment Studies have shown the value of using tumor microvessel density (MVD) as a prognostic indicator for a wide range of cancers. Measurement of MVD can facilitate assessment of the degree of angiogenic activity in a tumor and the prognosis, such as the likelihood of tumor recurrence, and it may even help guide treatment decisions.62 These measurements have been proven to be useful prognostic markers for several solid tumors and some hematologic malignancies. However, MVD by itself does not appear to be a good indicator of therapeutic efficacy.63 MVD is a measure of the number of vessels (or intercapillary distance) per high-power (microscope) field. In other words, MVD calculates the sum of all microvessel areas (in µm2) divided by the
total area of the stained tissue being analyzed (in µm2). Hence, quantitating microvessels by this method represents a relative area estimate of the vessels rather than a true vessel count. Image-analysis tools have been developed to help better measure MVD by evaluating immunohistochemically stained vessels, or vascular hot spots, by using CD34, CD31, CD105, or VEGF antibodies.64-67 Although endothelial stains are normally used to identify vessels, other secondary stains for muscle cells that are associated with these vessels can be incorporated into these algorithms. Typically, any stained endothelial cell or clusters separate from adjacent vessels are calculated as a single microvessel, even in the absence of a vessel lumen. The challenge in using MVD is the variability that exists in human samples. However, because tumor vasculature can range from well-defined normal vessels to complex neovascular structures that may be interconnected, obtaining reproducible results is often difficult. Finally, pathologists who perform these image analyses should be aware that MVD results might be influenced if they analyze stained microvessels in the periphery and not in the center of a tumor.
Summary Computer-assisted image analysis for quantitation of IHC stains in surgical pathology is currently in its infancy in the clinical arena. There is little question that reproducibility and quantitation of antigen targets will be improved substantially with widespread adoption of this modality in clinical practice. However, the current lack of standardization and dearth of comprehensive FDA-cleared imaging products for these theranostic applications are impediments to immediate widespread implementation. REFERENCES
The full reference list for this chapter is available at expertconsult.com.
References 1. Marchevsky A: Image analysis: A primer for pathologists: Raven; 1994. 2. Cregger M, Berger AJ, Rimm DL: Immunohistochemistry and quantitative analysis of protein expression. Arch Pathol Lab Med. 130:1026–1030, 2006. 3. Feldman MD: Beyond morphology: whole slide imaging, computer-aided detection, and other techniques. Arch Pathol Lab Med. 132:758–763, 2008. 4. Gurcan MN, Boucheron LE, Can A, Madabhushi A, et al: Histopathological image analysis: a review. IEEE Rev Biomed Eng. 2:147–171, 2009. 5. Parwani AV, Feldman M, Balis U, Pantanowitz L: Pathology Informatics: Theory & Practice, Chicago, 2012, ASCP Press. 6. Chantrain CF, DeClerck YA, Groshen S, McNamara G: Computerized quantification of tissue vascularization using highresolution slide scanning of whole tumor sections. J Histochem Cytochem. 151–158, 2003. 7. Yagi Y, Gilbertson JR: Digital imaging in pathology: the case for standardization. J Telemed Telecare. 11:109–116, 2005. 8. Bautista PA, Yagi Y: Improving the visualization and detection of tissue folds in whole slide images through color enhancement. J Pathol Inform. 1, 2010. 9. Nicolosi JS, Yoshida AO, Sarian LO, et al: Image compression impact on quantitative angiogenesis analysis of ovarian epithelial neoplasms. Appl Immunohistochem Mol Morphol. 20:91–95, 2012. 10. Lopez C, Lejeune M, Escriva P, et al: Effects of image compression on automatic count of immunohistochemically stained nuclei in digital images. J Am Med Inform Assoc. 15:794–798, 2008. 11. Zehntner SP, Chakravarty MM, Bolovan RJ, et al: Synergistic tissue counterstaining and image segmentation techniques for accurate, quantitative immunohistochemistry. J Histochem Cytochem. 56:873–880, 2008. 12. Keller B, Chen W, Gavrielides MA: Quantitative assessment and classification of tissue-based biomarker expression with color content analysis. Arch Pathol Lab Med. 136:539–550, 2012. 13. Beck AH, Sangoi AR, Leung S, et al: Systematic analysis of breast cancer morphology uncovers stromal features associated with survival. Sci Transl Med. 3:108ra13, 2011. 14. Brugmann A, Eld M, Lelkaitis G, et al: Digital image analysis of membrane connectivity is a robust measure of HER2 immunostains. Breast Cancer Res Treat. 132:41–49, 2012. 15. Ficarra E, Di Cataldo S, Acquaviva A, Macii E: Automated segmentation of cells with IHC membrane staining. IEEE Trans Biomed Eng. 58:1421–1429, 2011. 16. Berger AJ, Davis DW, Tellez C, et al: Automated quantitative analysis of activator protein-2alpha subcellular expression in melanoma tissue microarrays correlates with survival prediction. Cancer Res. 65:11185–11192, 2005. 17. Camp RL, Chung GG, Rimm DL: Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med. 8:1323–1327, 2002. 18. McCabe A, Dolled-Filhart M, Camp RL, et al: Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis. J Natl Cancer Inst. 97:1808–1815, 2005. 19. Welsh AW, Moeder CB, Kumar S, et al: Standardization of estrogen receptor measurement in breast cancer suggests false-negative results are a function of threshold intensity rather than percentage of positive cells. J Clin Oncol. 29:2978–2984, 2011. 20. Gustavson MD, Bourke-Martin B, Reilly D, et al: Standardization of HER2 immunohistochemistry in breast cancer by automated quantitative analysis. Arch Pathol Lab Med. 133:1413–1419, 2009. 21. Moeder CB, Giltnane JM, Moulis SP, et al: Quantitative, fluorescence-based in-situ assessment of protein expression. Methods Mol Biol. 520:163–175, 2009. 22. Nkoy FL, Hammond ME, Rees W, et al: Variable specimen handling affects hormone receptor test results in women with breast cancer: a large multihospital retrospective study. Arch Pathol Lab Med. 134:606–612, 2010. 23. Bai Y, Tolles J, Cheng H, et al: Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables. Lab Invest. 91:1253–1261, 2011. 24. Khoury T, Sait S, Hwang H, et al: Delay to formalin fixation effect on breast biomarkers. Mod Pathol. 22:1457–1467, 2009.
884.e1
25. Yildiz-Aktas IZ, Dabbs DJ, Bhargava R: The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol. 2012. 26. Pinhel IF, Macneill FA, Hills MJ, et al: Extreme loss of immunoreactive p-Akt and p-Erk1/2 during routine fixation of primary breast cancer. Breast Cancer Res. 12:R76, 2010. 27. Hammond ME, Hayes DF, Dowsett M, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Arch Pathol Lab Med. 134:907–922, 2010. 28. Anagnostou VK, Welsh AW, Giltnane JM, et al: Analytic variability in immunohistochemistry biomarker studies. Cancer Epidemiol Biomarkers Prev. 19:982–991, 2010. 29. Cheang MC, Treaba DO, Speers CH, et al: Immunohistochemical detection using the new rabbit monoclonal antibody SP1 of estrogen receptor in breast cancer is superior to mouse monoclonal antibody 1D5 in predicting survival. J Clin Oncol. 24:5637–5644, 2006. 30. Manion E, Hornick JL, Lester SC, et al: A comparison of equivocal immunohistochemical results with anti-HER2/neu antibodies A0485 and SP3 with corresponding FISH results in routine clinical practice. Am J Clin Pathol. 135:845–851, 2011. 31. Bordeaux J, Welsh A, Agarwal S, et al: Antibody validation. Biotechniques. 48:197–209, 2010. 32. Friedberg RC, Pantanowitz L: Practice evolution: decentralized computer-assisted immunohistochemical image analysis. Arch Pathol Lab Med. 133:597–600, 2009. 33. Bloom K, Harrington D: Enhanced accuracy and reliability of HER-2/neu immunohistochemical scoring using digital microscopy. Am J Clin Pathol. 121:620–630, 2004. 34. Jaraj SJ, Camparo P, Boyle H, et al: Intra- and interobserver reproducibility of interpretation of immunohistochemical stains of prostate cancer. Virchows Arch. 455:375–381, 2009. 35. Schnitt SJ: Estrogen receptor testing of breast cancer in current clinical practice: what’s the question? J Clin Oncol. 24:1797– 1799, 2006. 36. Rimm DL, Giltnane JM, Moeder C, et al: Bimodal population or pathologist artifact? J Clin Oncol. 25:2487–2488, 2007. 37. Chung GG, Zerkowski MP, Ghosh S, et al: Quantitative analysis of estrogen receptor heterogeneity in breast cancer. Lab Invest. 87:662–669, 2007. 38. Schultz DS, Katz RL, Patel S, Johnston D, et al: Comparison of visual and CAS-200 quantitation of immunocytochemical staining in breast carcinoma samples. Anal Quant Cytol Histol. 14:35– 40, 1992. 39. McClelland RA, Finlay P, Walker KJ, et al: Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer. Cancer Res. 50:3545–3550, 1990. 40. Wang S, Saboorian MH, Frenkel EP, et al: Assessment of HER-2/ neu status in breast cancer. Automated Cellular Imaging System (ACIS)-assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard. Am J Clin Pathol. 116:495– 503, 2001. 41. Markiewicz T, Wisniewski P, Osowski S, et al: Comparative analysis of methods for accurate recognition of cells through nuclei staining of Ki-67 in neuroblastoma and estrogen/progesterone status staining in breast cancer. Anal Quant Cytol Histol. 31:49– 62, 2009. 42. Gavrielides MA, Gallas BD, Lenz P, et al: Observer variability in the interpretation of HER2/neu immunohistochemical expression with unaided and computer-aided digital microscopy. Arch Pathol Lab Med. 135:233–242, 2011. 43. Nassar A, Cohen C, Agersborg SS, et al: Trainable immunohistochemical HER2/neu image analysis: a multisite performance study using 260 breast tissue specimens. Arch Pathol Lab Med. 135:896–902, 2011. 44. Fiore C, Bailey D, Conlon N, et al: Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry. J Clin Pathol. 65:496–502, 2012. 45. Gokhale S, Rosen D, Sneige N, et al: Assessment of two automated imaging systems in evaluating estrogen receptor status in breast carcinoma. Appl Immunohistochem Mol Morphol. 15:451– 455, 2007.
884.e2
Imaging and Quantitative Immunohistochemistry
46. Lloyd MC, Allam-Nandyala P, Purohit CN, et al: Using image analysis as a tool for assessment of prognostic and predictive biomarkers for breast cancer: How reliable is it? J Pathol Inform. 1:29, 2010. 47. Tolles J, Bai Y, Baquero M, et al: Optimal Tumor Sampling for Immunostaining of Biomarkers in Breast Carcinoma. Breast Cancer Res. 13:R51, 2011. 48. Chabot-Richards DS, Martin DR, Myers OB, et al: Quantitative image analysis in the assessment of diffuse large B-cell lymphoma. Mod Pathol. 24:1598–1605, 2011. 49. Simon RM, Paik S, Hayes DF: Use of archived specimens in evaluation of prognostic and predictive biomarkers. J Natl Cancer Inst. 101:1446–1452, 2009. 50. Decaestecker C, Lopez XM, D’Haene N, et al: Requirements for the valid quantification of immunostains on tissue microarray materials using image analysis. Proteomics. 9:4478–4494, 2009. 51. Dolled-Filhart M, Gustavson M, Camp RL, et al: Automated analysis of tissue microarrays. Methods Mol Biol. 664:151–162, 2010. 52. Lange H: Digital pathology: A regulatory overview. Lab Medicine. 42:588–591, 2011. 53. Rexhepaj E, Brennan DJ, Holloway P, et al: Novel image analysis approach for quantifying expression of nuclear proteins assessed by immunohistochemistry: application to measurement of oestrogen and progesterone receptor levels in breast cancer. Breast Cancer Res. 10:R89, 2008. 54. Laurinaviciene A, Dasevicius D, Ostapenko V, et al: Membrane connectivity estimated by digital image analysis of HER2 immunohistochemistry is concordant with visual scoring and fluorescence in situ hybridization results: algorithm evaluation on breast cancer tissue microarrays. Diagn Pathol. 6:87, 2011. 55. Wolff AC, Hammond ME, Schwartz JN, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 25:118–145, 2007. 56. Hanna W, O’Malley FP, Barnes P, et al: Updated recommendations from the Canadian National Consensus Meeting on HER2/ neu testing in breast cancer. Curr Oncol. 14:149–153, 2007.
57. Jeung J, Patel R, Vila L, et al: Quantitation of HER2/neu expression in primary gastroesophageal adenocarcinomas using conventional light microscopy and quantitative image analysis. Arch Pathol Lab Med. 136:610–617, 2012. 58. Spyratos F, Ferrero-Pous M, Trassard M, et al: Correlation between MIB-1 and other proliferation markers: clinical implications of the MIB-1 cutoff value. Cancer. 94:2151–2159, 2002. 59. Remes SM, Tuominen VJ, Helin H, et al: Grading of Neuroendocrine Tumors With Ki-67 Requires High-quality Assessment Practices. Am J Surg Pathol. 36:1359–1363, 2012. 60. Grala B, Markiewicz T, Kozlowski W, et al: New automated image analysis method for the assessment of Ki-67 labeling index in meningiomas. Folia Histochem Cytobiol. 47:587–592, 2009. 61. Goodell PP, Krasinskas AM, Davison JM, et al: Comparison of methods for proliferative index analysis for grading pancreatic well-differentiated neuroendocrine tumors. Am J Clin Pathol. 137:576–582, 2012. 62. Moreira LR, Schenka AA, Latuf-Filho P, et al: Immunohistochemical analysis of vascular density and area in colorectal carcinoma using different markers and comparison with clinicopathologic prognostic factors. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine. 32:527–534, 2011. 63. Hlatky L, Hahnfeldt P, Folkman J: Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn’t tell us. J Natl Cancer Inst. 94:883–893, 2002. 64. Lee JS, Jung JJ, Kim J: Quantification of angiogenesis by a computerized image analysis system in renal cell carcinoma. Anal Quant Cytol Histol. 22:469–474, 2000. 65. Krecicki T, Dus D, Kozlak J, et al: Quantitative evaluation of angiogenesis in laryngeal cancer by digital image measurement of the vessel density. Auris Nasus Larynx. 29:271–276, 2002. 66. Sullivan CA, Ghosh S, Ocal IT, et al: Microvessel area using automated image analysis is reproducible and is associated with prognosis in breast cancer. Hum Pathol. 40:156–165, 2009. 67. Fox SB, Harris AL: Histological quantitation of tumour angiogenesis. APMIS. 112:413–430, 2004.
INDEX
Page numbers followed by “f” indicate figures, “t” indicate tables, and “b” indicate boxes.
A
A6 (CD45RO), 765t, 769f A103. See Melan-A AAH, 586, 586t ABC. See Avidin-biotin conjugate Abdomen: metastatic carcinoma in, 239b ABI 3730 (Applied Biosystems) (Life Technologies), 46-47 Abscesses, 769-770, 769f, 819t, 827-828 Acanthamoeba, 66 Accuracy, 17-18 Achaete-scute complex-like 1 (ASCL1), 425 Acinar cell carcinoma, 552-553, 553b, 553f Acinar markers, 541 Acinar prostate carcinoma, 598f ACIS (Clarient/Chroma Vision), 883t Acquired immunodeficiency syndrome (AIDS), 772-773 Acral lentiginous melanoma, 189, 190f Actin(s) in breast tumors, 742, 745f in head and neck lesions, 246t-248t in hemangiopericytoma of dermis and subcutis, 496f in mesothelioma, 444t-446t in pleural neoplasms, 465t in sarcomatoid mesothelioma, 462f in tumors of soft tissue and bone, 77 Actinic keratosis, 199, 199f Activator protein 2 gamma, 644-645 Acute immune hyperplasia, 154-157 Acute lymphoblastic leukemia, B-cell, 174, 175f Acute lymphocytic leukemia antigen, 632-633 Acute respiratory distress syndrome, 60f Ad Hoc Committee on Immunohistochemistry Standardization, 15b recommendations for standardization of IHC, 15 Ad4BP. See Adrenal 4 binding protein Adamantinoma, 128-129, 129f Adenine (A), 39
Adenocarcinoma abdominal wall, 740f algorithmic approaches to, 237, 238f-239f ampullary, 563-565 intestinal-type, 563-564, 565f key diagnostic points, 565b anal gland, 526 CDX-2 in, 229-230, 229t CEA staining, 35f, 220-221, 221b ceruminous, 311, 312f colorectal, 522-525 anatomic molecular diagnostic applications to, 539 metastasis to ovary, 704f theranostic applications, 538 variants and subtypes, 525 cytokeratin 7 in, 390t cytokeratin 20 in, 391t endocervical, 662f intestinal-type, 661 invasive, 661, 661b minimal deviation, 662 endometrial, 428t endometrioid, 667-668 key differential diagnosis, 671t markers of emerging importance, 668f esophageal, 510-511, 511b gastric, 515-516 antibodies in, 516t Her2 immunohistochemistry, 538 key diagnostic points, 515b theranostic applications, 537-538 vs. lung adenocarcinoma, 519 vs. pancreatobiliary adenocarcinoma, 519 gastroesophageal, 538 gastrointestinal variants and subtypes, 525 intestinal-type, 272, 274f, 561 sinonasal, 272 types of, 272, 273f intestinal-type ampullary, 563-564, 565f invasive endocervical, 661, 661b invasive extrahepatic, 560-561 markers of, 840-843
Adenocarcinoma (Continued) metastatic appendiceal, 704-705, 704f metastatic colonic, 422t metastatic ovarian, 688t, 704f metastatic pleural, 450t, 842f metastatic prostatic, 360, 360f mucinous, 371 nonintestinal-type, 272 ovarian or fallopian tube, 688t pancreatic ductal, 543-545, 543f-544f differential diagnosis of, 546b key diagnostic points, 545b pancreatobiliary, 519, 561, 563-564 pancreatobiliary-type, 541 papillary, 282b, 283f polymorphous low-grade, 291t, 292, 296-298, 297f, 298b poorly differentiated, 852, 853f prostatic, 596f benign mimics, 590-594 diagnosis of, 587-589, 590f key diagnostic points, 82b metastatic, 360, 360f with neuroendocrine differentiation, 360, 360f, 597 posthormonal therapy histology, 595b posttherapy changes in, 594-596, 594f-595f pseudomesotheliomatous, 465-466, 465f pulmonary, 387, 387t, 844f, 847f, 853f cytokeratin profile, 454t immunohistochemical features of, 419t, 422t, 452t-453t, 469t, 471t immunohistochemical tests for, 459t, 470t mucinous, 421-422, 422t pseudomesotheliomatous, 465-466, 465f vs. breast carcinoma, 422 vs. colon cancer, 422 vs. metastatic carcinoma, 422-423
885
886
Index
Adenocarcinoma (Continued) rectal, 619, 620f salivary gland-type, 272 sinonasal, 272 sinonasal tract, 272 of small intestine, 519-520, 520f TTF-1 expression in, 393t tubulodesmoplastic pseudomesotheliomatous, 465-466, 465f of unknown primary, 852, 853f urinary bladder, 619, 620f, 622-624, 622f uterine, 671t villin in, 229-230, 229t Adenocarcinoma in situ, endocervical, 660, 660b, 661f Adenohypophysis, 326-328 Adenoid basal carcinoma, 663, 664f Adenoid clear cell carcinoma, 480-481 Adenoid cystic carcinoma cervical, 662-663 esophageal, 512-513, 513f head and neck, 249t, 291t, 292, 298-300, 298f-299f key diagnostic points, 300b thymic, 371 Adenoma alveolar, 393t basal cell, 291t bile duct, 580 canalicular, 291t β-catenin-activated, 573-574 ceruminous, 311, 311b, 311f follicular, 329 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t marker expression in, 334t hepatic, 559, 573-574 CD34 in, 221, 222f metanephric, 636, 636f middle ear, 315-318, 316f, 318b nephrogenic, 623, 624b pituitary, 265f key diagnostic points, 266b staining pattern, 256t pleomorphic, 290-292, 292f features of, 291t key diagnostic points, 292b Adenomatoid spindle cell thymoma, 368 Adenomatoid tumors, 473-474, 708 Adenosarcoma, müllerian, 675-676 Adenosis, 591, 593f key diagnostic points, 592b sclerosing, 591-592, 593f Adenosquamous carcinoma extrahepatic, 562 in pancreas, 547 Adenoviruses, 58t, 59-60, 60f Adenylyl cyclase, soluble, 196 Adhesion molecules, 542-543 Adipocytic tumors of mediastinum, 377 of soft tissue and bone, 115-117 Adnexal carcinoma, microcystic, 485, 487f
Adolescence: neoplasms of. See Pediatric neoplasms Adrenal 4 binding protein (Ad4BP) in adrenocortical tumors, 346, 346f in cancer of unknown primary site, 236 Adrenal cortical neoplasms, 428t Adrenal gland tumors, 345-351 Adrenal medullary tumors, 348-349, 351b Adrenal pheochromocytoma, 349, 349f Adrenocortical carcinoma, 346f Adrenocortical tumors, 345-348 clear cell carcinomas, 236b inhibin in, 235-236 key diagnostic points, 351b markers in, 346f melan-A in, 235-236 molecular diagnostic applications, 348 theranostic applications, 348 Adrenocorticotropic hormone big (ACTH[b]) in large cell neuroendocrine carcinoma, 357f in lung carcinoids, 356f in small cell carcinoma, 356f in endocrine tumors, 352t, 357f in head and neck lesions, 246t-248t in lung carcinoids, 356f in pituitary adenoma, 265-266, 266f in small cell carcinoma, 356f Adult T-cell leukemia/lymphoma, 178f, 184 AEC. See Amino-ethyl carbazole AFH. See Angiomatoid fibrous histiocytoma African tick bite fever, 65 Agendia, 747-748 Agendia BV, 755-756, 758t AGGRUS, 83 Agilent/Dako, 8, 10, 30 AIDS. See Acquired immunodeficiency syndrome AIP. See Autoimmune pancreatitis Air-dried smears, 832, 834 Albumin, 575, 577f ALK. See Anaplastic lymphoma kinase Alkaline phosphatase, 8-12 placental. See Placental alkaline phosphatase Alkaline phosphatase/anti-alkaline phosphatase (APAAP) method, 8-9, 9f, 24t Allele-specific hybridization (ASH), 45-46 Allele-specific PCR (AS-PCR), 45-46 Allred score, 747, 747f Alpha B-crystallin, 782 Alpha cells, 353-354 α-Fetoprotein (AFP) in cancer of unknown primary site, 236 in gynecologic pathology, 654t in hepatocellular carcinoma, 575-576, 577f
α-Fetoprotein (AFP) (Continued) in mediastinal tumors, 382t in ovarian and tubal tumors, 686 in testicular tumors, 645, 648t in yolk sac tumors, 701-702 Alveolar adenoma, 393t Alveolar rhabdomyosarcoma, 858, 859f-861f gene-expression analysis of, 861862 immunomarkers for, 91t, 859-861 in sinonasal tract, 266-267, 267f in soft tissue and bone, 74, 100, 100f Alveolar soft-part sarcoma differentiation of, 211t-212t in oral cavity, 285, 285f in soft tissue and bone, 121-122, 122f Alveolar soft-part sarcoma locus/ alveolar soft-part sarcoma chromosomal locus (ASPL/ ASPSCR1), 121-122 Alzheimer disease, 767t, 819-820, 820f AMACR. See α-Methylacyl-coenzyme A racemase American Society of Clinical Oncologists (ASCO), 15 Amino-ethyl carbazole, 24, 24t Ammon’s horn, 823f Amplification basic signal approaches, 37t chromosomal, 41 detection, 36-37, 37t methods of, 36-37 polymeric, 37t postdetection, 37, 37t predetection, 36, 37t stepwise, 37t tyramine-based, 37t AmpliSeq 1.0, 48f Ampulla, 563-568 autoimmune diseases of, 566-568 glandular psammomatous carcinoid of, 565-566, 566f premalignant lesions in, 563 Ampullary adenocarcinoma, 563-565 anatomic molecular diagnostic applications, 565 genomic applications, 565 intestinal-type, 563-564, 565f key diagnostic points, 565b Ampullary neuroendocrine neoplasms, 565-566, 566b Ampullary somatostatinoma, 565-566, 566f α-Amylase, 246t-248t Amyloid adenocarcinoma, 573t Anal gland adenocarcinoma, 526 Anal malignancy, 526-527, 528b Anaplasia, 859 Anaplastic astrocytoma, 776t-777t, 783 Anaplastic ependymoma, 776t-777t, 789 Anaplastic ganglioglioma, 796
Index
Anaplastic large cell lymphoma, 178-180, 179f-180f differential diagnosis of, 141t frequency of, 178f Hodgkin-like, 139-140, 141f markers, 188t mediastinal, 384 null cell type, 179 of skin, 488, 490f Anaplastic lymphoma, 427t Anaplastic lymphoma kinase -positive large B-cell lymphoma, 173 in anaplastic large cell lymphoma, 139-140, 179-180, 180f in bladder tumors, 625t in Hodgkin lymphoma, 138f, 138t, 141t in inflammatory myofibroblastic tumor, 624, 624f-625f in mediastinal hematopoietic tumors, 382-384 in non-Hodgkin lymphoma, 151, 173 in prostatic mesenchymal tumors, 603t in tumors of soft tissue and bone, 86 in urothelial carcinoma, 617 Anaplastic meningioma, 770t, 805t, 809 Anaplastic oligoastrocytoma, 779t, 791 Anaplastic oligodendroglioma, 766t, 790f, 791, 792f Anaplastic plasmacytoma, 188t Anaplastic thyroid carcinoma, 240-241 Ancient (sclerosing) thymoma, 368 Androgen receptor protein in head and neck lesions, 246t-248t in prostate, 587t in prostate carcinoma, 606f, 607t in skin carcinomas, 489f Angioendotheliosis proliferans, 412 Angiofibroma, nasopharyngeal, 280-282, 281f, 282b Angiogenesis, 607-608 Angioimmunoblastic lymphoma, 178f Angioimmunoblastic T-cell lymphoma, 142, 180, 181f Angioma, cavernous, 775t Angiomatoid fibrous histiocytoma, 106 Angiomatous meningioma, 807 Angiomyoadenomatous tumors, 640-641 Angiomyofibroblastoma, 102-103, 103b Angiomyolipoma, 583 immunohistology of, 641b renal, 639-640, 640f, 641b in soft tissue and bone, 109 Angiomyxoma, aggressive, 123-124 Angiosarcoma cutaneous, 504f key diagnostic points, 82b, 97b, 120b
Angiosarcoma (Continued) of lung, 406-407, 407f of mediastinum, 379, 379f, 380t postradiation, 209f of soft tissue and bone, 74, 95-97 spindle cell, 92t, 97f Annular tubules, 699 Anthrax, 69-70, 69f Anti-ALK protein, 90t Anti-h-Caldesmon, 90t Anti-β-catenin, 90t. See also β-Catenin Anti-CD31, 90t Anti-CD34, 90t Anti-CD68, 90t Anti-CD99, 90t Anti-CD163, 90t Anti-CDK4 protein, 90t Anti-claudin-1 protein, 90t Anti-EP, 37t Anti-epithelial membrane antigen, 90t. See also Epithelial membrane antigen Anti-ERG, 90t Anti-GFAP protein, 90t Anti-HMB-45, 90t Anti-INI1 protein, 90t Anti-MART-1, 90t Anti-MDM2 protein, 90t Anti-MUC4, 90t. See also Mucin 4 (MUC4) Anti-muscle-specific actin, 77, 90t Anti-MyoD1, 90t. See also MyoD1 Anti-myogenin/myf4, 90t Anti-Rb protein, 90t. See also Retinoblastoma Anti-S-100 protein, 90t Anti-(alpha) smooth muscle actin, 77 Anti-synaptophysin, 90t Anti-TFE3 protein, 90t Anti-TLE1 protein, 90t Antiadenocarcinoma antibodies, 684-686 Antiandrogen therapy, 594-595, 594f Antibody(ies). See also specific antibodies; specific sites of interest as antigens, 3, 3f biology of in endocrine tumors, 322-326 in Hodgkin lymphoma, 130-137 in lung neoplasms, 387-400 in mediastinum, 363 in melanocytic neoplasms, 189-196 in pediatric neoplasms, 854-855 in pleural neoplasms, 447-460 in prostate, 584-587 in soft tissue and bone neoplasms, 73-89 in testicular tumors, 644-645 in urothelial carcinoma, 616-618 in cholangiocarcinoma, 580f definition of, 3 detection systems, 5-14 direct-conjugate–labeled, 6, 6f, 12t fixation of, 832 in gynecologic pathology, 654t in hepatocellular carcinoma, 576, 577f
887
Antibody(ies) (Continued) incubation of, 23 keratin, 213t lung carcinoma panel, 240t mesothelioma panel, 240t monoclonal for Hodgkin lymphoma therapy, 145 for infectious and prion disease diagnosis, 58t melanocyte-specific, 193-196 PMEL-17–related, 193-195 for myoepithelial cells, 710-713, 711f, 711t, 714b neuroendocrine, 224-225 optimal dilution for, 12 in ovarian and tubal tumors, 684 paratope of, 3 polyclonal, 3-4 with postfixation, 833t in prostate, 584 in renal tumors, 632-635 as specific staining reagents, 3-4 surfactant, 429-430 titration of, 12-13 troubleshooting, 31t-32t Antibrachyury, 90t. See also Brachyury Antidesmin, 90t. See also Desmin Anti–Epstein-Barr virus therapy, 145 Antigen(s), 3, 3f antibodies as, 3, 3f B-cell–associated, 148-150 biology of in endocrine tumors, 322-326 gastrointestinal, 508-510 in Hodgkin lymphoma, 130-137 in lung neoplasms, 387-400 in mediastinum, 363 in melanocytic neoplasms, 189-196 in pancreas, 540-543 in pediatric neoplasms, 854-855 in pleural neoplasms, 447-460 in prostate, 584-587 in soft tissue and bone neoplasms, 73-89 in testicular tumors, 644-645 in urothelial carcinoma, 616-618 charge-shape profiles of, 3 enzyme-labeled, 11 in hematologic disorders, 148-153 keratin, 213t labeled antigen method, 12f mitochondrial, 336 prostate carcinoma, 232-233, 233b reference standards and standard curves for, 29-30 soft tissue, 85b in tissue sections, 29-30 Antigen determinants (epitopes), 3, 3f Antigen-presenting cell markers, 135 Antigen retrieval (AR), 2, 37t, 835, 882 automated systems, 29 heating conditions for, 20-21 for IHC, 20, 21f
888
Index
Antigen retrieval (AR) (Continued) by microwave heating method, 23-25 on-platform methods, 22 techniques for, 20-21, 21f test-battery approach for, 22, 22t Antigen retrieval (AR) solution, 21-22 Antikeratins, 90t Antiprion protein (PrP), 774 α1-Antitrypsin, 554, 554f, 556f, 572, 572f, 576, 577f Antoni A tissue, 789f Antrochoanal polyps, 282, 282f APAAP method. See Alkaline phosphatase/anti-alkaline phosphatase method APC, 607t Aperio, 878-879, 883t Apocrine cutaneous neoplasms, 506, 507f Apomucins, 515 Aponeuroses, 120-121 Appendiceal goblet cell carcinoid, 530, 531f Appendiceal neoplasms epithelial lesions, 520-521 metastatic adenocarcinoma, 704705, 704f mucinous, 521b, 521f neuroendocrine tumors, 530, 530b Applied Biosystems ABI 3730 (Life Technologies), 46-47 Applied Imaging, 883t AQUA (Genoptix), 878-879 AR. See Antigen retrieval Archiving, 878 Argentine hemorrhagic fever, 61 Arginase-1 in cancer of unknown primary site, 232 in hepatocellular carcinoma, 575 key diagnostic points, 232 ARIOL (Applied Imaging), 883t ARMS. See Alveolar rhabdomyosarcoma Arteriovenous malformations, 775, 775t Artifacts, 36, 36t, 836-837, 837f ARUP Laboratories, 758t ASCL1. See Achaete-scute complexlike 1 ASCO. See American Society of Clinical Oncologists ASH. See Allele-specific hybridization Aspergillus, 66, 70-72 Aspergillus fumigatus, 771 Aspirate smears, 830f ASPS. See Alveolar soft-part sarcoma Astroblastoma, 776t-777t, 784 Astrocytomas, 780-785, 795f anaplastic, 776t-777t, 783, 783f anatomic molecular diagnostic applications, 785 cystic, 819t differential features of, 776t-777t diffuse, 782, 782f fibrillary, 780f
Astrocytomas (Continued) gemistocytic, 782-783 differential features of, 776t-777t swollen GFAP-positive cells in, 826t giant cell, 784, 784f differential features of, 776t-777t subependymal, 784 grade II, 777f, 780f, 782-783, 782f-783f grade III, 783 key diagnostic points, 784b mixed oligodendrogliomaastrocytoma, 790-791 pilocytic, 780-782, 781f differential features of, 776t-777t optic nerve, 812 pleomorphic xanthoastrocytoma, 776t-777t theranostic applications, 785 variants, 784-785 Ataxia telangiectasia mutated gene (ATM), 355 ATL. See Adult T-cell leukemia/ lymphoma Atrophic gastritis, 513-514 Autoimmune diseases, 566-568 Autoimmune gastritis, 513-514, 514f Autoimmune pancreatitis, 566-567 diagnosis of, 568 IgG4–associated, 567 type 1, 568 type 2, 568 Autoimmune type atrophic gastritis, 513-514 Automated cell blocks, 830, 831f Automated quantitative analysis (AQUA), 878-881, 881f Automation, 27-30 Avian V-ETS erythroblastosis virus E26 oncogene homolog (ERG) ERG-ETS gene fusions, 608, 609f in prostate carcinoma, 606f, 607t, 608, 610f in tumors of soft tissue and bone, 81-82, 82b, 92t, 96, 97f Avidin-biotin conjugate (ABC), 1, 8, 8f
B
B-cell antigen, 130, 133f B-cell clone, 177t B-cell lymphoma aggressive, 168-176, 170f CD20-positive, 845f immunohistochemical features of, 155t lymphoblastic, 174, 175f MALT type, 409 marginal zone, 409, 410t mediastinal, 142, 142f, 168 T-cell–rich, 141t, 142 unclassifiable with features intermediate between DLBCL and BL, 176
B-cell lymphoma (Continued) with features intermediate between DLBCL and CHL, 176, 382-384 B-cell lymphoma 1 (BCL1/PRAD1) (cyclin D1), 152 B-cell lymphoma 2 (Bcl-2) in basal cell carcinoma, 485, 485f in breast carcinoma, 760-761 double staining for, 26f in head and neck lesions, 246t-248t in lung lymphomas, 410t in mediastinal tumors, 373-374, 374f, 382-384 in mesothelioma, 444t-446t, 476t in non-Hodgkin lymphoma, 152, 155t in pleural neoplasms, 465t in skin lesions, 492, 492f in spindle cell thymoma, 366, 367f in tumors of soft tissue and bone, 88 variable fixation artifacts, 33f B-cell lymphoma 6 (Bcl-6) double staining for, 26f-27f in Hodgkin lymphoma, 134, 138t in non-Hodgkin lymphoma, 150, 155t B-cell lymphoma 10 (Bcl-10), 410t B-cell prolymphocytic leukemia, 155t B-cell receptor (CD79a) in head and neck lesions, 246t-248t in Hodgkin lymphoma, 132 in lung lymphomas, 410t in mediastinal tumors, 382-384 in non-Hodgkin lymphoma, 150, 177t B-cell-specific activator protein (BSAP/PAX5). See also Paired box gene 5 (Pax-5) in Hodgkin lymphoma, 130-132, 141t in non-Hodgkin lymphoma, 150, 177t B-cell–associated antigens, 148-150 B-lymphoblastic lymphoma, 174, 175f B-lymphocyte-induced maturation protein (Blimp-1), 134 B-SA. See Biotin-streptavidin B2M, 191 B72.3, 840 in adenocarcinoma, 459t in cholangiocarcinoma, 580f in gastric adenocarcinoma, 516t in pancreatic ductal adenocarcinoma, 543f in pleural mesothelioma, 448f, 448t, 452t-453t, 468t-471t in pulmonary carcinomas, 395f Babesia, 66 Bacillus anthracis, 69-70 Background staining, 35-36 nonspecific, 4-5, 5f problems and solutions, 36t Bacterial infections, 62-65. See also specific infections BALT. See Bronchial-associated lymphoid tissue
Index
Barjoran purple, 24t Barrett esophagus, 510, 538f anatomic molecular diagnostic applications to, 539 definition of, 510 dysplasia in, 510, 511f key diagnostic points, 510b theranostic applications, 537 Bartonella, 64 Bartonella henselae, 58t, 64, 64f Bartonella quintana, 64 Basal cell adenoma, 291t, 292 Basal cell carcinoma, 479, 481, 481f, 507f adenoid clear cell, 480-481 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t hamartomatous, 480-481 infiltrative, 485 infundibulocystic, 480-481 metatypical, 480-481 morpheaform, 480-481 subtypes, 480-481 Basal-like carcinoma, 728-729, 729f Basaloid carcinoma, 420-421, 420f, 421t Basaloid salivary gland tumors, 291t Basaloid squamous cell carcinoma esophageal, 512, 513f head and neck, 249t, 250 keratinizing, 250 key diagnostic points, 252b from nasal cavity, 250-252, 252f nonkeratinizing, 250 nonkeratinizing with maturation, 250 Basic helix-loop-helix (BHLH) motif, 78 BCI. See Breast Cancer Index BCIP-NBT, 24t Bcl-2. See B-cell lymphoma 2 Benign cephalic histocytosis, 493 Benign cystic teratoma, 699 Benign fibroblastic polyps, 537 Benign lymphocytic angiitis and granulomatosis, 408-409 Benign tumors. See also Tumors; specific sites, types mixed tumors, 290 skeletal muscle tumors, 97-101 smooth muscle tumors, 107-110 vascular tumors, 91-94 BerEP4, 840 in adenocarcinoma, 459t, 842f in basal cell carcinoma, 481, 481f in carcinomas, 223, 223f in cholangiocarcinoma, 580f in colorectal adenocarcinoma, 524t in epithelial mesothelioma, 448f, 453t, 468t-470t in epithelioid mesothelioma, 471t, 475t in gastric adenocarcinoma, 516t key diagnostic points, 224b in melanocytic neoplasms, 191 in mesothelioma, 452t in peritoneal carcinoma, 841f
BerEP4 (Continued) in pleural neoplasms, 448t, 450t451t, 471t, 475t in pulmonary carcinomas, 395f in skin carcinomas, 489f Beta cells, 353-354 Bethesda panel, 52-54, 53f BG-7, 138t Bg8 in carcinomas, 223 key diagnostic points, 224b in pleural neoplasms, 448t, 452t, 459t, 468t-471t, 475t Bile duct adenomas, 580 Bile duct brushing, 834f Bile ducts extrahepatic, 559-563, 583 normal, 569 Biliary cirrhosis, primary, 570 Biliary intraepithelial neoplasia, 560 Biliary mucinous cystic neoplasms, 560, 582 Biliary tract, extrahepatic, 559-563 Biliary-type differentiation, 579 BilIN. See Biliary intraepithelial neoplasia Binding, nonspecific, 23 BioCare, 8, 10 Biological Stain Commission (BSC), 18, 832-834 Biopsy for metastatic melanoma, 197-198 for neurologic diseases, 767t specimen requirements, 42 specimen selection, 762 Bioterrorism, 68-70 bioTheranostics, 244, 757-759, 758t Biotin-avidin procedure, 7, 7f Biotin-streptavidin (B-SA), 1, 8 Biotinylated tyramine (tyramide), 10 Birbeck granules, 413, 429f, 815t BK virus, 58t, 61, 62f, 72f BL. See Burkitt lymphoma Bladder, urinary, 615-618 Bladder carcinoma anatomic molecular diagnostic applications, 627-631 benign mimics, 623 biomarkers of, 630 epigenetic alterations in, 630 gene expression in, 629 genomic analysis, 629 genomic applications, 624-627 ploidy and morphometric analysis, 630 predictive markers, 630-631 secondary rectal adenocarcinoma, 619, 620f small cell carcinoma, 622-623 targeted therapy for, 630-631 theranostic applications, 624-627 Bladder neoplasms, 619-631 inflammatory myofibroblastic tumors, 624, 624f-625f spindle cell neoplasms, 625t BLAG. See Benign lymphocytic angiitis and granulomatosis
889
Blastoma, 404-405, 467 Blastomyces dermatitidis, 66 Blimp-1. See B-lymphocyte-induced maturation protein Blocking, 31t BluePrint assay, 755-756 Bob-148, 150 BOB.1 in Hodgkin lymphoma, 132-133 in non-Hodgkin lymphoma, 150, 177t Bone-forming tumors, 125 Bone morphogenetic protein, 88-89 Bone tumors, 73-129 antibody reagents for study of, 90t antigens and antibodies in, 73-89 cartilaginous, 124 fibrous, 125 malignant small round cell tumors, 91t markers for, 85-89, 91t-92t spindle cell tumors, 92t Borrelia burgdorferi, 65, 771 Botryoid rhabdomyosarcoma, 858 Boutonneuse fever, 65 Bovine spongiform encephalopathy, 774 Bowen disease, 242 Brachyury in head and neck lesions, 246t-248t in tumors of soft tissue and bone, 89, 127, 127f BRAF PCR product of, 47f in thyroid cancer, 340t, 341 Brain abscesses, 769-770, 769f, 819t, 827-828 Brain cancer, 817 Brain lymphoma, 812-813, 813f Brain tissue diagnostic pitfalls, 824-828, 824f invasion of, 807 reactive changes in, 767-770, 768b Brain tumors, 817f clinical and radiologic perspective on, 764-766 differential diagnosis, 817, 817f distinct epithelial borders, 817 distinct tumor margin, 817 grading of, 775 infiltration of, 775, 778, 780f nonneoplastic lesions, 767-775 sarcoma, 810-811 Bratonella, 63 BRCA2, 344 Breast, 710-761 Breast Cancer Index (BCI), 757-759, 758t Breast carcinoma, 714f, 717f, 724f-725f 2-gene ratio model, 757-758, 758t 5-gene index, 758t 70-gene profile, 755-756, 758t 76-gene profile, 756, 758t analytic variables, 746 basal-like, 752-755, 754t CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t
890
Index
Breast carcinoma (Continued) classification of, 752-754, 754t ductal vs. lobular, 723 endocrine tumors, 361-362, 361f ERBB2, 752-755 gene expression models, 758t genomic applications, 752-758 HER2-enriched, 754t HOXB13:IL17RB index, 757-758 immunogenomics of, 752-758 immunostaining of, 846f “intrinsic” gene set, 752-754 intrinsic subtype, 758t invasive, 712f-713f, 721, 723f-725f key diagnostic points, 721b, 723 luminal tumors, 714, 752-754, 754t mesenchymal, 731 metaplastic, 731t metastatic, 740f to ear and temporal bone, 320-321, 320f to head and neck, 320-321 key diagnostic points, 739b sentinel lymph node micrometastatic disease, 738b systemic metastasis, 738-739 vs. gastric signet-ring cell carcinoma, 518-519, 519f molecular approaches to, 362 molecular classification of, 752-754, 754t molecular grade index, 757-758 myoepithelial, 731 myofibroblastoma, 731, 732f normal breast-like, 752-754 papillary, 719t postanalytic interpretation, 746-748 preanalytic variables, 745-746 prognosis for, 744, 760-761 proliferative lesions, 721b recurrence score model, 756-757, 758t in situ lesions, 721, 721b special types of, 725-731 spindle cell, 731, 731t theranostic applications, 744-752 triple-negative, 754-755 tumor markers, 758-761 type identification, 721-731, 732b vs. pulmonary adenocarcinoma, 422 wound response model, 756, 758t Breast carcinoma in situ, 716f, 721-723, 722f, 726f Breast markers, 846-848, 883 antibodies for myoepithelial cells, 710-713, 711f, 711t Brenner tumors, 693, 693f Broad ligament tumors, 706-708, 708b Bronchial-associated lymphoid tissue, 407-408 Bronchioloalveolar cell carcinoma, 429, 429f-430f BRST-2, 246t-248t, 846-847. See also Gross cystic disease fluid protein 15 (GCDFP-15) Brucellosis, 70
BSAP. See B-cell-specific activator protein BSC. See Biological Stain Commission BSCC. See Basaloid squamous cell carcinoma Bubble artifacts, 34f Burkitt lymphoma, 169, 170f, 174, 175f, 176
C
C-cell hyperplasia, 337, 337f C cells, 337-342, 337f c-erbB-1. See Epidermal growth factor receptor c-erbB-2, 506-507 c-Kit. See also CD117 in mediastinal tumors, 379-380, 381f, 382t in seminoma, 647, 647f in thymic carcinoma, 369-370, 369f-370f C-PATH, 879 C-reactive protein, 573t CA 15-3. See Carbohydrate antigen 15-3 CA 19-9. See Carbohydrate antigen 19-9 CA 125. See Carbohydrate antigen 125 Cadherin, kidney-specific (Kspcadherin), 633 E-Cadherin, 833t, 848 in breast carcinoma, 721-723, 722f-726f, 725, 728f, 848 in mesothelioma, 444t-446t, 468t-470t and p120, 722-723, 724f-725f in pancreas, 542 in pleural neoplasms, 448t N-Cadherin in mesothelioma, 444t-446t, 468t-469t in pleural neoplasms, 448t Cadherins in thyroid tumors, 335-336 in tumors of soft tissue and bone, 75-76 CAIA. See Computer-aided image analysis CAIX. See Carbonic anhydrase IX Calcifications, 827 Calcifying fibrous pseudotumor, 466-467 Calcitonin, 833t in endocrine tumors, 352t, 357f in head and neck lesions, 246t-248t in lung carcinoids, 356f in pheochromocytoma, 347f in sinonasal tract tumors, 256t in thyroid tumors, 338f Calcitonin gene-related peptide, 338, 338f Calcium-binding proteins, 192-193 Caldesmon in epithelial mesothelioma, 453t in gynecologic pathology, 654t in tumors of soft tissue and bone, 77
h-Caldesmon in breast tumors, 742, 745f in spindle cell tumors, 92t in tumors of soft tissue and bone, 77 in uterine tumors, 673t, 674-675, 675f Caliper/Perkin Elmer, 878-879 CALLA. See CD10/acute lymphocytic leukemia antigen Calponin in breast, 711t in head and neck lesions, 246t-248t, 274t in tumors of soft tissue and bone, 77-78 Calretinin, 838 in adenomatoid tumors, 708 in adrenocortical tumors, 346f in cancer of unknown primary site, 226-227 in colorectal adenocarcinoma, 524t in endometrioid carcinoma, 691t in female adnexal tumors, 708 in gastric adenocarcinoma, 516t in granulosa cell tumors, 696-697, 696t in gynecologic pathology, 654t in head and neck lesions, 246t-248t key diagnostic points, 227b in Leydig cell tumors, 699 in lung neoplasms, 389t in malignant mesothelioma, 839f in melanocytic neoplasms, 192-193 in mesothelioma, 444t-446t, 448f, 452t-453t, 453, 453f, 459t, 462f, 468t-471t, 475t in olfactory neuroblastoma, 258259, 258f in ovarian and tubal tumors, 685, 695-696 in pheochromocytoma, 347f, 348 in pleural neoplasms, 448t, 450t-451t in Sertoli cell tumors, 698-699 in Sertoli-Leydig cell tumors, 697-698 in sex cord tumors, 699 in sinonasal tumors, 256t in steroid cell tumors, 699 in sweat gland tumors, 483, 483f in thymic carcinoma, 369-370, 369f-370f in vulvar granular cell tumors, 656, 656f CAM5.2 in adrenocortical tumors, 346f in brain tumors, 783f, 799, 800f, 817f, 818 in cancer of unknown primary site, 213f in cholangiocarcinoma, 580f in colorectal adenocarcinoma, 522 in dendritic cells, 218, 219f in esophageal squamous cell carcinoma, 511, 511f in gastric adenocarcinoma, 515f in gliosarcoma, 794f
Index
CAM5.2 (Continued) in head and neck lesions, 246t-248t, 263-264 in hepatocellular carcinoma, 221f, 577f in malignant rhabdoid tumor, 870f in mesenchymal tumors, 218, 218f in nervous tissue, 765t in neuroblastoma, 350f in pancreatic tumors, 543f, 556, 556f in pheochromocytoma, 348 in pituitary adenoma, 265-266, 265f, 328f principal diagnostic use, 90t in sinonasal tract tumors, 256t, 259f, 260 in upper aerodigestive tract carcinomas, 249t Campanacci disease, 128-129 Canalicular adenoma, 291t Cancer. See Carcinoma; Neoplasms; specific cancers; specific sites Cancer Genome Project, 41 Cancer of unknown primary site, 852-853. See also Metastatic disease clinical aspects, 204-206 diagnosis of algorithmic approaches to, 237, 238f-239f combined antibody (panel) approach to, 237 molecular approach to, 242-244 molecular assays for, 242t multigene expression assays for, 244t screening immunohistochemistry for, 207-208 specimen preparation for, 206-207 stepwise approach to, 206-237 economic considerations, 206 poorly undifferentiated, 853f sarcomas that present as, 208, 211t-212t special presentations, 237-242 terminology, 204 CancerTYPE ID test, 243 Candida, 65 Candida albicans, 58t, 65 Candle guttering, 784 CAP, 14-15, 17 Capillary hemangioblastoma, 814-816 Capillary-like microvessels, 576 Capillary telangiectasia, 775t Carbohydrate antigen 15-3 (CA 15-3), 336 Carbohydrate antigen 19-9 (CA 19-9) in cholangiocarcinoma, 580f-581f, 581 in colorectal adenocarcinoma, 524t, 526f in epithelial mesothelioma, 468t-469t in gastric adenocarcinoma, 516t in pancreatic tumors, 543f, 556f in thyroid tumors, 336
Carbohydrate antigen 125 (CA 125) in cholangiocarcinoma, 580f in colorectal adenocarcinoma, 524t, 526f in gastric adenocarcinoma, 516t in gynecologic pathology, 654t in ovarian and fallopian tube tumors, 685, 688f Carbonic anhydrase IX, 633-634, 635f Carcinoembryonic antigen, 840 in adenocarcinoma, 35f, 220-221, 221b, 459t in adenoid cystic carcinoma, 299f, 300 in cancer of unknown primary site, 220-221 in colorectal adenocarcinoma, 526f in endocervical adenocarcinoma, 662, 663f in extrahepatic biliary tract carcinoma, 561, 561f in gastric adenocarcinoma, 516t in head and neck lesions, 246t-248t in hepatocellular carcinoma, 221, 221f, 574, 574f key diagnostic points, 222b in lung neoplasms, 421t-422t, 427t in melanocytic neoplasms, 190-191 in meningioma, 805f in mesothelioma, 453t, 468t-471t monoclonal (mCEA), 833t, 840 in colorectal adenocarcinoma, 524t in endocervical adenocarcinoma, 662f in gastric carcinoma, 519f in gynecologic pathology, 654t in large cell neuroendocrine carcinoma, 357f in lung carcinoids, 356f in mesothelioma, 452t in pancreatic tumors, 556, 556f in pleural neoplasms, 448t in small cell carcinoma, 356f in thyroid tumors, 338f in upper aerodigestive tract carcinomas, 249t in pancreatic carcinoma, 544, 544f, 548, 548f in pleural neoplasms, 448t polyclonal (pCEA) in cholangiocarcinoma, 580f in epithelial mesothelioma, 448f in hepatocellular carcinoma, 577f in lung neoplasms, 388t-389t in mesothelioma, 444t-446t, 452t in neuroblastoma, 350f in pancreatic ductal adenocarcinoma, 543f in pleural neoplasms, 448t, 450t-451t polyclonal (pCEA), 395f in sinonasal tract lesions, 273t-274t in skin carcinomas, 489f in small cell lung carcinoma, 402, 403f in thyroid tumors, 336
891
Carcinoid, 358-362 appendiceal goblet cell carcinoid, 530, 531f atypical, 356 definition of, 400 presenting as mesothelioma, 471-472, 472f-473f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t in gallbladder, 562, 562f glandular psammomatous, 565-566 ileal, 352f-353f laryngeal atypical, 287, 288f-289f differential diagnosis of, 289-290, 289t typical, 287, 287f lung, 356f, 402, 404f malignant, 400 molecular diagnostic approaches to, 353 in ovary, 703, 703f thymic, 372f TTF-1 expression in, 393t typical, 356, 400 Carcinoma. See also specific sites, types cytokeratin 7 in, 389-390, 390t cytokeratin 20 in, 389-390, 391t diagnosis of, 764f lymphoma mimics, 185t markers, 185t metastatic immunomarkers of, 92t of unknown primary site, 204-244 metastatic to CNS, 817-818, 817f metastatic to pleura and abdomen, 237, 239b poorly differentiated algorithmic approaches to, 237, 238f-239f of liver, 582 undifferentiated classification of, 207 of liver, 582 of unknown primary site, 204 vimentin coexpression in, 219, 219b-220b, 220f Carcinoma in situ in breast, 716f, 721b classification of, 248 pleomorphic lobular, 727f Carcinomatosis, intraabdominal, 543 Carcinosarcoma head and neck lesions, 252-253 of liver, 582 in lung, 404-405 uterine, 675 CARD. See Catalyzed reporter deposition technique Cardiac muscle-α, 77 Cartilaginous tumors of bone, 124 Castleman disease, 381-382 Cat-scratch disease, 64, 64f Catalogue of Somatic Mutations in Cancer (COSMIC) database, 41
892
Index
Catalyzed reporter deposition technique (CARD), 10 β-Catenin -activated adenomas, 573-574 in desmoid-type fibromatosis, 534, 534f in focal nodular hyperplasia, 573t in GI tract, 508, 509t in head and neck lesions, 246t-248t, 275, 276f in hepatocellular adenoma, 573t in pancreas, 542-543 in pancreatoblastoma, 554, 554f in solid-pseudopapillary neoplasms, 554f-555f, 555 in spindle cell tumors, 92t in thyroid tumors, 335-336, 335f, 340t, 341 in tumors of soft tissue and bone, 85-86, 92t in uterine carcinomas, 666, 668f Cavernous angioma, 775t Cavitary gliosis, 819t Cbp/p300-interacting transactivator 1, 334t, 335 CCC. See Clear cell chondrosarcoma CCR4. See Chemokine receptor 4 CCR7. See Chemokine receptor 7 CCS. See Clear cell sarcoma CD markers, 833t CD1a in CNS tumors, 815t in head and neck lesions, 246t-248t in Langerhans cell histiocytosis of the skin, 493, 494f CD2 in Hodgkin lymphoma, 134-135 in non-Hodgkin lymphoma, 152 in nonhematopoietic neoplasms, 428t CD3, 833t in Hodgkin lymphoma, 134-135, 138t, 141t in mediastinal hematopoietic tumors, 382-384 in mesothelioma, 444t-446t in non-Hodgkin lymphoma, 152, 156f in nonhematopoietic neoplasms, 428t in peripheral T-cell lymphoma, 178, 179f CD4, 833t in Hodgkin lymphoma, 134-135 in mycosis fungoides, 491-492, 491f in non-Hodgkin lymphoma, 152-153 in nonhematopoietic neoplasms, 428t CD5, 833t in cancer of unknown primary site, 237 in diffuse large B-cell lymphoma, 169-170, 170f in Hodgkin lymphoma, 134-135 key diagnostic points, 237b in lung lymphomas, 410t
CD5 (Continued) in mediastinal hematopoietic tumors, 382-384 in non-Hodgkin lymphoma, 153, 155t in nonhematopoietic neoplasms, 428t in thymic carcinoma, 369-370, 369f-370f CD7 in non-Hodgkin lymphoma, 153 in nonhematopoietic neoplasms, 428t CD8, 833t in Hodgkin lymphoma, 134-135 in non-Hodgkin lymphoma, 152-153 in nonhematopoietic neoplasms, 428t CD10, 632-633, 833t in cancer of unknown primary site, 234-235 in CD20-positive B-cell lymphoma, 845f in endometrial tumors, 673-675, 674f-675f in female adnexal tumors, 708 in gynecologic pathology, 654t in hepatocellular carcinoma, 577f in lung lymphomas, 410t in mediastinal hematopoietic tumors, 382-384 in non-Hodgkin lymphoma, 155t in skin tumors, 485, 486f, 491f, 498, 500f in solid-pseudopapillary neoplasms, 554f in uterine tumors, 666-667, 673t CD10/acute lymphocytic leukemia antigen, 632-633 CD11c, 155t CD15, 833t, 840-843 in adenocarcinoma, 459t in cholangiocarcinoma, 580f in clear cell carcinoma, 692f in epithelial mesothelioma, 448f, 453t, 469t-470t in Hodgkin lymphoma, 130-132, 133f, 138, 138t, 139f, 141t, 143, 177t in lung neoplasms, 388t-389t, 395f, 4 10t in mediastinal hematopoietic tumors, 382-384 in mesothelioma, 444t-446t, 452t, 471t, 475t in pleural neoplasms, 448t, 450t-451t in thyroid tumors, 336 CD20, 833t, 852 -positive B-cell lymphoma, 845f anti-CD20 monoclonal antibodies, 145 bubble artifacts, 34f in encephalitis, 769f in Hodgkin lymphoma, 130-132, 133f, 138f, 138t, 139, 141t, 158f, 177t
CD20 (Continued) in lung neoplasms, 410t, 411-412, 412f, 427t in mediastinal tumors, 382-384, 384f in mesothelioma, 444t-446t in nervous tissue, 765t in non-Hodgkin lymphoma, 148-150, 155t, 156f, 171f, 177t staining, 34f weak or absent, 33f CD21, 382-384 CD23 in lung lymphomas, 410t in non-Hodgkin lymphoma, 155t CD25 in Hodgkin lymphoma, 137 in non-Hodgkin lymphoma, 155t CD30, 833t anti-CD30 monoclonal antibodies, 145 in dysgerminoma, 701t in embryonal carcinoma, 702 in gynecologic pathology, 654t in Hodgkin lymphoma, 130-132, 133f, 138f, 138t, 139, 140f, 141t, 142-143 in large cell undifferentiated malignancies, 202f in lung neoplasms, 410t, 427t in mediastinal tumors, 382-384, 382t in mesothelioma, 444t-446t in non-Hodgkin lymphoma, 151, 170, 177t, 179, 179f, 183, 488, 490f in skin tumors, 493, 495f in testicular tumors, 648t CD30 lymphoproliferative disorders, 493-494, 495f CD31 in cutaneous angiosarcoma, 502, 504f in epithelioid hemangioendothelioma, 504, 505f, 583, 583f in head and neck lesions, 246t-248t in mediastinal neoplasms, 379, 379f, 380t in mesothelioma, 444t-446t in serous cystadenoma, 552, 552f in spindle cell tumors, 92t in tumors of soft tissue and bone, 81 in vascular proliferations, 775 CD34 in fibrous tumors, 806, 806f in head and neck lesions, 246t-248t in hepatocellular carcinoma, 575, 577f in liver neoplasia, 221, 222f in mediastinal tumors, 373-374, 374f, 380t, 382-384 in meningiomas, 806, 806f in mesothelioma, 444t-446t in non-Hodgkin lymphoma, 151-152 in perineurioma, 111-113, 112f in pleural neoplasms, 465t
Index
CD34 (Continued) in prostatic tumors, 603t, 604-605, 604f in skin tumors, 485, 486f, 496-497, 497f-499f in tumors of soft tissue and bone, 80-81, 92t in upper aerodigestive tract carcinomas, 249t in vascular proliferations, 775 CD40 anti-CD40 monoclonal antibodies, 145 in Hodgkin lymphoma, 134, 134f, 138t, 140f, 141t CD43, 33f in lung lymphomas, 410t in non-Hodgkin lymphoma, 152, 155t, 156f CD44, 507 CD44S, 468t-470t CD44v6 (heparan sulfate proteoglycan), 336 CD45, 833t in dysgerminoma, 701t in granulosa cell tumors, 696t in Hodgkin lymphoma, 130-132, 132f, 138f, 138t, 139, 141t in mediastinal tumors, 382-384, 384f in mesothelioma, 444t-446t in non-Hodgkin lymphoma, 148, 149f, 177t in ovarian and tubal tumors, 687 in tumors of soft tissue and bone, 91, 91t variable fixation artifacts with, 32f CD45RB, 246t-248t, 256t CD45RO, 138t, 765t, 769f CD56, 833t in adrenocortical tumors, 346f in cancer of unknown primary site, 224 discriminative value, 402t in endocrine tumors, 324-325 in gynecologic pathology, 654t in head and neck lesions, 246t-249t, 256t, 263-264, 263f, 267-269, 267f-268f in lung neoplasms, 402t in non-Hodgkin lymphoma, 153 in nonhematopoietic neoplasms, 428t in olfactory neuroblastoma, 258259, 258f in ovarian tumors, 693-694 in pancreatic tumors, 554f, 556f in pituitary adenoma, 265-266, 265f in skin carcinomas, 489f in tumors of soft tissue and bone, 79 CD57 in cancer of unknown primary site, 224 discriminative value, 402t in endocrine tumors, 324 in head and neck lesions, 246t-248t in Hodgkin lymphoma, 134-135, 134f, 138t
CD57 (Continued) in lung neoplasms, 402t, 403f, 425, 426f in melanocytic neoplasms, 200, 201f-202f in nervous tissue, 765t in non-Hodgkin lymphoma, 153 in tumors of soft tissue and bone, 79 CD68 in CNS tumors, 765t, 815t in encephalitis, 769f in head and neck lesions, 246t-248t in neurothekeoma, 501-502, 503f in tumors of soft tissue and bone, 84, 90t in xanthoma, 592-594, 594f CD74, 136, 138t CD79a. See B-cell receptor CD95 (Fas), 134 CD99 in desmoplastic small round cell tumor, 865-866, 866f in Ewing sarcoma, 126, 126f, 863, 863f-864f in gastric adenocarcinoma, 516t in granulosa cell tumors, 696t in gynecologic pathology, 654t in head and neck lesions, 246t-248t in malignant rhabdoid tumor, 869, 869f in neuroblastoma, 351 in pediatric neoplasms, 855 in PNETs, 378, 378f, 488, 489f in sinonasal tract tumors, 256t in tumors of soft tissue and bone, 86, 91, 91t CD117, 851 in adenoid cystic carcinoma, 299f, 300 in dysgerminoma, 700, 700f, 701t in gastrointestinal tumors, 509, 509t, 532-533, 533f, 605-606, 605f in gynecologic pathology, 654t in head and neck lesions, 246t-248t in lung neoplasms, 402t, 403f in mediastinal tumors, 379-380, 381f, 382t in non-Hodgkin lymphoma, 152 in ovarian tumors, 686 in prostatic tumors, 603t, 605-606, 605f in seminoma, 647, 647f in sinonasal tract tumors, 256t in testicular tumors, 644, 648t in thymic carcinoma, 369-370, 369f-370f in upper aerodigestive tract carcinomas, 249t CD133, 351 CD138 in head and neck lesions, 246t-248t in Hodgkin lymphoma, 134 in mediastinal hematopoietic tumors, 382-384 in non-Hodgkin lymphoma, 151 in nonhematopoietic neoplasms, 428t
893
CD141, 83. See also Thrombomodulin CD163 in head and neck lesions, 246t-248t key diagnostic points, 84b in nervous tissue, 765t in tumors of soft tissue and bone, 84 CD207, 246t-248t CD246, 833t CDK4. See Cyclin-dependent kinase 4 CDKN1B, 607t CDKN2A, 848-849 in pancreatic neoplasms, 545 in urothelial carcinoma, 618 CDw75, 138t CDX2, 843 in adenocarcinomas, 229-230, 229t, 844f in ampullary adenocarcinoma, 563-564, 565f in appendiceal neoplasms, 520-521, 521f in cancer of unknown primary site, 229-230 in colon carcinoma, 230, 231f in colorectal carcinoma, 522-523, 524t, 525f-526f, 843 in endocervical adenocarcinoma, 661, 663f in endocrine tumors, 353, 353f in endometrioid tumors, 690-692 in gastric carcinoma, 515, 516t, 519f in gastrointestinal tumors, 508, 509t, 521f, 532f, 843 in gynecologic pathology, 654t in head and neck lesions, 246t-248t immunoreactivity of, 427 in intraampullary neoplasms, 563, 564f in intraductal neoplasms, 549, 549f in lung neoplasms, 388t-389t, 422, 422t in mesothelioma, 444t-446t in ovarian and tubal tumors, 688t, 704-705, 704f in pancreatic carcinoma, 543f, 548, 548f in sinonasal tract lesions, 273t-274t in small intestinal adenocarcinoma, 519-520, 520f in urachal cysts, 230, 230f CEA. See Carcinoembryonic antigen Cecal adenocarcinoma, invasive, 524f Celiac disease, 519 refractory, 537 Cell blocks, 829-832, 830f automated, 830, 831f cytoscrape, 830-831 false-negative results with, 832, 834f formalin-fixed, paraffin-embedded, 830f problems with, 837f rapid, 830 Cell-cycle markers, 325-326 Cell-cycle regulators, 626f, 628-629 Cell differentiation. See Tumor differentiation Cell membrane proteins, 189-192 Cell-specific products, 224-237
894
Index
Cell type identification, 775 Cellient, 830, 831f Cellular density, 827 Cellular neurothekeoma, 199-200, 201f Centers for Medicare and Medicaid Services (CMS), 14-15 Central nervous system carcinoma metastatic to, 817, 817f cells infiltrating parenchyma, 768t demyelination in, 821-822 Central neurocytoma, 776t-777t, 788f, 799 Centrosome abnormalities, 607t Cephalic histocytosis, benign, 493 Cerebellar gangliocytoma, dysplastic, 797 Cerebellar hemangioblastoma, 814-815, 815f Cerebellar liponeurocytoma, 799 Cerebral rhabdomyosarcoma, 811 Cerebral tumors, 762-764, 764f Cerebritis, 768t, 770 Cerebrovascular diseases, 774-775 Ceruminous adenocarcinoma, 311, 312f Ceruminous adenoma, 311, 311b, 311f-312f Cervical carcinoma adenoid basal, 663, 664f adenoid cystic, 662-663 neuroendocrine, 663-664 small cell, 663-664 Cervical endocrine tumors, 358-359, 359f Cervical squamous intraepithelial lesions, 658-659, 659b, 659f-660f Cervix, 658-664, 659f CGCTs. See Cutaneous granular cell tumors CGH. See Comparative genomic hybridization Check Sample program (CAP), 17 Checkerboard titration, 12, 14t Chemodectoma-like bodies, minute, 418 Chemokine receptor 4 (CCR4), 145 Chemokine receptor 7 (CCR7), 137, 137f, 138t Chemokines, 137 Chemotherapy susceptibility, 539 Chest cavity lymphomas, 411-413 Childhood neoplasms. See Pediatric neoplasms Chlamydia, 58t, 64-65 4-Chloro-1-naphthol (4-CN), 13, 24t Cholangiocarcinoma, 581, 581f antibodies in, 580f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t combined with hepatocellular carcinoma, 579-580 cytokeratin 7 in, 390t cytokeratin 20 in, 391t differential diagnosis, 582 intrahepatic, 581 key diagnostic points, 582b peripheral, 581
Cholangiocarcinoma (Continued) prognosis for, 582 spindle cell (sarcomatoid), 581-582 treatment of, 582 variants, 581-582 vs. hepatocellular carcinoma, 580, 582 vs. metastatic lesions, 582 Cholangiocellular carcinoma, 579-580 Cholangitis IgG4–associated, 570 IgG4–related, 570 lymphoplasmacytic sclerosing pancreatitis with, 567 sclerosing, 570 Chondroblastoma, 124 Chondrocytes, 75 Chondroid chordoma, 810 Chondromyxoid tumor, ectomesenchymal, 286-287, 286f, 287b Chondrosarcoma, 124, 811 clear cell, 125 extraskeletal myxoid, 122-123, 211t-212t mesenchymal, 91t, 124-125 Chordoid meningioma, 807 Chordoid sarcoma, 122 Chordoma chondroid, 810 CNS, 810, 810f differential diagnosis, 211t-212t, 766t key diagnostic points, 128b in soft tissue and bone, 127-128, 127f Choriocarcinoma in female genital tract, 680, 702 in gastric adenocarcinoma, 517-518 immunohistochemistry of, 648t immunohistology of, 648b markers for, 237b Choroid plexus tumors anatomic molecular diagnostic applications, 800 carcinoma, 766t, 799-800 epithelial tumors, 799-800 key diagnostic points, 799b papilloma, 766t, 799, 800f Chromogen freckles, 31f Chromogen staining, 36t Chromogenic in situ hybridization (CISH), 10, 751, 751t, 851 Chromogens, 24, 24t, 32t Chromogranin-A in adrenocortical tumors, 346f, 349, 349f in colorectal adenocarcinoma, 524t in desmoplastic trichoepithelioma, 485, 486f discriminative value, 402t in endocrine tumors, 323, 352-353, 352f-353f, 357f in esophageal carcinomas, 513f in extraadrenal neuroblastoma, 350, 350f in GI tract, 508-509, 509t in gynecologic pathology, 654t
Chromogranin-A (Continued) in head and neck lesions, 246t-249t in lung neoplasms, 356f, 357-358, 402, 402t, 403f-404f, 419t, 421t, 427t, 471-472, 473f in mesothelioma, 444t-446t in nervous tissue, 765t in neuroblastoma, 350f in neuroendocrine carcinoma, 372f, 402t, 403f in non-NE neoplasms, 425, 426f in pancreas, 542, 542f in pancreatic tumors, 556f in parathyroid adenomas, 343f in pituitary adenoma, 328f in skin carcinomas, 489f in small cell carcinoma, 356f, 402, 404f, 665f in thymic carcinoma, 372f in thyroid tumors, 330f, 338f Chromogranin-B, 323, 338f Chromogranin-C, 323 Chromogranins in cancer of unknown primary site, 224 in endocrine tumors, 323-324 in granulosa cell tumors, 696t in head and neck lesions, 256t, 258-259, 258f, 263f, 273t in lung neoplasms, 356f, 401 in ovarian tumors, 693-694 in pheochromocytoma, 347f in pituitary adenoma, 265-266, 265f Chromophobe renal cell carcinoma, 637-638, 638b Chromosomal alterations, 41 in bladder carcinoma, 627 in seminoma, 649, 650t Chromosomal deletions, 41, 51-52 Chromosomal rearrangement, 41, 50-51 Chromosome 1p deletions in brain tumors, 791, 792f detection of, 51-52, 52f Chronic inflammatory demyelinating polyneuropathy, 822, 822f Chronic lymphocytic leukemia, 154, 160-161, 409-411 diagnostic pitfalls, 161 Hodgkin-like cells in, 143 Hodgkin lymphoma in, 143 immunohistochemical features of, 155t, 410t prognostic and therapeutic studies, 161 proliferation centers in, 160, 161f Chronic lymphoid hyperplasia, 154 Chronic pancreatitis, nonalcoholic duct-destructive, 567 Chronic sclerosing pancreatitis, 567 Cirrhosis, 570-571 CISH. See Chromogenic in situ hybridization CITED1, 335 Clarient/Chroma Vision, 883t Claudin-1, 79-80, 111-113, 112f Claudin-5, 82-83, 380t
Index
Clear cell adenocarcinoma, 525 Clear cell carcinoma, 692f adenoid, 480-481 endometrial, 667t, 671 hepatocellular, 579 hyalinizing, 303, 303f-304f differential diagnosis of, 296t key diagnostic points, 304b key diagnostic points, 236b, 671b in lung, 414, 414f-415f in ovary and fallopian tubes, 692-693, 692f papillary renal cell carcinoma, 640-641, 641b renal cell carcinoma, 636-637, 637b thymic, 371 vs. yolk sac tumor, 693t Clear cell chondrosarcoma, 125 Clear cell ependymoma, 787 Clear cell lesions, 763f Clear cell meningioma, 807-808, 808f Clear cell myoepithelioma, 296t Clear cell salivary gland tumors, 296t Clear cell sarcoma, 121f differentiation from CUPS, 211t-212t of tendons and aponeuroses, 120-121 Clear cell sarcoma–like tumors, 121 Clinical Laboratory Standards Institute (CLSI), 15, 832-834 CLIP-170/Restin, 136 Closed systems, 28 Clostridium, 58t, 65 CLSI. See Clinical Laboratory Standards Institute CMS. See Centers for Medicare and Medicaid Services Coagulant fixatives, 20 Codons, 41 Cofilin, 353 Cold ischemic time, 745-746 Collagen type IV in breast carcinoma, 713f, 717t, 719, 719f in nervous system tumors, 789f in nervous tissue, 765t in tumors of soft tissue and bone, 79 Collecting duct carcinoma, 638b, 639f Colloid carcinoma, 547-548, 548f Colloid cysts, 818-819 differential features of, 816t of the third ventricle, 818-819 Colon cancer. See also Colorectal adenocarcinoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t hereditary nonpolyposis colon cancer syndrome (Lynch syndrome), 524f KRAS mutation in, 49, 50f metastatic, 422 Colonic polyps, 522
Colorectal adenocarcinoma, 522-525 anatomic molecular diagnostic applications to, 539 antibodies in, 524t cytokeratins in, 522, 522f-523f genomic applications, 537 key diagnostic panels for, 525-526 key diagnostic points, 525b metastatic differential diagnosis of, 273t immunohistochemical features of, 422t to ovary, 704f with microsatellite instability, 523-525, 525f mucinous, 521f, 523f secondary involvement of prostate by, 602 theranostic applications, 538 vs. lung adenocarcinoma, 525, 525f vs. müllerian endometrioid adenocarcinoma, 525, 526f vs. prostatic adenocarcinoma, 526, 526f vs. urothelial carcinoma, 526, 526f Colorectal neuroendocrine tumors, 531, 531b Combined antibody (panel) approach, 237 Combined small cell carcinoma, 400 Common acute lymphoblastic leukemia antigen, 666-667 Comparative genomic hybridization (CGH), 49 Computer-aided image analysis, 877, 882, 884 Congenital granular cell tumor, 113 Congenital nevi, 198 Congenital self-healing reticulohistiocytosis, 493 Congenital superficial hemangiopericytoma, 494-495 Contamination, microbiologic, 36t Control materials, 30 Controls, 15-19, 835 Cornu ammonis, 823f Cornu ammonis region 1, 822, 823f-824f Coronavirus, SARS-associated, 68, 69f Corpora amylacea, 824f Cortical carcinoma, 390t-391t Cortical malformations, 824 COSMIC database. See Catalogue of Somatic Mutations in Cancer database Counterstaining, 24-25, 36t Cowdry type A bodies, 771 Coxiella burnetii, 63-65 Craniopharyngioma, 816, 816f cystic, 816t differential diagnosis, 766t Creutzfeldt-Jakob disease, 767t, 768f, 773-774, 774f, 819-820 CRI, 878-879 Cribriform cystadenocarcinoma, low-grade, 307-308, 308b, 308f Crimean-Congo hemorrhagic fever, 61, 71f
895
Crohn-like reaction, 523 Cross-linking fixatives, 20 Crow’s-feet vessels, 115-117 Cryptococcus neoformans, 65-66, 771 Cryptosporidium, 58t, 66 Crystalloid material, 469-471, 471f Crystalloids of Reinke, 699 CTNNB1 gene, 554, 554f CUPS. See Cancer of unknown primary site Cushing syndrome, 371-373 Cutaneous granular cell tumors, 202 Cutaneous lymphohematopoietic disorders, 488-494 Cutaneous lymphoproliferative disorders, 183 Cutaneous melanoma, 192, 192f Cutaneous neoplasms eccrine/apocrine, 506, 507f indeterminate fibrohistiocytic lesions, 496-497 large cell lymphoid proliferations, 492-493 special topics, 506-507 Cyclin B1, 355 Cyclin D1, 833t in lung lymphomas, 410t in non-Hodgkin lymphoma, 152, 155t, 170, 171f in solid-pseudopapillary neoplasms, 554f Cyclin-dependent kinase 4 (CDK4), 87, 92t Cyclooxygenase 1 (COX-1), 336 Cyclooxygenase 2 (COX-2) in GI tract, 509, 509t in thyroid tumors, 336 Cystadenocarcinoma biliary, 582 cribriform, low-grade, 307-308, 308b, 308f TTF-1 expression in, 393t Cystadenoma biliary, 582 serous, 551-552, 552f Cystic astrocytoma, 819t Cystic carcinoma. See Adenoid cystic carcinoma Cystic craniopharyngioma, 816t Cystic mesothelioma, 473, 474f Cystic neoplasms, mucinous biliary, 582 extrahepatic, 560 in pancreas, 550-551, 551f Cystic teratoma, benign, 699 Cysts colloid, 818-819 differential features of, 816t of the third ventricle, 818-819 dermoid, 816t, 819 enterogenous, 816t, 819 ependymal, 816t, 818 epidermoid, 816t, 819 epithelial-lined, 816t glial, 818, 819t meningeal, 819, 819t of nervous system, 818-819 neuroepithelial, 818
896
Index
Cysts (Continued) pineal, 818, 819t simple, 818, 819t urachal, 230, 230f with walls of fibrillar cells, 818, 819t Cytokeratin 1 (CK1) antibodies, 213t distribution in tissue, 212t in spindle cell squamous cell carcinoma, 253-254, 254f in thyroid tumors, 331t Cytokeratin 2 (CK2), 212t Cytokeratin 3 (CK3), 212t Cytokeratin 4 (CK4) distribution in tissue, 212t in head and neck lesions, 246t-248t in thyroid tumors, 331t Cytokeratin 5 (CK5) antibodies, 213t in cancer of unknown primary site, 216-217 distribution in tissue, 212t key diagnostic points, 217b in thyroid tumors, 331t Cytokeratin 5/6 (CK5/6), 838 in adenoid cystic carcinoma, 299f, 300 in anal squamous cell carcinoma, 526, 527f in breast carcinoma, 728-730, 729f-730f, 731t in cancer of unknown primary site, 216-217, 217f in colorectal adenocarcinoma, 522 in epithelial neoplasms, 217t in esophageal carcinomas, 511, 511f-513f in gastric adenocarcinoma, 515f in head and neck lesions, 246t-248t key diagnostic points, 217b in lung neoplasms, 388t-389t, 395f, 419, 419t, 421t-422t, 422 in mesothelioma, 444t-446t, 448f, 452t-453t, 468t-471t, 475t, 839f in pleural neoplasms, 448t, 471t, 475t in sinonasal tract tumors, 256t in skin carcinomas, 489f in upper aerodigestive tract carcinomas, 249t Cytokeratin 6 (CK6) distribution in tissue, 212t in thyroid tumors, 331t Cytokeratin 7 (CK7), 833t antibodies, 213t in Brenner tumors, 693, 693f in cancer of unknown primary site, 209f, 210-214, 214f in carcinomas, 389-390, 390t in cholangiocarcinoma, 580f in clear cell carcinoma, 692-693, 692f in colorectal adenocarcinoma, 522, 525f-526f coordinate expression with CK20 in carcinomas, 389-390, 392t in gastric adenocarcinoma, 515
Cytokeratin 7 (CK7) (Continued) diagnostic utility of, 214 distribution in tissue, 212t in endocervical adenocarcinoma, 661, 663f in endocervical-like mucinous tumors, 690, 690f in endometrioid tumors, 690-692 in epithelial neoplasms, 215b, 215t, 448f, 453t in esophageal carcinomas, 511f, 513f in female adnexal tumors, 708 in gastric adenocarcinoma, 515, 515f, 517f in gastrointestinal tumors, 521f in GI tract, 508, 509t in gynecologic pathology, 654t in head and neck lesions, 246t-248t in hepatocellular carcinoma, 577f immunostaining patterns, 214 in intestinal-type mucinous tumors, 689, 689f in lung neoplasms, 387-390, 388t-389t, 395f, 398-400, 399t, 403f, 419t, 421t-422t, 422, 427t in mesothelioma, 444t-446t, 448f, 453t, 462f, 839f in ovarian and tubal tumors, 684, 687-690, 688t in pancreas, 540, 541f in pancreatic ductal adenocarcinoma, 543, 543f in pituitary adenoma, 328f in pleural neoplasms, 465t in sinonasal tract lesions, 256t, 273t-274t in small cell carcinoma, 358t in small intestinal adenocarcinoma, 519-520, 520f in thyroid tumors, 330f, 331t, 338f in upper aerodigestive tract carcinomas, 249t in urothelial carcinoma, 616, 616t in vulvar Paget disease, 653-655, 655f in yolk sac tumors, 701-702 Cytokeratin 8 (CK8) antibodies, 213t distribution in tissue, 212t in head and neck lesions, 246t-248t in pancreatic ductal adenocarcinoma, 543f in thyroid tumors, 331t in upper aerodigestive tract carcinomas, 249t Cytokeratin 10 (CK10) antibodies, 213t in head and neck lesions, 246t-248t in thyroid tumors, 331t Cytokeratin 11 (CK11) antibodies, 213t in thyroid tumors, 331t
Cytokeratin 13 (CK13) antibodies, 213t in head and neck lesions, 246t-248t in thyroid tumors, 331t in upper aerodigestive tract carcinomas, 249t Cytokeratin 14 (CK14) antibodies, 213t in breast carcinoma, 729-730, 730f in esophageal squamous cell carcinoma, 511 in head and neck lesions, 246t-248t in spindle cell squamous cell carcinoma, 253-254, 254f in thyroid tumors, 331t in upper aerodigestive tract carcinomas, 249t Cytokeratin 15 (CK15), 213t Cytokeratin 16 (CK16), 213t Cytokeratin 17 (CK17) in breast carcinoma, 729-730, 730f in colorectal adenocarcinoma, 522 in esophageal squamous cell carcinoma, 511f in gastric adenocarcinoma, 515f in spindle cell squamous cell carcinoma, 253-254, 254f in thyroid tumors, 331t Cytokeratin 18 (CK18) in angiosarcomas, 96 antibodies, 213t in colorectal adenocarcinoma, 522 in gastric adenocarcinoma, 515f in head and neck lesions, 246t-248t in pancreatic tumors, 543f, 556 in spindle cell squamous cell carcinoma, 253-254, 253f in thyroid tumors, 331t in upper aerodigestive tract carcinomas, 249t Cytokeratin 19 (CK19) antibodies, 213t in cancer of unknown primary site, 210, 213t in cholangiocarcinoma, 580f in colorectal adenocarcinoma, 522 in esophageal carcinomas, 511, 511f-513f in gastric adenocarcinoma, 515f in head and neck lesions, 246t-248t in pancreatic tumors, 543f, 556f557f, 557 in pituitary adenoma, 328f in thyroid tumors, 330f, 331t, 332f, 334t, 335 Cytokeratin 20 (CK20), 833t antibodies, 213t in appendiceal mucinous neoplasms, 520-521, 521f in Brenner tumors, 693 in cancer of unknown primary site, 213t, 214-215, 214f in carcinomas, 389-390, 391t in cholangiocarcinoma, 580f in clear cell carcinoma, 692-693 in colorectal adenocarcinoma, 522, 523f, 525f-526f
Index
Cytokeratin 20 (CK20) (Continued) coordinate expression with CK7 in carcinomas, 389-390, 392t in gastric adenocarcinoma, 515 in desmoplastic trichoepithelioma, 485, 486f in endocervical adenocarcinoma, 661, 663f in endometrioid tumors, 690-692 in epithelial neoplasms, 215b, 216t, 448f, 453t in esophageal squamous cell carcinoma, 511f in gastric tumors, 515, 515f, 517f, 519f in gastrointestinal tumors, 521f in GI tract, 508, 509t in gynecologic pathology, 654t in head and neck lesions, 246t-248t in hepatocellular carcinoma, 577f in intestinal-type mucinous tumors, 689, 689f in lung neoplasms, 388t-389t, 398-400, 399t, 419t, 421t-422t, 422, 427t in mesothelioma, 444t-446t, 448f, 453t in ovarian and tubal tumors, 684, 688-690, 688t in pancreatic ductal adenocarcinoma, 543, 543f in rectal adenocarcinoma, 844f in sinonasal tract lesions, 273t-274t in small cell carcinoma, 356f, 358t in small intestinal adenocarcinoma, 519-520, 520f in thyroid tumors, 330f, 331t, 338f in upper aerodigestive tract carcinomas, 249t in urothelial carcinoma, 616, 616t, 618f, 620f, 621t in uterine carcinomas, 671t Cytokeratin 34βE12 (HMWK, K903) in breast, 719-721, 720f in colorectal adenocarcinoma, 522 discriminative value, 402t in ductal hyperplasia, 720f in esophageal carcinomas, 511, 511f-513f in gastric adenocarcinoma, 515f in gynecologic pathology, 654t in head and neck lesions, 246t-248t in lung carcinomas, 395f, 403f in prostate, 587t in prostate adenocarcinoma, 594-595, 594f in sarcomatoid mesothelioma, 462f in sinonasal tract lesions, 274t Cytokeratin 35βH11 in colorectal adenocarcinoma, 522 in esophageal squamous cell carcinoma, 511f in gastric adenocarcinoma, 515f in lung carcinomas, 395f, 403f in sarcomatoid mesothelioma, 462f Cytokeratin AE1/AE3, 833t in brain tumors, 783f in breast carcinoma, 712f
Cytokeratin AE1/AE3 (Continued) in cholangiocarcinoma, 581, 581f in choriocarcinoma, 702 in colorectal adenocarcinoma, 522 in embryonal carcinoma, 702 in esophageal squamous cell carcinoma, 511, 511f in gastric adenocarcinoma, 515f in gynecologic pathology, 654t in head and neck tumors, 253-254, 253f, 267-269, 268f, 273t in lung tumors, 395f, 403f, 427t in mesothelioma, 448f, 453t, 462f in neuroblastoma, 350f in neuroendocrine carcinoma, 263-264, 263f, 403f, 556, 556f in pancreatic tumors, 543f, 556, 556f in pituitary adenoma, 265-266, 265f, 328f in pleural neoplasms, 448t, 465t principal diagnostic use, 90t in spindle cell squamous cell carcinoma, 253-254, 253f in testicular tumors, 648t in yolk sac tumors, 701-702 Cytokeratin AE13, 485, 487f, 489f Cytokeratin AE14, 489f Cytokeratins in adenomatoid tumors, 708 in adrenocortical tumors, 346f basal, 728-729 in breast carcinoma metastasis, 738, 740f in clear cell carcinoma, 692-693 coexpression in carcinomas, 219, 219b, 220f in colorectal adenocarcinoma, 522 in desmoplastic small round cell tumor, 865-866, 866f in dysgerminoma, 700, 701t in endocrine tumors, 325, 353f in endometrioid tumors, 690-692, 691t in epithelial tumors, 688f in esophageal squamous cell carcinoma, 511, 511f in gastric adenocarcinoma, 515, 515f in GI tract, 508 in granulosa cell tumors, 696-697, 696t in head and neck lesions, 245 in hepatocellular carcinoma, 574 high-molecular-weight in adenosis, 591-592, 593f in prostate, 586 in prostate carcinoma, 587-589, 588f, 591f, 595-596, 595f596f, 621t in prostatic atrophy, 590, 592f-593f in prostatic intraepithelial neoplasia, 587-589, 588f-589f in urothelial carcinoma, 598-599, 601f-602f, 617, 621f, 621t identification of, 208-219 in Leydig cell tumors, 699
897
Cytokeratins (Continued) in lung tumors, 356f, 454t in malignant rhabdoid tumor, 869f in mediastinal neoplasms, 380t, 382t in meningioma, 805f in mesothelioma, 454t in neuroendocrine carcinoma, 225, 357f in ovarian tumors, 684, 693-694 in pancreatic tumors, 353f in papillary tumors, 330f in pheochromocytoma, 347f in prostate adenocarcinoma, 590f in Sertoli cell tumors, 698-699 in Sertoli-Leydig cell tumors, 697-698 in sex cord tumors, 699 simple, 208-219, 215b in spindle cell thymoma, 366, 367f in steroid cell tumors, 699 in thymus, 364, 364f, 369-370 in thyroid tumors, 331-332, 331t in tumors of soft tissue and bone, 74 in upper aerodigestive tract tumors, 249t, 250 in uterine tumors, 664-666, 673t Cytokines, 137 CytoLyt (Cytyc), 829-830 Cytomegalovirus, 58-60, 58t, 59f Cytomegalovirus lymphadenitis, 143, 143f Cytopathology, 829, 830b Cytoscrape cell blocks, 830-831 Cytosine (C), 39 Cytospins, 830f Cytotoxic markers, 134-135, 135f Cytyc, 829-830
D
D2-40, 838-840 in dysgerminoma, 700 in gynecologic pathology, 654t in lung neoplasms, 389t in lymphatic channels, 734, 735f in mediastinal tumors, 380t, 382t in mesothelioma, 444t-446t, 448f, 452t-453t, 471t, 475t in pleural neoplasms, 448t in sarcomatoid mesothelioma, 462f in testicular tumors, 644 in tumors of soft tissue and bone, 83 DAB. See Diaminobenzidine Dabska tumor, 94 Dako, 10 DAS-1, 516t DBA44, 155t DDLPS. See Dedifferentiated liposarcoma de Perrot staging system, 464b Deciduoid mesothelioma, 462 Dedifferentiated liposarcoma, 76, 87, 92t, 115, 116f Deep lymphoid infiltrates, 492 Deep space black, 24t Definiens, 878-879
898
Index
Dehydration, incomplete, 32t Deleted in pancreatic carcinoma, locus 4 (DPC4). See SMAD4 Deletion and insertion mutations, 41 Deletions chromosomal, 41 detection of, 51-52 small, 41 Dementia, 819-821 frontotemporal, 772-773 multiinfarct vascular, 772-773 Dementia/Alzheimer disease confirmatory features of, 767t Dementia/Creutzfeldt-Jakob disease confirmatory features of, 767t Demyelinating polyneuropathy chronic inflammatory, 822, 822f Demyelination, 821-822 central nervous system, 821-822 confirmatory features of, 767t peripheral nervous system, 822 primary, 821, 821f secondary, 821 Dendritic cell tumors mediastinal, 385 Dendritic or antigen-presenting cell markers in Hodgkin lymphoma, 135 Dengue hemorrhagic fever, 61 Deoxyribonucleic acid (DNA), 39-40 Dermatofibromas differential diagnosis of, 496-497, 498f Dermatofibrosarcoma protuberans, 79-80, 495, 497f Dermoid cysts, 819 differential features of, 816t Desmin, 833t in bladder tumors, 625t in desmoplastic small round cell tumor, 865-866, 866f-867f in epithelial mesothelioma, 476t in gynecologic pathology, 654t in head and neck lesions, 246t-248t, 267-269, 267f-268f in leiomyosarcoma of subcutis, 499, 500f in malignant rhabdoid tumor, 869, 869f in mesothelioma, 444t-446t in myofibroblastoma of breast, 732f in pleural neoplasms, 465t in prostatic mesenchymal tumors, 603t in rhabdomyosarcoma, 859-861, 859f-860f in sarcomatoid mesothelioma, 462f in tumors of soft tissue and bone, 73, 76-77, 91t-92t, 111f in uterine tumors, 673t Desmoglein, 75-76 Desmoglein 3 (DSG3), 393-394 Desmoid fibromatosis, 102, 102f Desmoid tumors of pleura, 467 Desmoid-type fibromatosis, 534, 534f genomic applications, 537 Desmoplakin, 75-76
Desmoplasia, 802 Desmoplastic carcinoma differential features of, 779t Desmoplastic fibroblastoma, 79-80, 102 Desmoplastic fibroma of bone, 125 Desmoplastic infantile ganglioglioma, 797f, 799 Desmoplastic malignant melanoma, 202f Desmoplastic medulloblastoma, 802, 802f differential features of, 779t Desmoplastic mesothelioma differential diagnosis of, 477t vs. reactive fibrosing pleuritis, 477, 477t Desmoplastic round cell tumors, 269, 270f Desmoplastic small round cell tumors, 865-868, 866f differentiation from CUPS, 211t-212t immunohistochemical profile, 865-866, 866f immunomarkers for, 91t key diagnostic points, 117b, 868b key features of, 875t in lung, 417 in ovary, 694, 695f in soft tissue and bone, 76, 117, 118f Desmoplastic spindle cell thymoma, 368-369 Desmoplastic trichoepithelioma, 485, 486f Detection, 37t alkaline phosphatase/anti-alkaline phosphatase (APAAP) method, 8-9, 9f avidin-biotin conjugate (ABC) procedure, 8, 8f biotin-avidin methods, 7, 7f biotin-streptavidin (B-SA) method, 8 biotin-streptavidin (B-SA) systems, 8 direct-conjugate–labeled antibody method, 6, 6f representative dilution titration to determine optimal titer of antibody for, 12t titration in, 12, 12t enzyme bridge method, 6, 7f enzyme bridge technique, 6 enzyme-labeled antigen method, 11 indirect-conjugate (sandwich) method, 6, 6f determination of optimal titers for, 12, 14t labeled antigen double stain, 12f labeled antigen method, 12f multistep systems, 37t peroxidase-antiperoxidase (PAP) method, 7, 7f, 9f polymer-based methods, 9-10, 10f
Detection (Continued) polyvalent systems, 8-12 Protein A conjugate method, 11f Protein A-peroxidase-antiperoxidase (PAP) method, 11f titration of primary antibody and, 12-13 unlabeled antibody methods, 6-7 Detection-phase amplification, 36-37 Detection reagents incubation of, 24 Detection systems, 5-14 troubleshooting variables, 31t Dextran-coated charcoal drawbacks with, 744-745 Diagnosis molecular applications, 70-72 Diagnostic immunohistochemistry of GI tract, 510-537 in Hodgkin lymphoma, 139-144 of prostate lesions, 587-606 Diaminobenzidine (DAB), 13, 24, 37t commonly used chromogens or reaction products, 24t Differentiating neuroblastoma, 855-856 Diffuse fibrillary astrocytomas, grade II, 777f Diffuse large B-cell lymphoma (DLBCL), 187f activated–B-cell type, 168 CD5-positive, 169-170, 170f CD30 in, 170 classification of, 168, 169f cyclin D1 in, 170, 171f differential diagnosis of, 177t EBV-associated, of the elderly, 170-171, 171f Epstein-Barr virus in, 170-171 features intermediate between Burkitt lymphoma and, 176 features intermediate between CHL and, 176 germinal center–derived, 168, 169f Digital fibroma/digital fibromatosis infantile, 494-495 Digital imaging, 877 FDA-approved applications, 883t process, 878 Digital Pathology Association, 26-27 Dilution optimal for antibodies, 12 Direct-conjugate–labeled antibody method, 6, 6f representative dilution titration to determine optimal titer of antibody for, 12t titration in, 12, 12t Dirty necrosis, 523, 602 DLBCL. See Diffuse large B-cell lymphoma DNA. See Deoxyribonucleic acid DNA-based tissue identity testing, 54-55, 54f DNA microarrays, 49
Index
DNA sequencing conventional analysis, 46-47, 47f next-generation, 47-48 next-generation sequencing (NGS), 48f paired-end sequencing, 47 pyrosequencing, 47 DNTs. See Dysembryoplastic neuroepithelial tumors DOG-1 in gastrointestinal stromal tumor, 533 Dot-blot analysis, 45-46 “Double”, 20 Double-hit lymphoma, 176 Double (multiplex) staining, 8-12, 9f, 25-27, 25f-27f DPC4 (deleted in pancreatic carcinoma, locus 4). See SMAD4 Driver mutations, 41 DSRCTs. See Desmoplastic small round cell tumors Duct ectasia, 733, 734f Ductal adenocarcinoma pancreatic, 543-545, 543f-544f prostatic, 596-597, 596f Ductal carcinoma cytokeratin 7 in, 390t cytokeratin 20 in, 391t invasive, 753f key diagnostic points, 723 Ductal carcinoma in situ, 712f-713f, 713, 716f, 718-721, 724f-725f Ductal epithelial hyperplasia, 719-721 Ductal epithelial lesions, proliferative, 719-721 Ductal hyperplasia atypical, 719-721, 720f florid, 720f Ductal of pancreas 2 (DUPAN-2), 540, 556, 556f Duodenal gangliocytic paraganglioma, 566 Duodenal neuroendocrine tumors, 529, 529f-530f Dysembryoplastic neuroepithelial tumors, 797-798, 798f Dysgerminoma, 700, 700f-701f, 701t Dysplasia in Barrett esophagus, 510 classification of, 248 extrahepatic, 560 in gallbladder, 560, 560f in head and neck, 245-250 in inflammatory bowel disease, 522 markers of, 848-850 mild, 248 moderate, 248 severe, 248 vs. neoplasms, 828 Dysplastic gangliocytoma, 797
E
E-cadherin, 75-76, 246t-248t Ear tumors, 310-321, 320f Eastern equine encephalitis, 70, 70f EBER. See EBV-encoded small RNAs
Ebola hemorrhagic fever, 68, 70 Ebola virus, 61f, 68 EBV-encoded small RNAs (EBER) in head and neck lesions, 246t-248t in Hodgkin lymphoma, 135, 136f in nasopharyngeal carcinoma, 279-280, 280f in sinonasal tract lesions, 256t in upper aerodigestive tract carcinomas, 249t Eccrine carcinoma, 483f, 506, 507f Ectomesenchymal chondromyxoid tumor, 286-287, 286f, 287b Editing, 878 EDNRB, 607t Effusion cytology, 837-838 EGFR. See Epidermal growth factor receptor Ehrlichia chaffeensis, 68, 72f Ehrlichia ewingii, 68 Ehrlichia muris-like (EML) organism, 68 Ehrlichioses, 68 EIDs. See Emerging infectious diseases ELST. See Endolymphatic sac tumor EMA. See Epithelial membrane antigen Embryonal carcinoma, 702, 702f immunohistochemistry of, 648t immunohistology of, 648b key diagnostic points, 380b markers for, 237b Embryonal rhabdomyosarcoma, 858, 860f-861f extrahepatic, 563 gene-expression analysis of, 861-862 immunomarkers for, 859-861 in sinonasal tract, 267, 267f in soft tissues and bone, 98-99, 99f Embryonal sarcoma, of liver, 583 Embryonal tumors, 801-804 key diagnostic points, 803b rare, 803-804 EMCS. See Extraskeletal myxoid chondrosarcoma Emerging infectious diseases, 67-68 EMM, 202f Emulsion PCR (emPCR), 47 EMZL. See Extranodal marginal zone lymphoma Encephalitis, 769f, 770-771 definition of, 767, 770-771 differential features of, 768t herpes simplex, 767t, 771 perivenous, 768-769 subacute sclerosing panencephalitis, 772 viral, 70 Encephalopathy, 771 progressive multifocal leukoencephalopathy, 773f spongiform, 773-774 Endocervical adenocarcinoma, 662f intestinal-type, 661, 663f invasive, 661, 661b minimal deviation, 662, 663f
899
Endocervical adenocarcinoma in situ, 660, 660b, 661f Endocervical-like (seromucinous) tumors, 690, 690f Endocrine tumors, 358-362 antigens and antibodies in, 322-326 of breast, 361-362, 361f cervical, 358-359 diagnostic pitfalls, 326 gastrointestinal, 352-353, 352f, 352t of head and neck, 339f immunohistology of, 322-362 key diagnostic points, 488b molecular approaches to, 328, 353 pancreatic, 353-355 markers in, 353, 353f molecular diagnostic applications, 355 in prostate, 359-360 pulmonary, 356-358 of skin, 360-361, 487-488 of specific sites, 326-358 of thymus, 362 Endolymphatic sac tumor, 316-319, 318f, 319b Endometrial adenocarcinoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t genomic applications for, 708 Endometrial carcinoma, 667-672 cytokeratin 7 in, 390t cytokeratin 20 in, 391t diagnostic pitfalls, 680-681 DNA mismatch repair proteins in, 680-682, 681f metastatic, 671-672 microsatellite instability testing in, 681-682, 682f mismatch repair proteins in, 682f secondary, 671 staging, 672 subtypes, 671 synchronous with ovarian, tubal, and peritoneal carcinomas, 672-673 theranostic applications, 682-683 Endometrial clear cell carcinoma, 667t, 671 Endometrial serous carcinoma, 668-670, 669f-670f key diagnostic points, 671b key differential diagnosis, 667t, 671t Endometrial stromal nodules, 673-675, 674f Endometrial stromal sarcoma, 674f differentiation from CUPS, 211t-212t low-grade, 673-675 Endometrial stromal tumors, 675f key diagnostic points, 675b mixed, 674-675 Endometrial tumors, mesenchymal, 676-678
900
Index
Endometrioid adenocarcinoma, 667-668 key diagnostic points, 668b key differential diagnosis, 667t, 671t markers of emerging importance, 668f müllerian vs. colon adenocarcinoma, 525 vs. colorectal adenocarcinoma, 526f Endometrioid carcinoma, 691, 691f key diagnostic points, 668b vs. sex cord-stromal tumors, 691t Endometrioid serous carcinoma, 668f Endometrioid tumors, 690-692 Endothelial differentiation markers, 80-83 Enhanced polymer one-step staining (EPOS) system (Dako), 10 Enolase muscle (β-β), 323 neuron-specific. See Neuron-specific enolase neuronal (γ-γ), 323 nonneuronal (α-α), 323 Entamoeba histolytica, 66 Enterogenous cysts, 816t, 819 Enteropathy-type T-cell lymphoma, 178f, 182, 182f Enterovirus 71 (EV71), 67-68, 68f Enterovirus 71 encephalomyelitis, 67-68 Enzymatic (acinar) markers, 541 Enzyme bridge method, 6, 7f Enzyme bridge technique, 6 Enzyme digestion, 2 Enzyme-labeled antigen method, 11, 12f Enzymes, 322-323 Eosinophilic granular bodies, 782 Eosinophilic granuloma, 413-414, 493 Ependymal cysts, 816t, 818 Ependymoblastoma, 793t, 804 Ependymomas, 785-789, 786f anaplastic, 776t-777t, 789 clear cell, 787 differential features of, 766t, 776t-777t low-grade, 787 malignant, 766t, 789 myxopapillary, 766t, 785-789, 786f subependymoma, 787 tanycytic, 776t-777t, 787 Epidermal growth factor receptor (EGFR, HER-1, c-erbB-1), 833t in breast carcinoma, 728-729, 729f, 760 in glioblastoma, 795, 795f in head and neck lesions, 246t-248t, 250 in lung cancer, 432-433, 434t in phyllodes tumor, 741, 744f in prostate basal cell layer, 587t in skin carcinoma, 507, 507f Epidermal tumors, 479-481 Epidermoid cysts, 816t, 819 Epidermotropic infiltrates, 492
Epilepsy, 822-824 confirmatory features of, 767t temporal lobe, 822 Epithelial atypia, 560 Epithelial cell-adhesion molecule, 587t, 633 Epithelial differentiation, 208 Epithelial lesions anal, 526-527 appendiceal, 520-521 of esophagus, 510-513 gastrointestinal, 510-527 anatomic molecular diagnostic applications to, 539 genomic applications, 537 theranostic applications to, 537-538 proliferative ductal, 719-721 of small intestine, 519-520 of stomach, 513-519 Epithelial-lined cysts, 816t Epithelial markers in neuroendocrine carcinoma, 263f in pancreas, 540 supplemental, 220-223 in tumors of soft tissue and bone, 75-76 used with postfixation, 833t in uterus, 664-666 Epithelial membrane antigen, 833t in adenoid cystic carcinoma, 299f, 300 in adrenocortical tumors, 346f in angiosarcomas, 96 in bladder tumors, 625t in brain tumors, 817-818, 817f in breast carcinoma, 725-728, 728f in cancer of unknown primary site, 222-223, 223t in cholangiocarcinoma, 580f in clear cell carcinoma, 692-693 in cutaneous perineurioma, 501, 503f in desmoplastic small round cell tumor, 865-866, 866f-867f in dysgerminoma, 700 in embryonal carcinoma, 702 in endometrioid tumors, 690-692, 691f, 691t in female adnexal tumors, 708 in gastric adenocarcinoma, 516t in granulosa cell tumors, 696-697, 696t in gynecologic pathology, 654t in hepatocellular carcinoma, 576, 577f in Hodgkin lymphoma, 134, 135f, 138f, 138t key diagnostic points, 223b in Leydig cell tumors, 699 in lung neoplasms, 388t-389t, 395f, 416f, 416t, 427t in malignant rhabdoid tumor, 869, 869f-870f in mediastinal hematopoietic tumors, 382-384 in melanocytic neoplasms, 190, 191f in meningioma, 805-806, 805f-806f
Epithelial membrane antigen (Continued) in mesothelioma, 444t-446t, 448f, 453t, 455, 455f, 462f, 468t469t, 476t in nervous tissue, 765t in nonepithelial tissues, 222, 223t in ovarian tumors, 693-694 in pancreatic ductal adenocarcinoma, 543f in papillary thyroid carcinoma, 330f in perineurioma, 111-113, 112f in pleural neoplasms, 448t in sclerosing hemangioma, 415, 416f in Sertoli cell tumors, 698-699 in Sertoli-Leydig cell tumors, 697-698 in sex cord tumors, 699 in sinonasal tract tumors, 256t in skin carcinomas, 489f in spindle cell tumors, 92t, 253-254, 253f in steroid cell tumors, 699 in synovial sarcoma, 117-118, 119f in tumors of soft tissue and bone, 75, 91t in upper aerodigestive tract carcinomas, 249t in uterus, 664-666 in yolk sac tumors, 701-702 Epithelial membrane antigen E29, 246t-248t Epithelial mesothelioma antibodies in, 468-469, 468t cytokeratin profile, 454t differential diagnosis of, 237, 239b, 468t histologic variants or subtypes, 438, 438b, 439f-443f immunohistochemical features of, 453-455, 453t, 455f, 469t, 476t immunohistochemical tests in, 470t immunohistology of, 447, 448f markers in, 452t mucin-positive, 469-471, 471f well-differentiated papillary, 472-473, 473f Epithelial metaplasia glioblastoma/gliosarcoma with, 779t gliosarcoma with, 795 Epithelial-myoepithelial carcinoma, 300f-302f, 301-302, 302b Epithelial-myoepithelial neoplasms, 417 Epithelial neoplasms CK5/6 expression in, 217t CK7 expression in, 215b, 215t CK20 expression in, 215b, 216t extrahepatic, 559-562 thymic, 365-371 Epithelial tumors choroid plexus, 799-800 cytokeratins in, 688f key diagnostic points, 480b, 687b ovarian and fallopian tube neoplasms, 687-694, 688f renal, 633b of skin, 479-488
Index
Epithelioid cell tumors, 763f, 766t Epithelioid fibrosarcoma sclerosing, 88, 105, 105f, 494-495 in skin, 494-495 Epithelioid fibrous histiocytoma, 199, 200f Epithelioid hemangioendothelioma, 94-95, 96f, 583, 583f mediastinal, 380t mimicking mesothelioma, 466 pseudomesotheliomatous, 466, 466f-467f Epithelioid inflammatory myofibroblastic sarcoma, 86 Epithelioid leiomyosarcoma, 109 Epithelioid mesothelioma differential diagnosis of, 475t immunohistochemical features of, 454t malignant, 448t markers for, 471t, 475t mucin-expressing and with crystalloid features, 464-468 Epithelioid rhabdomyosarcoma, 101, 859 Epithelioid sarcoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t differentiation from CUPS, 208, 211t-212t differentiation of, 208, 211t-212t key diagnostic points, 120b in skin, 505-506 in soft tissue and bone, 119-120, 120f of subcutis, 505-506, 505f Epithelioid sarcoma–like hemangioendothelioma, 504, 505f Epithelioid tumors algorithmic approaches to, 238f-239f malignant PNSTs, 114 trophoblastic, 679-680 uterine, 673t Epitope retrieval solutions, 22 Epitopes (antigen determinants), 3, 18 EPOS system. See Enhanced polymer one-step staining system Epstein-Barr virus, 59, 60f, 70-72 anti-EBV therapy, 145 –associated DLBCL of the elderly, 170-171, 171f –associated gastric carcinoma, 539 –associated smooth muscle tumors, 109, 110f in diffuse large B-cell lymphoma, 170-171, 171f in Hodgkin lymphoma, 135, 136f, 138f, 138t, 141t, 177t in lung lymphomas, 410t in non-Hodgkin lymphoma, 154-156, 159f Erasmus University Medical Center, 756
ERBB2, 833t, 851 -enriched breast carcinoma, 754t -enriched tumors, 755 analytic issues, 749 in breast cancer, 748-750, 752, 753f in endometrial carcinoma, 682-683 in extramammary Paget disease of skin, 482-483, 482f FISH assays, 750-752, 752b in gastric and gastroesophageal adenocarcinoma, 538 guidelines for testing, 15 in head and neck lesions, 246t-248t immunohistochemistry of, 750b ISH assays, 750-752 in mesothelioma, 444t-446t in Paget disease of nipple, 732, 733f postanalytic issues, 749-750 preanalytic issues, 749 ERBB2 breast carcinoma, 752-754 Erdheim-Chester disease, 128, 413, 815t ERG. See Avian V-ETS erythroblastosis virus E26 oncogene homolog ERMS. See Embryonal rhabdomyosarcoma Esophageal adenocarcinoma, 510-511, 511b Esophageal carcinoma basaloid-patterned, 513f with spindle cell or mesenchymal differentiation, 513 Esophageal melanoma, 535, 535f Esophageal squamous cell carcinoma, 511-512, 512f cytokeratin reactivity in, 511, 511f key diagnostic points, 512b Esophagus epithelial lesions of, 510-513 neuroendocrine tumors of, 528, 528b Estrogen receptor, 833t, 841f-842f, 846, 846f-847f in breast tumors, 720f in cancer of unknown primary site, 228-229 in colorectal adenocarcinoma, 524t, 526f in cutaneous neoplasms, 506, 507f in gastric adenocarcinoma, 515-516 key diagnostic points, 229b in lung neoplasms, 389t in mesothelioma, 444t-446t quantitative measurement of, 878f in small cell carcinoma, 358t in thyroid carcinoma, 330f, 338f in uterine tumors, 654t, 667t, 671t, 673-674, 674f, 682-683 ETV1-4, 607t EvaGreen, 44 Ewing sarcoma/PNETs. See also Primitive neuroectodermal tumors in children and adolescents, 862-865 differential diagnosis, 865 differentiation from CUPS, 211t-212t extraskeletal, 210f in head and neck, 271f
901
Ewing sarcoma/PNETs (Continued) immunohistochemical profile, 863f immunomarkers for, 91, 91t key diagnostic points, 82b, 127b, 272b, 865b key features of, 875t sinonasal, 256t, 270-272 in soft tissue and bone, 74, 79, 126-127, 126f Exons, 40 Exserohilum rostratum, 771 Extraadrenal paraganglia, 348-349 Extrahepatic bile duct tumors, 583 Extrahepatic biliary tract, 559-563 dysplasia in, 560 epithelial neoplasms in, 559-562 intraductal neoplasms, 559-560 invasive adenocarcinoma of, 560-561 invasive carcinoma of, 561-562 neuroendocrine neoplasms in, 562-563 nonepithelial neoplasms in, 563 Extramedullary myelogenous leukemia (granulocytic sarcoma), 385 Extranodal marginal zone lymphoma, 163-165 features of, 155t, 165f genetic abnormalities in, 163, 165t Extranodal natural killer/T-cell lymphoma, nasal type, 181-182, 181f, 256t Extraosseous plasmacytoma, 385 Extraskeletal myxoid chondrosarcoma, 122-123
F
β-F1, 153 Factor VIII, 775 Factor VIII antigen, 444t-446t, 577f Factor VIII-related antigen in head and neck lesions, 246t-248t in mediastinal neoplasms, 380t in tumors of soft tissue and bone, 80 Factor XIIIa in hepatocellular carcinoma, 576 in skin tumors, 496-497, 498f-499f Factor XIIIA, 84 Fallopian tube neoplasms, 706-708 carcinoma, 683-684 epithelial tumors, 687-694 key diagnostic points, 708b mucinous tumors, 688-690 serous tumors, 687-688, 688f-689f theranostic applications for, 708-709 Fallopian tubes, 683-706 Familial insomnia, fatal, 773 Fas (CD95), 134 Fascin in Hodgkin lymphoma, 135-136, 136f, 138t, 140f, 141t in nonneuroendocrine lung neoplasms, 388t-389t Fast blue BB, 24t Fast red TR, 24t Fat-forming solitary fibrous tumor, 103-104
902
Index
Fatal familial insomnia, 773 FATWO. See Female adnexal tumor of wolffian origin Faux tissues, 29-30, 30f Female adnexal tumor of wolffian origin, 708 Female genital tract, 653-709 Female reproductive system neoplasms, 428t Fetal lung, 356f FFPE tissue. See Formalin-fixed paraffin-embedded tissue Fibrillar cell masses, 776t-777t, 779f Fibrillar cell walls: cysts with, 818, 819t Fibroadenoma, 739-742, 741f-742f Fibroblastic osteosarcoma, 92t Fibroblastic polyps, benign, 537 Fibroblastic sarcoma, myxoinflammatory, 105-106 Fibroblastic tumors gastric lesions, 537 skin neoplasms, 494-495 in soft tissue and bone, 102-107 Fibroblastoma, desmoplastic, 79-80, 102 Fibroepithelial lesions, 742 Fibroepithelial tumors, 739-742, 742b Fibrohistiocytic differentiation, 199-200 Fibrohistiocytic lesions indeterminate, 496-497, 499f of unknown malignant potential, 499f Fibrohistiocytic markers, 83-84 Fibrohistiocytic neoplasms, 495-498, 498b Fibrolamellar hepatocellular carcinoma, 579 Fibroma, 125, 695-699 Fibromatosis, 79-80, 102 desmoid, 102, 102f desmoid-type, 534, 534f, 537 immunomarkers of, 92t Fibromyxoid sarcoma, low-grade, 79-80, 88, 92t, 104f Fibromyxoid tumors, 76 ossifying, 78-79, 88, 117 sarcoma, low-grade, 79-80, 88, 92t, 104-105, 104f Fibronectin 1, 334t, 335 Fibroplastic meningioma, 776t-777t, 805-806 Fibrosarcoma, 811 differential features of, 776t-777t infantile, 107 sclerosing epithelioid, 88, 105, 105f of skin, 495, 496f Fibrosing pleuritis differential diagnosis of, 477t reactive, 477 Fibrosis in brain tissue, 769-770, 769f differential features of, 776t-777t Fibrous histiocytoma, 76 angiomatoid, 106 malignant, 776t-777t myxoid malignant, 107
Fibrous meningioma, 776t-777t, 805-806, 806f Fibrous pseudotumor, calcifying, 466-467 Fibrous tumors. See also Solitary fibrous tumors of bone, 125 markers in, 806f pleural, 465t Fibroxanthoma, atypical, 498, 500f Filamentous proteins, 189 Fine needle aspiration, 42 FISH. See Fluorescent in situ hybridization 5-gene index, 758t Fixation, 832 definition of, 15 false-negative results with, 832, 834f inadequate, 32t troubleshooting variables, 31t Fixation artifacts, 32f-33f Fixatives, 20 Flexner-Wintersteiner types, 255-258 Fli-1. See Friend leukemia integration 1 (FLi-1) Floating neurons, 797-798 Fluorescent in situ hybridization (FISH), 48-49 benefits and limitations of, 751, 751t for determining site of origin, 242t ERG fusion analysis, 608, 609f HER2 assays, 750-752, 752b FMC7, 155t FN1, 335 Focal nodular hyperplasia, 571-572, 573t Follicle-stimulating hormone, 246t248t, 265-266, 266f Follicular adenoma, 329 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t marker expression in, 334t Follicular bronchitis/bronchiolitis, 408 Follicular carcinoma, 240-241, 329-336 cytokeratins in, 331t genetic alterations in, 340t marker expression in, 334t well-differentiated, 331, 331f, 333, 333f Follicular cells, 329-336 Follicular lymphoma, 160 diagnostic pitfalls, 160 features of, 155t, 157f grade 1 to 2/3 with increased proliferation rate, 160 of lung, 410t, 411 prognostic and therapeutic studies, 160 of skin, 492, 492f Folliculostellate cells, 326-327 Food and Drug Administration (FDA), 18, 832-834 Formalin, 19-20, 35 Formalin-fixed paraffin-embedded (FFPE) tissue, 1 specimen requirements, 42
FOXA1, 761 FOXL2 in endometrioid carcinoma, 691t in granulosa cell tumors, 696-697, 696t in ovarian and tubal tumors, 685 in Sertoli-Leydig cell tumors, 697-698 FOXN1, 369f-370f in thymic carcinoma, 369-370 Fresh- or snap-frozen tissue, 42 specimen requirements, 42 Friend leukemia integration 1 (FLi-1), 855 in Ewing sarcoma/PNETs, 863, 863f-864f in head and neck lesions, 246t-248t, 256t, 263-264, 264f in solid-pseudopapillary neoplasms, 554f in tumors of soft tissue and bone, 81 Frontotemporal dementia, 772-773 Frozen tissue sections, 42 Fundic gland polyps, 514 Fungal infections, 65-66. See also specific infections Fusarium, 66 FXIIIA. See Factor XIIIA
G
Galectin, 145 Galectin-1, 137 Galectin-3 (GAL-3) in medullary thyroid carcinoma, 338f in thyroid tumors, 330f, 334-335, 334t, 335f Gallbladder, 559-563 carcinoids in, 562, 562f dysplastic, 560, 560f intracholecystic papillary-tubular neoplasms of, 559, 559f mucinous carcinoma in, 561-562 neuroendocrine neoplasms in, 562 Gangliocytic paraganglioma, 566 Gangliocytoma, 796 dysplastic, 797 Ganglioglioma, 796, 796f anaplastic, 796 desmoplastic infantile, 797f, 799 Ganglion cell neoplasms differential features of, 776t-777t, 779t identification and evaluation of, 795-796 Ganglioneuroblastoma, 123, 795, 799, 855 intermixed, 855-856 nodular, 855-856 Ganglioneuroma, 536f, 855 Gardner fibroma, 102 Gastric adenocarcinoma, 515-516 antibodies in, 516t CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t choriocarcinomatous differentiation in, 517-518
Index
Gastric adenocarcinoma (Continued) cytokeratins in, 515, 515f, 517f hepatoid differentiation in, 517-518 Her2 immunohistochemistry, 538 intestinal (glandular) type, 517f key diagnostic panels, 518-519 key diagnostic points, 515b neuroendocrine differentiation, 518 spindle cell differentiation, 517 theranostic applications, 537-538 variants, 516-518, 519b vs. lung adenocarcinoma, 519 vs. pancreatobiliary adenocarcinoma, 519 yolk sac differentiation, 517-518 Gastric carcinoma Epstein-Barr virus–associated, 539 lymphoepithelioma-like, 518f Gastric cardia, 510 Gastric neuroendocrine tumors, 528, 528b Gastric pyloric glandular mucin, 541 Gastric signet-ring cell carcinoma, 517f, 518-519, 519f Gastric-type mucins, 541 Gastric-type papillae, 549-550, 549f Gastrin, 352t Gastrin-releasing peptide, 356, 356f-357f Gastrinomas, 354, 354f, 556 Gastritis atrophic, 513-514 autoimmune, 513-514, 514f lymphocytic, 513 Gastroenteropancreatic tumors, 355 Gastroesophageal adenocarcinoma, 538 Gastrointestinal endocrine tumors, 352, 352f, 352t Gastrointestinal markers, 843 Gastrointestinal stromal tumors, 78-79, 532-534, 533f CD117 staining in, 532-533 differentiation from CUPS, 211t-212t DOG-1 staining in, 533 gastric, 533f genomic applications, 537 key diagnostic points, 534b prostatic, 603t, 605-606, 605f uterine, 676-678 Gastrointestinal tract anatomic molecular diagnostic applications to, 539 antigens in, 508-510 assays for antibodies in, 509t CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t clear cell sarcoma–like tumor of, 121 diagnostic immunohistochemistry of, 510-537 epithelial lesions in, 510-527 anatomic molecular diagnostic applications to, 539 genomic applications, 537 theranostic applications, 537-538
Gastrointestinal tract (Continued) genomic applications to, 537 immunohistology of, 508-539 mesenchymal lesions in, 532-537 anatomic molecular diagnostic applications to, 539 theranostic applications, 538-539 neuroendocrine lesions in, 527-532, 537 theranostic applications to, 537-539 GATA binding protein 3 (GATA3) in breast carcinoma, 761 in prostate carcinoma, 621t in urothelial carcinoma, 617, 621t GCDFP. See Gross cystic disease fluid protein GCTs. See Germ cell tumors Gemistocytic astrocytoma, 782-783 differential features of, 776t-777t swollen GFAP-positive cells in, 826t Gene-expression profiling in breast carcinoma, 758t in Hodgkin lymphoma, 144 markers identified through, 88 of rhabdomyosarcoma, 861-862 Genes, 40, 40f. See also Oncogenes; specific genes Genetic Imagery Exploitation (Genie), 878-879 Genetic polymorphisms, 41 PCR-RFLP analysis, 45 single-nucleotide, 45 single-strand conformation, 45 Genital tract, female, 653-709 Genitourinary tract neoplasms, 428t Genomic Health, 747-748, 758t Genomics, integrated, 610-611 Genoptix, 878-879 GEP. See Gastroenteropancreatic tumors Germ cell markers, 431 Germ cell tumors, 699-706 CD2, 3, 4, 5, 7, 8, 56, and 138 in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t extratesticular, 647-648 global methylation in, 649-651, 651f immunohistology of, 648b intracranial, 812 intratubular neoplasia, 645-646, 648t key diagnostic points, 700b lymphoma mimics, 185t malignant, 699 markers for, 185t, 236, 237b mediastinal, 379-380, 382t metastatic, 647-648 mixed, 701-702, 702f nonseminomatous, 646-647 pathogenesis of, 649, 650f testicular, 645-648 Germinal center markers, 134 Germinomas, 812 Germline mutations, 41 Gerstmann-Sträussler-Scheinker syndrome, 773
903
Gestational trophoblastic disease, 678-680, 683 GFAP. See Glial fibrillary acidic protein Giant cell astrocytoma, 784, 784f differential features of, 776t-777t subependymal, 784 Giant cell carcinoma of lung, 426, 427t osteoclastic, 562 Giant cell glioblastomas, 793f-794f Giant cell tumors of bone, 128 of soft tissue, 128 of tendon sheath, 128 Giant cells, osteoclast-like, 546-547 Giardia intestinalis, 58t Giardia lamblia, 66 GISTs. See Gastrointestinal stromal tumors Gitter cells, 766t, 767, 769f Glandular/ductal markers, 540-541 Glandular psammomatous carcinoid, 565-566, 566f Glial cysts, 818, 819t Glial fibrillary acidic protein in brain tumors, 781f, 783, 783f, 786f, 788f, 792, 792f, 796, 797f in breast carcinoma, 731t diagnostic pitfalls with, 825 in glial heterotopia, 310, 310f in glioblastoma, 793f in gliosarcoma, 794f in gliosis, 767, 768f in head and neck lesions, 246t-248t in nervous tissue, 765t in olfactory neuroblastoma, 258259, 258f in oligodendroglioma, 793f in pheochromocytoma, 347f in pituitary adenoma, 328f in sinonasal tract tumors, 256t in tumors of soft tissue and bone, 73, 75 types of swollen cells with, 826t Glial heterotopia, 310, 310b, 310f Glioblastoma, 779f, 790f, 791-795, 793f anatomic molecular diagnostic applications, 795 with epithelial metaplasia, 779t giant cell, 793f-794f key diagnostic points, 794b markers in, 795f primary, 795 secondary, 795 theranostic applications, 795 Glioblastoma multiforme, 776t-777t, 791-795 Gliomas, 778-795, 779f infiltrating vs. noninfiltrating cells, 827 infiltration into brain, 778, 780f key diagnostic points, 778b low grade, 778 malignant, 201-202 tumor, 778-780 tumor margin, 768t, 778-780, 780f vs. gliosis, 825-827, 826f
904
Index
Glioneuronal tumors, 797f, 798-799 Gliosarcoma, 792-794, 794f, 811 differential features of, 776t-777t with epithelial metaplasia, 779t, 795 Gliosis, 767, 768f, 779f-780f cavitary, 819t differential features of, 768t in epilepsy, 822, 823f-824f reactive, 826t trapped, 790f vs. glioma, 825-827, 826f Glomangiopericytoma, 272, 275-277, 275f, 277b Glomus tumors, 378, 379f, 380t Glucagon, 352t Glucagonoma, 556 Glucose oxidase method, 24t Glucose transporter type 1 (GLUT-1), 840 in epithelial mesothelioma, 476t in serous cystadenoma, 551-552, 552f in tumors of soft tissue and bone, 82, 94 Glutamine synthetase, 573t Glutathione S-transferase alpha (GST-α), 634-635 Glutathione S-transferase pi (GSTP1), 587t, 606f, 607t p-Glycoprotein, 476t Glycoprotein 36 (gp36), 83 Glycoprotein 100 (gp100) antibodies, 193-195, 195b Glycoprotein 130 (gp130), 574 Glycoprotein 200 (gp200), 234 Glycoproteins mucin-related, 540-541 myelin-associated, 79 Glypican-3 in clear cell carcinoma, 692-693 in gynecologic pathology, 654t in hepatocellular carcinoma, 575, 575f, 577f in mediastinal germ cell tumors, 382t in ovarian and tubal tumors, 686 in testicular tumors, 645, 648t in yolk sac tumors, 701-702 Goblet cell carcinoid, 530, 531f Goblet cells, 549f Goblet-type mucin, 541 Gonadoblastoma, 703 Granular cell tumors congenital, 113 cutaneous, 202 extrahepatic, 563 in GI tract, 534 in head and neck, 284-285, 284f key diagnostic points, 285b in lung, 417 primitive nonneural, 113 in skin, 501, 502f in soft tissue and bone, 113 vulvar, 656, 656f Granular cells, 767 Granule proteins, 323-324 Granulocytic epithelial lesion–forming pancreatitis, 568
Granulocytic sarcoma, 385 Granuloma differential features of, 776t-777t eosinophilic, 413-414, 493 Granulomatosis, lymphomatoid, 411-412, 412f, 813 Granulomatous lymphadenitis, 144, 144f Granulosa cell tumors, 694, 696-697 adult type, 694, 696, 697f differential diagnosis, 696t juvenile type, 694, 697, 697f Granzyme B, 134-135, 153 Gray-zone lymphoma, 176, 382-384 Gross cystic disease fluid protein in breast carcinoma, 738-739 in cancer of unknown primary site, 227-228, 228f in extramammary Paget disease of skin, 482-483, 482f key diagnostic points, 228b in lung neoplasms, 389t in vulvar Paget disease, 653-655, 655f Gross cystic disease fluid protein 15 (GCDFP-15), 846-847 in breast adenocarcinoma, 846f in colorectal adenocarcinoma, 524t in gastric adenocarcinoma, 516t in gynecologic pathology, 654t in mesothelioma, 444t-446t Groucho, 88 Growth hormone, 246t-248t, 328f GSS syndrome. See GerstmannSträussler-Scheinker syndrome GST-α. See Glutathione S-transferase alpha Guanine (G), 39 Guillain-Barré syndrome, 822, 822f Gynecologic cytology markers, 848 Gynecologic tumors antibodies in, 654t molecular diagnostic applications, 709
H
H score (histochemical score), 747-748, 747f Haemophilus influenzae, 65, 70-72 Hairy cell leukemia, 155t, 165-166, 166f Hairy cell leukemia variant, 167 Hallmark cells, 183 Hamartoma, mesenchymal, 583 Hanker-Yates reagent, 24t Hantavirus, 67, 67f Hantavirus pulmonary syndrome, 67, 67f HASH-1. See Human achaete-scute complex homolog 1 Hassall corpuscles, 364, 364f HBME-1. See Hector Battifora mesothelial cell 1 HCL. See Hairy cell leukemia Head and neck definition of, 245 dysplasia in, 245-250
Head and neck lesions, 245 antibodies for diagnosis of, 246t-248t endocrine tumors, 339f immunohistology of, 245-321 metastatic tumors, 319-321 key diagnostic points, 320b squamous cell carcinoma, 850f molecular markers for, 277-278 squamoproliferative, 245-254 squamous cell carcinoma, 245-250 Heat-induced epitope retrieval, 2 Heating conditions, 20-21, 32t Hector Battifora mesothelial cell 1 (HBME-1), 838 in mesothelioma, 448f, 452t-453t, 453-455, 455f, 459t, 468t-470t in pituitary adenoma, 328f in pleural neoplasms, 448t in thyroid tumors, 330f, 334-335, 334f, 334t Hector Battifora mesothelial cell 2 (HBME-2), 459t Helicobacter pylori, 58t, 62-63, 63f in extranodal marginal zone lymphoma, 163-165, 165t gastric, 513, 513f immunohistochemical staining for, 163-165, 166f Helix pomotia agglutinin (HPAgg), 458 Hemangioblastoma, 814-816 anatomic molecular diagnostic applications, 815-816 capillary, 814-816 cerebellar, 814-815, 815f differential features of, 779t, 819t Hemangioendothelioma, 504 epithelioid mimicking mesothelioma, 466 pseudomesotheliomatous, 466, 466f-467f pulmonary, 406, 406f-407f in soft tissue and bone, 94-95, 96f epithelioid sarcoma–like, 504, 505f Kaposiform, 94 pseudomyogenic (epithelioid sarcoma-like), 94, 95f pulmonary, 406 Hemangioma, 91-94 mediastinal, 380t sclerosing, 415, 415f-416f Hemangiopericytomas, 809 congenital, 494-495, 496f differential features of, 793t, 805t meningeal, 809, 810f sinonasal type, 275-277 Hematologic disorders. See also specific disorders antigens for evaluation of, 148-153 important markers of, 151-152 Hematologic specimens, 42 Hematopoietic markers, 191-192 Hematopoietic neoplasms of CNS, 812-814 mediastinal tumors, 383t Hematoxylin and eosin (H&E) slides, 42
Index
Hemorrhage, brain, 768t Hemorrhagic fevers, viral, 60-61, 70 Hemosiderin, 473, 474f Hendra virus encephalitis, 68 Heparan sulfate proteoglycan (CD44v6), 334t Hepatic adenoma, 573-574 CD34 in, 221, 222f Hepatic clear cell carcinomas, 236b Hepatic interstitium, normal, 569 Hepatic parenchyma, normal, 568-569 Hepatic tissue, benign, 580 Hepatitis B, 57 Hepatitis B core antigen (HBcAg), 57, 58t, 571, 571f Hepatitis B surface antigen (HBsAg), 57, 58t, 571, 571f Hepatitis B virus, 57 Hepatitis C virus, 62 Hepatoblastoma, 582-583 Hepatocellular adenoma, 573t, 574 Hepatocellular carcinoma, 574-579, 574f-576f antibodies in, 576, 577f biliary-type differentiation in, 579 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t CD34 in, 221, 222f CEA in, 221, 221f clear cell, 579 combined with cholangiocarcinoma, 579-580 cytokeratin 20 in, 391t differential diagnosis, 580 fibrolamellar, 579 genomic applications, 576-579 HepPar1 in, 231, 232f key diagnostic points, 580b markers in, 576 molecular applications, 576-579 spindle cell, 579 variants, 579-580 vs. benign hepatic tissue, 580 vs. cholangiocarcinoma, 580, 582 vs. metastatic lesions, 580 Hepatocyte nuclear factor 1β (HNF1β), 654t, 692-693, 692f, 848 Hepatocyte paraffin 1 (HepPar1), 231-232, 232f in gastric carcinoma, 516t, 519f in hepatocellular carcinoma, 574-575, 575f, 577f in lung neoplasms, 389t Hepatocytes, normal, 568-569 Hepatoid carcinoma, 562 Hepatoid differentiation, 517-518 Hepatoid thymic carcinoma, 371 Hepatoma, 390t Hepatosplenic T-cell lymphoma, 178f, 183 HER1. See Epidermal growth factor receptor HER2 Hercept test (Agilent/Dako), 30 HER2/neu. See ERBB2 Herceptin (trastuzumab), 851
Hereditary nonpolyposis colon cancer syndrome (Lynch syndrome), 524f Hereditary nonpolyposis colorectal cancer, 709 Herpes simplex encephalitis, 767t, 771 Herpes simplex virus, 57, 59-60, 59f, 851f Herpes simplex virus 1 (HSV-1), 57, 58t Herpes simplex virus 2 (HSV-2), 57, 58t Herpesvirus antigen, 765t Herpesviruses, 57-59 Heterotopia, glial, 310 Heterotopic neuroglial tissue, 310 HHF-35. See Muscle-specific actin HIER. See Heat-induced epitope retrieval High-grade (poorly differentiated) neuroendocrine carcinoma. See also Small cell carcinoma in colon, 532, 532f esophageal, 513f in GI tract, 531-532 key diagnostic points, 532b Hilus cell tumors, 699 Hippocampus, 822, 823f cornu ammonis, 823f cornu ammonis region 1, 822, 823f-824f neuronal loss in, 822, 823f-824f Hirschsprung disease, 522 Histaminase (diamine oxidase), 323 Histiocytic markers, 83-84 Histiocytic proliferations, 199-200 Histiocytic tumors, 380 Histiocytoid carcinoma, 725 Histiocytoma, fibrous, 76 malignant, 107, 776t-777t Histiocytosis benign cephalic, 493 of central nervous system, 813-814, 814f, 815t differential features of, 776t-777t regressing atypical, 493-494 regressive atypical, 493 Histiocytosis X pulmonary, 413-414 of skin, 493, 494f Histochemical score (H score), 747-748, 747f Histoids, 29-30, 30f Histoplasma capsulatum, 65 HistoQuant (3DHistech), 878-879 HistoQuest (TissueGnostics), 878-879 HistoRx, 878-879 HIV. See Human immunodeficiency virus HL. See Hodgkin lymphoma HMB-45. See Human melanoma black 45 HMFGPs. See Human milk fat globule proteins HNF1A gene, 574 Hodgkin-like cells, 143
905
Hodgkin lymphoma, 130 anatomic molecular diagnostic applications, 144-145 antibodies in, 130-139, 138f antigens in, 130-137, 138f biologic markers, 136-137, 137t characteristics of, 130 classic, 130, 131f-132f in CLL, 143 diagnostic algorithm for, 146f diagnostic immunohistochemistry, 139-140, 140f differential diagnosis of, 141t, 177t features intermediate between DLBCL and, 176 lymphocyte-depleted type, 130 lymphocyte-rich type, 130 mixed cellularity type, 130 nodular sclerosis type, 130 diagnostic immunohistochemistry, 139-144 differential diagnosis of, 141t granulomas in, 144, 144f immunohistology of, 130-147 immunostaining pitfalls, 138-139 macrophages in, 145 mediastinal, 384 molecular anatomic pathology of, 144 nodular lymphocyte-predominant, 130, 132f-133f diagnostic algorithm for, 146f differential diagnosis of, 141t prognosis for, 145 pseudoneoplastic look-alikes, 143-144 pulmonary, 410t, 411 syncytial, 188t, 384 theranostic applications, 145 Hodgkin lymphoma-associated antigens, 138t Hodgkin/Reed-Sternberg cells, 135f-137f, 139f in Hodgkin lymphoma, 132, 132f-134f new biologic markers for, 137t Hodgkin/Reed-Sternberg–like cells, 140-141, 142f-143f, 143 Hologic, 829 Home brewing, 13, 13t Homeobox, 229 Homeodomains, 229 Homer-Wright rosettes, 787f Honolulu Consensus Document, 568 Hormonal therapy antiandrogen therapy, 594-595, 594f posttherapy histology, 595b Hormone receptors Allred score, 747, 747f analytic variables, 746 in breast carcinoma, 744-748 in cancer of unknown primary site, 228-229 histochemical score (H score), 747-748, 747f key diagnostic points, 229b, 748b in lung neoplasms, 430-431
906
Index
Hormone receptors (Continued) postanalytic interpretation, 746-748 preanalytic variables, 745-746 in thyroid tumors, 336 Hormones in endocrine tumors, 322 in neuroendocrine lung neoplasms, 401b Horseradish peroxidase (HRP), 1 HOXB13:IL17RB index, 757-758 HPAgg. See Helix pomotia agglutinin HRP. See Horseradish peroxidase HRPT2 gene, 344 Human achaete-scute complex homolog 1 (HASH-1), 425 Human B-lymphocyte antigen, 444t-446t Human chorionic gonadotropin (hCG) in choriocarcinoma, 702 in dysgerminoma, 700 in gynecologic pathology, 654t in ovarian and tubal tumors, 686-687 in testicular tumors, 645, 648t Human chorionic gonadotropin alpha (hCGα), 353f, 354, 355f Human chorionic gonadotropin beta (hCGβ), 382t Human epithelial antigen, 388t-389t, 444t-446t Human epithelial-related antigen, 444t-446t Human herpesvirus 6, 59 Human herpesvirus 8, 57-58, 59f diagnosis of, 58t in head and neck lesions, 246t-248t Human herpesvirus 8 latent nuclear antigen 1, 504, 504f Human immunodeficiency virus (HIV), 57-58, 67f, 772. See also Acquired immunodeficiency syndrome (AIDS) Human immunodeficiency virus p24 antigen, 772, 772f Human leukocyte antigen, 444t-446t Human leukocyte antigen-DR (HLA-DR), 249t Human melanoma black 45 (HMB45), 833t in clear cell tumors, 121, 121f, 414, 414f commercial distribution of, 194 in gynecologic pathology, 654t in head and neck lesions, 246t-248t in lung neoplasms, 427t in melanocytic neoplasms, 194, 194f, 202f in mucosal melanoma, 260-262, 261f in renal angiomyolipoma, 640, 640f in sinonasal tract tumors, 256t in tumors of soft tissue and bone, 84-85, 109-110, 111f, 121, 121f Human melanoma black 50 (HMB50), 194
Human milk fat globule 2 (HMFG-2) in adenocarcinoma, 459t in lung neoplasms, 388t-389t, 395f in mesothelioma, 444t-446t, 448f, 453t, 462f in pleural neoplasms, 448t Human milk fat globule proteins, 75, 455 Human papillomavirus, 59-60, 70-72, 72f in head and neck lesions, 245-248, 246t-248t in upper aerodigestive tract carcinomas, 249t Human placental lactogen in gynecologic pathology, 654t in testicular tumors, 645 Human T-lymphocyte antigen, 444t-446t Hürthle cell carcinoma, 240-241, 336 Hyalinizing clear cell carcinoma, 303, 303f-304f differential diagnosis of, 296t key diagnostic points, 304b Hyalinizing trabecular tumors, 329, 330f Hybrid schwannoma/perineurioma, 113 Hybridization, allele-specific, 45-46 Hybridoma technique, 1-2 Hydatidiform mole, complete, 678-679 Hydrops fetalis, 60f Hypopharynx: tumors of, 287-290
I
IAPNs. See Intraampullary papillarytubular neoplasms ICPNs. See Intracholecystic papillarytubular neoplasms Identity testing, 54-55, 54f IDF. See Infantile digital fibroma/ digital fibromatosis IDH1 mutant, 765t IDHs. See Isocitrate dehydrogenases IFPs. See Intermediate filament proteins IGF-1R. See Insulin-like growth factor receptor 1 IHC. See Immunohistochemistry Ileal carcinoid tumor, 352f-353f Illumina, 47 Image acquisition, 878 Image analysis, 877 automated quantitative analysis (AQUA) method, 880-881, 881f computer-aided, 877 process of, 879, 879f quantification, 879, 880f software algorithms for, 878-881 Image quantification, 880f ImageJ, 878-879 Imaging feature extraction, 879 feature selection, 879 multispectral microscopic, 878 whole-slide, 877-878
Imaging systems, 877-878 Imidazole, 37t Immune hyperplasia, acute, 154-157 Immunocytochemistry, 829, 830f Immunocytology, 829-853 applications, 829, 837-851 in cytopathology, 829, 830b interpretation of, 836-837, 837f limitations of, 836-837 techniques, 829-836 theranostic applications, 851-852 Immunoenzymatic techniques, double or multiplex, 25-27, 26f-27f Immunoglobulin D, 132-134 Immunoglobulin G4–associated autoimmune pancreatitis, 567 Immunoglobulin G4–associated cholangitis, 570 Immunoglobulin G4–associated immune complex multiorgan autoimmune disease, 567 Immunoglobulin G4–related cholangitis, 570 Immunoglobulin G4–related disease, 567 Immunoglobulin G4–related sclerosing cholangitis, 570 Immunoglobulins, 132-134 Immunogold-silver staining, 24t, 26, 37t Immunohistochemistry advantages of, 56, 57b, 744-745 artifacts, 837f automated, 27-30 basic principles of, 2-3 combined with ISH, 27f cutaneous, 506-507 detection systems, 5-14 diagnostic in gastric adenocarcinoma, 518-519 of GI tract, 510-537 of infectious diseases, 56, 57b factors that affect outcome, 15, 16t first-line panels, abbreviated, 207 internal tissue control for, 824, 824f interpretation of, 837f line of differentiation panels epithelial, 208 lymphoid, 207-208 melanocytic, 208 mesenchymal, 208 overview of, 1-2 protocols for, 22-37 sample preparation for, 14-15 screening, 207-208 specimen selection for, 762 techniques for, 22-37 total test in, 14, 14t troubleshooting, 22-37, 31t-32t Immunohistology of breast, 710-761 of endocrine tumors, 322-362 of female genital tract, 653-709 of gastrointestinal tract, 508-539 genomic applications, 89b of head and neck lesions, 245-321 of Hodgkin lymphoma, 130-147
Index
Immunohistology (Continued) of infectious diseases, 56-72 of lung and pleural neoplasms, 386-478 of mediastinum, 363-385 of melanocytic neoplasms, 189-203 of neoplasms of soft tissue and bone, 73-129 of nervous system, 762-828 of non-Hodgkin lymphoma, 148-188 of pediatric neoplasms, 854-876 of pleural neoplasms, 386-478 of prostate, 584-614 of renal neoplasms, 631-644 of renal tumors, 635-641 of skin tumors, 479-507 of testicular tumors, 644-652 of urinary bladder, 615-618 ImmunoMembrane, 878-879 Immunoperoxidase double or multiplex staining, 25f indirect determination of optimal titers for, 14t indirect-conjugate (sandwich) method, 12 ImmunoRatio, 878-879 Immunostaining, 850-851, 851f. See also Staining automated, 27-30 double, 9, 9f false-negative, 832, 834f, 836 false-positive, 836 pitfalls in Hodgkin lymphoma, 138-139 ways to maximize tissue for, 835b IMT. See Inflammatory myofibroblastic tumor In situ hybridization (ISH) benefits and limitations of, 751t combined with IHC, 27f for determining site of origin, 242t HER2 assays, 750-752 in non-Hodgkin lymphoma, 154 In situ proteomics, quantitative, 30 Inclusion-like cells, 130 Incubation of detection reagents, 24 of primary antibody, 23 troubleshooting, 32t Indeterminate cutaneous fibrohistiocytic lesions, 496-497 Indirect-conjugate (sandwich) method, 6, 6f determination of optimal titers for, 12, 14t Infantile digital fibroma/digital fibromatosis (IDF), 494-495 Infantile fibrosarcoma, 107 Infantile ganglioglioma, desmoplastic, 797f, 799 Infections. See also specific infections bacterial, 62-65 fungal, 65-66 protozoal, 66 secondary to AIDS, 772-773 viral, 56-62, 571
Infectious disease markers, 850-851 Infectious diseases. See also specific diseases diagnosis of, 56, 57b, 58t emerging, 67-68 immunohistology of, 56-72 key diagnostic points, 773b of nervous system, 770-772 Infectious mononucleosis, 143 Infiltrating cells, 827 Infiltrative basal cell carcinoma, 485 Inflammation, perivascular, 768-769 Inflammatory bowel disease, 522 Inflammatory demyelinating polyneuropathy, chronic, 822, 822f Inflammatory hepatocellular adenoma, 574 Inflammatory leiomyosarcoma, 109 Inflammatory myofibroblastic tumors of bladder, 624, 624f-625f, 625t epithelioid sarcoma, 86 prostatic, 603t of soft tissue and bone, 106-107, 106f uterine, 678 Inflammatory pseudotumors of lung, 408, 417 mediastinal, 374-375 Influenza A, 71f Inform (Caliper/Perkin Elmer), 878-879 Inhibin in adrenocortical tumors, 235-236, 346f, 347 in endometrioid tumors, 690-692, 691t in female adnexal tumors, 708 in granulosa cell tumors, 696-697 in gynecologic pathology, 654t in Leydig cell tumors, 699 in lung neoplasms, 389t in ovarian and tubal tumors, 685, 695-696 in pheochromocytoma, 347f in Sertoli cell tumors, 698-699 in Sertoli-Leydig cell tumors, 697-698 in sex cord tumors, 699 in steroid cell tumors, 699 in uterine tumors, 673t in vulvar granular cell tumors, 656, 656f Inhibin A, 645 Inhibin-α, 430, 516t INI1. See Integrase interactor 1 INI1 gene. See SMARCB1 gene Insertions, small, 41 Insomnia, fatal familial, 773 Institute of Medicine (IOM), 67 Insulin-like growth factor receptor 1, 760 Insulinomas, 354 Insulioma, 556 Integrase interactor 1 (INI1), 87-88 Interferon regulatory family 4 (IRF4), 150 Interfollicular lymphadenitis, 144
907
Interleukin-2 receptor (CD25), 145 Intermediate filament proteins, 73-75 in endocrine tumors, 325 in follicular tumors, 331-332 in melanocytic neoplasms, 189 Internal reference standards, 17, 19 quantitative, 30 Intestinal-type adenocarcinoma, 272, 274f, 561 sinonasal, 272, 275b types of, 272, 273f Intestinal-type endocervical adenocarcinoma, 661, 663f Intestinal-type mucinous tumors, 689, 689f Intestinal-type papillae, 549-550, 550f Intestinal-type secretory mucin, 541 Intraabdominal carcinomatosis, 543 Intraampullary papillary-tubular neoplasms, 563, 564f Intracholecystic neoplasms, 559-560 Intracholecystic papillary-tubular neoplasms, 559 anatomic molecular diagnostic applications, 560 biliary type, 559 foveolar type, 559 of gallbladder, 559, 559f gastric type, 559 intestinal type, 559 pyloric type, 559, 559f Intracranial masses, 814-816 Intracranial tumors, 807-808 Intracutaneous melanoma, 198-199 Intracystic papillary carcinoma, 718-719, 719f Intracystic papillary neoplasms, 559 Intraductal carcinoma, low-grade, 307 Intraductal neoplasms, 559-560 Intraductal oncocytic papillary neoplasms, 549 Intraductal papillary mucinous neoplasms, 549-550, 549f-550f, 563 anatomic molecular diagnostic applications, 550 antibodies in, 549, 549f morphologic patterns, 549 Intraductal papillary neoplasms, 559 Intraductal papilloma, 718, 718f Intraductal tubulopapillary neoplasms, 551, 551f Intraepithelial carcinoma, tubal, 707, 707f Intraepithelial lesions, vulvar squamous, 656-658 Intraepithelial neoplasia biliary, 560 pancreatic, 548-549 tumoral, 559-560 vulvar, 656-658, 658f Intrahepatic cholangiocarcinoma, 581 Intraoperative molecular testing, 737-738, 738f Intrapulmonary thymoma, 417-418
908
Index
Intratubular germ cell neoplasia, 645-646, 646f, 648t Intravascular bronchioloalveolar tumors, 406 Intravascular large B-cell lymphoma, 174 Intravascular lymphoma, 410t, 412, 813 Introns, 40 Invasive adenocarcinoma extrahepatic, 560-561 key differential diagnosis, 561b Invasive carcinoma, extrahepatic, 561-562 Invasive ductal carcinoma, 724f-725f, 753f Invasive endocervical adenocarcinoma, 661, 661b Invasive micropapillary carcinoma, 725-728, 728f Ion sphere particles, 47 Ion Torrent (Life Technologies), 47, 48f IOPNs. See Intraductal oncocytic papillary neoplasms IPMNs. See Intraductal papillary mucinous neoplasms IPNs. See Intraductal papillary neoplasms Irf4/MUM1, 134 ISH. See In situ hybridization Isocitrate dehydrogenases, 765t, 778 ITPNs. See Intraductal tubulopapillary neoplasms IVLBCL. See Intravascular large B-cell lymphoma
J
J-chain, 133-134 JAM. See Junctional adhesion molecule JAZF1-SUZ12 translocation, 683 JC virus, 61, 62f, 765t JunB, 133, 134f, 138t Junctional adhesion molecule, 79-80
K
Kaposi sarcoma, 57-58, 59f, 97, 98f immunomarkers of, 92t, 94 in lung, 405-406, 406f nodular stage, 504 patch stage, 504 of skin, 504, 504f Kaposiform hemangioendothelioma, 94 KBA, 260-262, 261f KER(PAN). See Pancytokeratin Keratin 5, 450t-451t Keratin 903 (K903) in breast carcinoma, 731t in cancer of unknown primary site, 216, 216f in head and neck lesions, 246t-248t in upper aerodigestive tract carcinomas, 249t
Keratins aberrant, 217-218 anomalous, 217-218 antigens and antibodies, 213t in bladder tumors, 625t in breast carcinoma, 729-731 complex, 216-217, 216b in desmoplastic small round cell tumor, 865-866, 866f distribution in tissues, 209-219, 212t, 213f in epithelial mesothelioma, 453t in epithelioid sarcoma of subcutis, 505-506, 505f hard, 485 in large cell undifferentiated malignancies, 202f in lung neoplasms, 388t-389t, 396t, 402, 404-406, 404f-406f, 416t, 419t, 421t, 427t in malignant small round cell tumors, 91t in melanocytic neoplasms, 191f in Merkel cell carcinoma, 487, 487f in mesenchymal tumors, 218, 218f in mesothelioma, 444t-446t, 447 in neuroblastoma, 350f in nonepithelial cells, 217-219, 219b pilar type, 485, 486f in pleural neoplasms, 465t in sentinel lymph nodes, 737, 737f simple epithelial, 209-215 in skin tumors, 479, 480f in small cell carcinoma of cervix, 665f in spindle cell tumors, 92t spurious, 217-218 of stratified epithelia, 216-217 in thymoma, 365-366, 366f in tumors of soft tissue and bone, 73-74 unexpected, 217-218 KHE. See Kaposiform hemangioendothelioma Ki-1, 444t-446t Ki-67, 833t, 850. See also Molecular immunology Borstel 1 (MIB-1) in breast carcinoma, 759 in CD20-positive B-cell lymphoma, 845f in cervical squamous intraepithelial lesions, 658, 660f in cervix, 658, 659f in endocervical adenocarcinoma, 660, 661f in endometrial serous carcinoma, 669-670, 669f in fibroadenoma, 740, 741f-742f in gynecologic pathology, 654t in head and neck lesions, 246t-249t, 248-250, 248f, 263-264, 264f in lung neoplasms, 402t, 403f in melanocytic neoplasms, 196, 197f in pancreatic tumors, 557 in phyllodes tumor, 740-741, 741f-743f in uterine carcinomas, 667t in uterine sarcoma, 683
Ki-67 (Continued) in vulvar intraepithelial neoplasia, 656-658, 658f in vulvar lesions, 656, 657f Kidney-specific cadherin (Kspcadherin), 633 Kidney tumors. See also under Renal secondary, 641 Kikuchi disease, 492 Klebsiella pneumoniae, 58t KM2760, 145 KP1, 765t, 769f. See also CD68 KRAS mutations and chemotherapy susceptibility, 539 detection of, 49-50, 50f in GI tumors, 539 in pancreatic ductal adenocarcinoma, 545 KSA. See Epithelial cell-adhesion molecule KSA1/4. See Epithelial cell-adhesion molecule Ksp-cadherin. See Kidney-specific cadherin Kulchitzky cell carcinoma II, 400 Kupffer cells, 75 Kuru, 773 Kuttner tumors, 567
L
L26. See also CD20 in encephalitis, 769f in nervous tissue, 765t Labeled antigen double stain, 11-12, 12f Labeled antigen method, 11, 12f Labeling alkaline phosphatase labels, 8-12 polymer-based methods, 9-10, 10f Labeling index (LI), 775-777 Laboratories, 15, 17t LAM. See Lymphangioleiomyomatosis Laminins in head and neck lesions, 246t-248t in tumors of soft tissue and bone, 73, 79 Langerhans cell granules, 413, 429f Langerhans cell histiocytosis/ granulomatosis pulmonary, 413-414, 414f of skin, 493, 494f in soft tissue and bone, 79, 128 Langerhans cells, 413, 413f Langerhans histiocytosis, 815t Large B-cell lymphoma, 168-176 ALK-positive, 173 cutaneous large cell lymphoid proliferations that simulate, 492-493 intravascular, 174 key diagnostic points, 177b markers, 188t mediastinal, 173, 174f, 382-384, 384f subtypes, 171-173 T-cell/histiocyte-rich, 172, 173f
Index
Large cell anaplastic lymphoma diagnostic immunohistochemistry, 139-140 differential diagnosis of, 141t of lung, 426-427 mediastinal, 384 of skin, 488, 490f Large cell carcinoma, 393t Large cell lymphoid proliferations, cutaneous, 492-493 Large cell neuroendocrine carcinoma of cervix, 359, 359f description of, 400 diagnostic criteria for, 400 immunohistochemical features of, 427t markers in, 357f of prostate, 597-598 pulmonary, 356, 357f undifferentiated, 224 Large-cell non-Hodgkin lymphoma, 202f Large cell undifferentiated carcinoma classification of, 207 of lung, 427t neuroendocrine, 224 Large cell undifferentiated neoplasms, 426-427, 427t Larynx carcinoid of atypical, 287, 288f typical, 287, 287f metastasis in, 289t tumors of, 287-290, 290b Lassa fever, 61, 61f Latent associated nuclear antigen 1 (LANA-1), 57-58 Latent membrane protein 1 (LMP-1), 59, 60f LC Green, 44 LCC, 202f LCH. See Langerhans cell histiocytosis LCNHL. See Large-cell non-Hodgkin lymphoma Legionella, 70-72 Legionella dumoffii, 65 Legionella pneumophila, 65 Leica, 8 Leiomyoma, 76, 603-604, 673 Leiomyomatous proliferation, prominent, 640-641 Leiomyosarcoma differentiation from CUPS, 211t-212t epithelioid, 109 immunomarkers of, 92t inflammatory, 109 key diagnostic points, 673b mediastinal, 375, 376f morphologic variants, 109 pleomorphic, 109 prostatic, 603-604, 603t in soft tissue and bone, 74, 76, 108-109, 108f in subcutis, 499, 500f of urinary bladder, 625t uterine, 673 Leishmania, 66
Leishmaniasis, 66 Length polymorphisms, 41 Lennert lymphoma, 140-141 Lentigo maligna-type melanoma, 189, 190f Leptospira, 65f Leptospirosis, 65, 68 Leu-7. See also CD57 in cancer of unknown primary site, 224 discriminative value, 402t in lung neoplasms, 402t, 403f, 425, 426f in nervous tissue, 765t in tumors of soft tissue and bone, 79 Leu-M1, 468t LEU19, 79. See also CD57 Leukemia acute lymphoblastic, B-cell, 174, 175f adult T-cell, 178f, 184 chronic lymphocytic. See Chronic lymphocytic leukemia craniospinal, 813 extramedullary myelogenous, 385 hairy cell, 155t, 165-166, 166f hairy cell variant, 167 myeloid leukemia cutis, 488, 490f in skin, 488-492 small lymphocytic, 154, 155t, 160-161 T-cell large granular lymphocytic, 183-184 T-cell prolymphocytic, 182-183 Leukemic infiltrates, 413-414 Leukocyte common antigen in dysgerminoma, 701t in granulosa cell tumors, 696t in Hodgkin lymphoma, 138t, 139 Leukoencephalopathy, 771 progressive multifocal, 767t, 773 LeuM1, 840-843. See also CD15 Lewis-Bg8, 452t Lewis-X type 2 chain (BG-7), 138t Lewis Y antigen, 444t-446t Lewy body diseases, 772 Leydig cell tumors, 648b, 649, 699 LGFMS. See Low-grade fibromyxoid sarcoma Lhermitte-Duclos disease, 797 LI. See Labeling index Life Technologies, 46-47, 48f Ligand-binding assay, 744-745 LightCycler probe, 44 LIP. See Lymphocytic interstitial pneumonia-pneumonitis Lipoma, 115 Lipomatous or fat-forming solitary fibrous tumor, 103-104 Lipomatous tumor, atypical, 115 Liponeurocytoma, cerebellar, 799 Liposarcoma, 78-79 dedifferentiated, 76, 87, 115, 116f immunomarkers of, 92t myxoid, 115-117 pleomorphic, 115 round cell, 115-117 well-differentiated, 115, 116f
909
Listeria monocytogenes, 58t, 65, 771 Liver, 568-583. See also under Hepatic CD34 in, 221, 222f medical diseases of, 568-573 metabolic disorders of, 572 metastatic carcinoid tumor in, 352f neoplastic diseases of, 573-583 neuroendocrine neoplasms in, 583 nonneoplastic diseases of, 573 normal vasculature, 569 normal vasculature of, 569 tumors in, 583 undifferentiated or embryonal sarcoma of, 583 viral infections of, 571 Liver fatty acid-binding protein, 573t Liver transplantation, 572-573 LK2H10, 224 Lobular capillary hemangioma, 275-277, 276f Lobular carcinoma, 722f-725f cytokeratin 20 in, 391t invasive, 723f key diagnostic points, 723 pleomorphic, 723, 725 variants, 725 Lobular carcinoma in situ, 723f, 726f cell adhesion in, 721-723 pleomorphic, 723, 727f Lobular hyperplasia, 722-723 Lobular neoplasia, 726f LOH. See Loss of heterozygosity Loss of heterozygosity, 41, 51-52, 52f Loss of SMAD4, 543f Low-grade cribriform cystadenocarcinoma, 307-308, 308b, 308f Low-grade fibromyxoid sarcoma, 79-80, 88, 92t, 104-105, 104f Low-grade intraductal carcinoma, 307 Low-grade salivary duct carcinoma, 307 LPL. See Lymphoplasmacytic lymphoma LPS. See Liposarcoma Luminal tumors, 752-754 Lung fetal, 356f lymphomas of, 409, 410t lymphoproliferative disorders of, 407-414 metastatic carcinoma in, 426b multiple myeloma in, 409 peripheral small cell carcinoma resembling carcinoid tumor in, 400 placental transmogrification of, 418 rare primary lymphomas in, 411-413 secondary lymphomas that involve, 413-414 Lung adenocarcinoma, 387, 387t, 844f, 847f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t metastic, 319-320, 319f mutational profiles, 432, 433f poorly differentiated, 852, 853f
910
Index
Lung adenocarcinoma (Continued) vs. colon adenocarcinoma, 525 vs. colorectal adenocarcinoma, 525f vs. gastric adenocarcinoma, 519 vs. gastric signet-ring cell carcinoma, 519f Lung carcinoid, 356f Lung carcinoma antibody panel for, 240t basaloid, 420-421, 420f, 421t clear cell, 236b immunohistologic pitfalls, 426b markers for, 225-226, 226b, 395f metastatic to head and neck, 319-320, 319f metastatic to larynx, 289t pseudomesotheliomatous, 465-466 sarcomatoid, 219, 220f small cell, 419t, 693-694 squamous cell, 419-420 cytokeratin profile, 454t metastatic, 420 Lung markers, 843 Lung neoplasms antigens and antibodies in, 387-400 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t clear cell neoplasms, 414, 414f diagnosis of, 432-437 diagnostic pitfalls, 418-431 differential diagnosis of, 418-431 immunohistology of, 386-478 large cell undifferentiated, 426-427, 427t neuroendocrine, 400-404, 423-425 nonneuroendocrine, 387-400, 388t-389t primary, 387-418 rare, 404-407 sarcoma, 405, 426-427 theranostic applications in, 431-437 tumor-specific markers for, 389t Luteinizing hormone, 246t-248t LYG. See Lymphomatoid granulomatosis Lyme disease, 65, 771 Lymph nodes endometrial carcinoma metastasis in, 672 mesothelial cells in, 474 metastatic neuroblastoma in, 350f sentinel. See Sentinel lymph nodes Lymphadenitis cytomegalovirus, 143, 143f granulomatous, 144, 144f interfollicular, 144 Lymphangioleiomyomatosis, 415 Lymphangioma, 91-94, 380t Lymphatic space invasion, 734, 735f Lymphoblastic lymphoma, 382, 488, 491f Lymphocytic and histiocytic cells (L&H cells), 130 Lymphocytic gastritis, 513 Lymphocytic interstitial pneumoniapneumonitis, 408 Lymphoepithelial carcinoma, 278-279
Lymphoepithelial-like carcinoma, 516 Lymphoepithelioma, 278-279 Lymphoepithelioma-like gastric carcinoma, 516, 518f Lymphoepithelioma-like lung carcinoma, 427t Lymphoepithelioma-like medullary carcinoma, 579 Lymphohematopoietic disorders, 488-494 Lymphohistiocytoid mesothelioma, 472, 473f Lymphoid differentiation, 207-208 Lymphoid hyperplasia micronodular thymic carcinoma with, 370-371 micronodular thymoma with, 368 Lymphoid interstitial pneumonitis, 408 Lymphoid lesions, pseudoneoplastic, 492-493 Lymphoid markers, 843-846 Lymphoid neoplasms, 812-814 Lymphoid proliferations, pseudoneoplastic, 493b Lymphoma. See also Hodgkin lymphoma; Non-Hodgkin lymphoma B-cell, 142f B-lymphoblastic, 174 brain, 812-813, 813f Burkitt, 174, 175f CD30-positive cutaneous, 142 CNS, 812-813 diagnosis of, 154-157, 184-187 differential diagnosis, 154-157, 158f, 768t, 793t double-hit, 176 follicular, 155t, 157f, 160 gray-zone, 176 immunomarkers for, 91t intravascular, 410t, 412, 813 large B-cell, 141t in liver, 583 low-grade, 154 in lung, 409, 426-427 immunohistochemical features of, 410t rare primary, 411-413 lymphoblastic, 382 lymphoplasmacytic, 155t, 162-163 malignant high-grade, 154-157 M rule for differential diagnosis of, 766 malignant mimics, 185t mantle cells, 161-162 marginal zone, 164f extranodal, 155t, 163-165, 165f, 165t nodal, 163 markers associated with other neoplasms, 186-187 metastatic carcinoma mimicry, 186-187, 187f mimicry, 184-187, 188t neoplasms that express markers associated with, 185-186 of ovary, 694, 694f
Lymphoma (Continued) peripheral T-cell, 140-141 plasmablastic, 171-172, 172f pleural, 467-468 posttransplant, 813, 814f primary effusion lymphoma, 173 pseudolymphoma, 408, 583 pyothorax-associated, 410t, 413 rare, in lung and chest cavity, 411-413 secondary, that involve lung and leukemic infiltrates, 413-414 in skin, 488-492 small B-cell, 157-167 small cell, 492 Lymphomatoid granulomatosis, 411-412, 412f, 813 Lymphomatoid granulomatosis lymphoma, 410t Lymphomatoid papulosis of skin, 493, 495f type A, 493-494 type B, 493-494 type C, 493-494 Lymphoplasmacytic lymphoma, 162-163 diagnostic pitfalls, 163 immunohistochemical features of, 155t prognostic and therapeutic studies, 162 Lymphoplasmacytic sclerosing pancreatitis with cholangitis, 567 Lymphoproliferative disorders CD-30, 183, 493-494, 495f of lung, 407-414 mediastinal, 380-385 posttransplant, 813 Lymphovascular invasion, 672 Lynch syndrome, 524f genomic applications for, 708 molecular diagnostic applications, 709 testing for, 681-682, 682f Lynch syndrome–associated tumors, 681-682 LYP. See Lymphomatoid papulosis Lysozyme (muramidase), 84
M
M rule, 766 M2A, 83 Macrophages in brain lesions, 767, 768t, 815t in Hodgkin lymphoma, 145 Mad cow disease, 773 MAGEC2, 382t MAGs. See Myelin-associated glycoproteins Malaria, 66 Malignancy epithelioid, 200-201 grading malignant potential, 775-778 poorly differentiated, 237, 238f-239f
Index
Malignant carcinoids, 400 Malignant ependymoma, 766t, 789 Malignant fibrous histiocytoma differential features of, 776t-777t myxoid, 107 Malignant germ cell tumors, 699 Malignant glioma, 201-202 Malignant lymphoma high-grade, 154-157 M rule for differential diagnosis of, 766 Malignant melanoma, 203 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t desmoplastic, 202f immunohistochemical features of, 427t metastatic, 92t, 834f microscopic phenotypes of, 189 morphologic variants, 189 rhabdoid, 200 S-100 protein staining in, 34f sarcomatoid, 200, 202f Malignant meningioma, 807-809 Malignant mesothelial cell proliferations, 475-477 Malignant mesothelioma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t diffuse, 477-478 epithelioid, 448t immunostain profile, 839f localized, 462 markers for, 450t, 452t vs. reactive mesothelial hyperplasia, 476-477, 476t Malignant mixed oligodendrogliomaastrocytoma, 791 Malignant oligodendroglioma, 791 Malignant peripheral nerve sheath tumor, 75, 113-114, 114f, 812 differentiation from CUPS, 211t-212t epithelioid, 114 immunomarkers of, 92t Malignant phyllodes tumor, 741, 744f Malignant proliferating pilar tumors, 486 Malignant rhabdoid tumor, 868-871, 868f extrarenal, 124 immunohistochemical profile, 869, 869f key diagnostic points, 871b key features of, 875t Malignant schwannoma, 113 Malignant small round cell tumors, 91t Malignant Triton tumor, 76, 113 Malignant vascular tumors, 94-97 Mallory bodies, 569-570, 569f MALT. See Mucosa-associated lymphoid tissue Mammaglobin, 227-228, 847 in breast carcinoma, 228, 228f, 739, 740f, 846f key diagnostic points, 228b in lung neoplasms, 389t
Mammalian achaete-scute complex homolog 1 (MASH-1), 425 Mammalian achaete-scute complexlike protein (MASH), 355 MammaPrint assay (Agendia BV), 747-748, 755-756, 758t Mammary analog secretory carcinoma, 308, 308b, 309f Mammostrat panel, 759 Mantle cell lymphoma, 161-162, 162f blastoid, 176 diagnostic pitfalls, 162 immunohistochemical features of, 155t in lung, 410t, 411 markers of, 152 prognostic and therapeutic studies, 162 Marburg disease, 61, 70 Marginal zone B-cell lymphoma immunohistochemical features, 410t MALT type, 409 Marginal zone lymphoma, 154, 164f extranodal, 163-165, 165f, 165t genetic abnormalities in, 163, 165t nodal, 163 splenic, 155t, 166-167 MART-1. See also Melan-A in melanocytic neoplasms, 193-194, 193f, 202f in tumors of soft tissue and bone, 85 MASH. See Mammalian achaete-scute complex homolog Masking, 3 Mast cells, 533f MCM. See Minichromosome maintenance protein MCS. See Mesenchymal chondrosarcoma MDM2. See Murine double-minute type 2 MDR1, 607t MEAT. See Middle ear adenomatous tumor Mediastinal B-cell lymphoma, 142, 168 Mediastinal gray-zone lymphoma, 382-384 Mediastinal large B-cell lymphoma, 142f, 173, 174f, 382-384, 384f Mediastinal tumors, 385 adipocytic neoplasms, 377 angiosarcoma, 379, 379f dendritic cell tumors, 385 fibrohistiocytic proliferations, 373-375 fibrous proliferations, 373-375 germ cell tumors, 379-380, 382t glomus tumors, 378, 379f hematopoietic, 383t histiocytic, 380 leiomyosarcoma, 375, 376f lymphoproliferative disorders, 380-385 mesenchymal neoplasms, 373-379 mesothelial neoplasms, 373 myofibroblastic proliferations, 373-375
911
Mediastinal tumors (Continued) myogenic neoplasms, 375-376 neuroectodermal, 377-378, 378f neuroendocrine carcinomas, 371-373, 372t paraganglioma, 372t, 373 parathyroid, 373 perivascular neoplasms, 378-379 primitive neuroectodermal tumors, 378, 378f prognostic markers for, 385 rhabdomyosarcoma, 375, 376f schwannoma, 377, 377f seminoma, 379-380, 381f sites of origin, 240-242 solitary fibrous tumor, 373-374, 374f types of, 240-242 vascular neoplasms, 378-379 Mediastinitis, sclerosing, 373-374 Mediastinum, 363 antigens and antibodies in, 363 immunohistology of, 363-385 Medical liver diseases, 568-573 Medullary carcinoma, 337-342, 338f-339f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t key diagnostic points, 340b lymphoepithelioma-like, 579 markers in, 338f molecular approach to, 340-342 in pancreas, 547 Medulloblastoma, 787f, 801-802 classic, 801 desmoplastic, 779t, 802 diagnosis of, 764f differential diagnosis of, 766 differential features of, 793t Medulloepithelioma, 766t, 803-804, 805f Medullomyoblastoma, 793t, 802 Melan-A (A103) in adrenocortical tumors, 235-236, 346-347, 346f in cancer of unknown primary site, 236 in head and neck lesions, 246t-248t in lung neoplasms, 389t in melanocytic neoplasms, 193-194, 193f, 202f in mucosal melanoma, 260-262, 261f in pheochromocytoma, 347f in tumors of soft tissue and bone, 85, 111f Melanocyte-specific monoclonal antibodies, 193-196 Melanocytic differentiation determination of, 208 markers of, 84-85 Melanocytic lesions, 196-197 Melanocytic markers in mucosal melanoma, 260-262, 261f in spindle cell melanoma, 261f
912
Index
Melanocytic neoplasms antigens and antibodies in, 189-196 differential diagnosis of, 198 filamentous proteins in, 189 immunohistology of, 189-203 metastatic to nervous system, 818 Melanocytic nevus, 198 Melanoma acral lentiginous, 189, 190f amelanotic, 200-201 cell types, 260, 260f cutaneous, 192, 192f differential diagnosis, 198-200, 200f, 202, 289t, 766, 766t differential features of, 776t-777t, 779t esophageal, 535, 535f in GI tract, 535 immunohistology of, 189, 197f intracutaneous, 198-199 invasive, 195f lentigo maligna-type, 189, 190f in lung, 426-427 malignant, 203 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t desmoplastic, 202f lymphoma mimics, 185t markers, 185t metastatic, 834f microscopic phenotypes of, 189 morphologic variants, 189 rhabdoid, 200 S-100 protein staining in, 34f sarcomatoid, 200, 202f markers of, 729-731, 811f in mesothelioma, 444t-446t metastatic, 193f, 195f-196f differential diagnosis of, 201-202 sentinel lymph node biopsies for, 197-198 mucosal, 260-262, 260f differential diagnosis of, 262, 262f, 289-290, 289t key diagnostic points, 262b sinonasal, 256t nodular, 189, 190f prognostic markers for, 202-203 sarcomatoid, 201f of soft parts (clear cell sarcoma), 211t-212t superficial spreading, 189, 190f targeted therapy for, 202-203 Melanoma associated antigen (mutated) 1 (MUM1/IRF4), 150 Melanoma in situ, 199, 199f Melanoma markers, 729-731, 811f Melanophages, pigmented, 31f Melanosomes, 414, 415f Melanotic schwannoma, 196-197, 197f Melting temperature (Tm), 44 Membrane-associated mucins, 541 Meningeal cysts, 819, 819t Meningeal hemangiopericytomas, 809, 810f Meningeal tumors, 764-766, 804-810
Meningiomas, 804-807 aggressive, 807-809 anaplastic, 770t, 805t, 809 anatomic molecular diagnostic applications, 809 angiomatous, 807 antibodies in, 805, 805f atypical, 770t, 808-809 chordoid, 807 clear cell, 807-808, 808f differential diagnosis, 766t differential features of, 805t ectopic, 314-315, 314f-315f, 315b fibrous (fibroplastic), 805-806 differential features of, 776t-777t immunostaining characteristics of, 806, 806f grade I, 770t, 807 grading of, 770t key diagnostic points, 809b malignant, 807-809 meningothelial, 762-764, 764f meningotheliomatous, 806, 806f mixed, 806 papillary, 809 psammomatous, 806-807 rhabdoid, 809 secretory, 807, 807f syncytial, 806 theranostic applications, 809 transitional, 779t, 806 Meningitis, 770 Meningothelial-like nodules, minute pulmonary, 418 Meningothelial meningioma, 762-764, 764f Meningothelial nodules, 418 Meningotheliomatous meningiomas, 806, 806f Merkel cell carcinoma, 360-361, 360f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t in skin, 487, 487f, 489f in soft tissue and bone, 74-75 vs. small cell lung carcinoma, 425-426 Merlin, 812 Mesenchymal chondrosarcoma, 124-125 differential diagnosis of, 262, 262f immunomarkers for, 91t Mesenchymal differentiation determination of, 208 esophageal carcinoma with, 513 Mesenchymal hamartoma, 583 Mesenchymal lesions genomic applications, 537 of GI tract, 532-537 anatomic molecular diagnostic applications to, 539 differential diagnosis of, 535 presenting as mural masses, 532-535 theranostic applications, 538-539 immunohistochemical staining of, 536f
Mesenchymal lesions (Continued) keratin in, 218, 218f of mediastinum, 373-379 mural in GI tract, 532-535 key diagnostic points, 535b presenting as polypoid lesions, 536-537 small round cell, 506 vulvovaginal, 655-656, 656b Mesenchymal markers, 833t Mesenchymal skin tumors, 494-506 Mesenchymal tumors in breast, 731 endometrial, 676-678 prostatic, 602-606 uterine, 673-678, 673t Mesonephric remnants, 658 Mesothelial cells in lymph nodes, 474 separation of benign and malignant proliferations, 475-477 Mesothelial hyperplasia, reactive, 476-477, 476t Mesothelial markers, 838-840 Mesothelial neoplasms, 373 Mesothelin in lung neoplasms, 389t in mesothelioma, 444t-446t, 448f, 452t-453t, 468t-469t, 475t in pancreatic ductal adenocarcinoma, 543f in pleural neoplasms, 448t Mesothelioma antibodies in, 438, 444t-446t antibody panel for, 240t atypical carcinoid presenting as, 471-472, 472f-473f cystic, 473, 474f deciduoid, 462 desmoplastic, 477, 477t epithelial antibodies in, 468-469, 468t cytokeratin profile, 454t differential diagnosis of, 237, 239b, 468t histologic variants or subtypes, 438, 438b, 439f-443f immunohistochemical features of, 453-455, 453t, 455f, 469t, 476t immunohistochemical tests in, 470t immunohistology of, 447, 448f markers in, 452t mucin-positive, 469-471, 471f well-differentiated papillary, 472-473, 473f epithelioid differential diagnosis of, 475t immunohistochemical features of, 454t immunohistochemical markers for, 471t, 475t mucin-expressing and with crystalloid features, 464-468 epithelioid hemangioendothelioma mimicking, 466
Index
Mesothelioma (Continued) grading, 478 immunohistochemical tests for, 459t immunohistochemistry of, 454t, 469b key diagnostic points, 236b lymphohistiocytoid, 472, 473f malignant CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t diffuse, 477-478 immunostain profile, 839f localized, 462 markers for, 448t, 450t, 452t vs. reactive mesothelial hyperplasia, 476-477, 476t markers for, 448t, 451t metastatic, 462 peritoneal, 706-709, 706t, 707f pleomorphic, 463 prognosis for, 478 with rhabdoid features, 463-464 round cell, 463 sarcomatoid diagnostic considerations, 460461, 461f immunohistochemical features of, 453, 453f, 454t, 462f markers in, 452t small cell, 463 transitional, 462 TTF-1 expression in, 393t Mesothelioma antigen, 444t-446t Mesothelioma-like tumors, 465 Mesothelioma pattern, 842f Metabolic liver disorders, 572 Metanephric adenoma, 636, 636f Metaplastic carcinoma, 729-731 differential diagnosis, 731t spindle cell, 729-730, 730f Metastatic disease. See also Cancer of unknown primary site from appendix, 704-705, 704f breast carcinoma key diagnostic points, 739b vs. gastric signet-ring cell carcinoma, 518-519, 519f in CNS, 817-818 colon adenocarcinoma, 273t colon cancer vs. pulmonary adenocarcinoma, 422 germ cell tumors, 647-648 in head and neck, 319-321, 320b immunomarkers of, 92t in lung, 426b lymphoma mimicry, 186-187, 187f M rule for differential diagnosis of, 766 mesothelioma, 462 micrometastatic disease, 738b mucinous adenocarcinoma of rectum, 844f in ovary, 704-706, 704f, 706b in peritoneal fluid, 841f
Metastatic disease (Continued) in pleura and abdomen, 237 key diagnostic points, 239b markers for, 450t renal cell carcinoma vs. pulmonary adenocarcinoma, 422-423 serous carcinoma, high-grade, 841f small cell neuroendocrine carcinoma, 487-488, 488f of unknown primary site, 204-244 vs. cholangiocarcinoma, 582 vs. gastric signet-ring cell carcinoma, 518-519, 519f vs. hepatocellular carcinoma, 580 vs. pulmonary adenocarcinoma, 423 well-differentiated neuroendocrine tumors, 531 Metastatic melanoma, 92t, 834f differential diagnosis of, 201-202 MART-1/melan-A protein in, 193f sentinel lymph node biopsies for, 197-198 α-Methylacyl-coenzyme A racemase in cancer of unknown primary site, 233 in gastric adenocarcinoma, 516t in prostate tumors, 586-589, 586t, 588f, 594-595, 594f in prostatic atrophy, 590, 592f Methylation-specific multiplex ligation-dependent probe amplification assay, 630 O6-Methylguanine DNA methyltransferase, 323 MF. See Mycosis fungoides MHC2A proteins, 191 MIB-1. See Molecular immunology Borstel 1 MIC2 protein, 86 Micro-RNA (miRNA), 39-40, 243-244 Microarrays, 49, 342 Microcystic adnexal carcinoma, 485, 487f Microgemistocytes, 788f, 825, 826t Microglandular adenosis, 717t Micrometastatic disease, 738b Micronodular thymic carcinoma, 370-371 Micronodular thymoma, 368 Micropapillary carcinoma invasive, 725-728 salivary duct carcinoma, 253-254 Microphthalmia-associated transcription factor (MITF) in head and neck lesions, 246t-248t in melanocytic neoplasms, 195-196, 199, 199f in tumors of soft tissue and bone, 85 Microsatellite instability, 52-54 colorectal adenocarcinoma with, 523-525, 525f detection of, 52-54, 53f in endometrial carcinoma, 681-682, 682f high-frequency (MSI-H), 52-54 low-frequency (MSI-H), 52-54 in pancreatic ductal adenocarcinoma, 545
913
Microsatellite-stable cancer, 523, 525f Microvessels capillary-like, 576 density assessment, 884 sinusoid-like, 576 Microwave heating method, 23-25 Middle ear adenoma, 315-318, 316f key diagnostic points, 318b neuroendocrine, 313-314, 318b Middle ear adenomatous tumor, 315 Minichromosome maintenance protein 2 (MCM2), 246t-248t, 299f, 300 Minute chemodectoma-like bodies, 418 Minute pulmonary meningothelial-like nodules, 418 MiSeq (Illumina), 47 Mismatch repair deficiency, 52-54 Mismatch repair proteins in endometrial carcinoma, 680-682, 681f-682f in GI tract, 509 Missense mutations, 41 MITF. See Microphthalmia-associated transcription factor Mitochondrial antigens, 336 Mitotic activity, 775 Mixed adenoNEC, 530 Mixed B-cell lymphoma, 492 Mixed tumors benign, 290 endometrial epithelial carcinoma, 672 endometrial stromal tumor, 674-675 meningiomas, 806 oligodendroglioma-astrocytoma, 790-791 MLH1 in colorectal adenocarcinoma, 523, 525f in GI tract, 509, 509t in gynecologic pathology, 654t in pancreatic carcinoma, 545 MOC-31, 840 in carcinomas, 223 in cholangiocarcinoma, 580f in colorectal adenocarcinoma, 524t in GI tract, 509, 509t key diagnostic points, 224b in melanocytic neoplasms, 191 in mesothelioma, 452t, 468t-471t, 475t in peritoneal carcinoma, 841f in pleural neoplasms, 448t, 450t-451t Molecular biology anatomic pathology, 39-55 concepts, 611 general principles, 39-41 morphology, 38 Molecular colocalization, 880-881 Molecular grade index, 757-758 Molecular immunology Borstel 1 (MIB-1), 850. See also Ki-67 in astrocytomas, 783, 783f, 785 in brain tumors, 783 in nervous system tumors, 765t, 775, 802f
914
Index
Molecular testing diagnostic applications, 70-72, 71f-72f specimen requirements for, 42 techniques for, 42-49 Monoclonal antibodies. See also Antibody(ies); specific antibodies for Hodgkin lymphoma therapy, 145 for infectious and prion disease diagnosis, 58t melanocyte-specific, 193-196 PMEL-17–related, 193-195 Monoclonal light chain, 410t Monocytoid cells, 409 Monstrocellular sarcomas, 793f Mothers against decapentaplegic homolog 4. See SMAD4 Mounting slides, 24-25 MPMNs. See Minute pulmonary meningothelial-like nodules MRT. See Malignant rhabdoid tumor MS-MLAP. See Methylation-specific multiplex ligation-dependent probe amplification assay MSA. See Muscle-specific actin MSI. See Microsatellite instability Mucicarmine, 469-471, 471f Mucin(s), 541 -related glycoproteins and oncoproteins, 540-541 gastric pyloric glandular, 541 gastric-type, 541 gel-forming, 541 in GI tract, 509-510 goblet-type, 541 mammary-type, 541 membrane-associated, 541 in mesothelioma, 464-471, 471f pyloric-type, 541 in salivary duct carcinoma, 253-254 soluble, 541 Mucin 1 (MUC1), 541 in colorectal adenocarcinoma, 524t in gastric adenocarcinoma, 516t in GI tract, 509-510, 509t in head and neck lesions, 246t-248t in intraampullary papillary-tubular neoplasms, 563, 564f in intraductal papillary mucinous neoplasms, 549, 549f in pancreatic ductal adenocarcinoma, 543-544, 543f-544f in tumors of soft tissue and bone, 75 Mucin 2 (MUC2), 541 in colorectal adenocarcinoma, 524t in gastric adenocarcinoma, 516t in GI tract, 509-510, 509t in head and neck lesions, 246t-248t, 273t in intraampullary papillary-tubular neoplasms, 563, 564f in intraductal papillary mucinous neoplasms, 549-550, 549f-550f in mucinous adenocarcinoma, 521f in pancreatic carcinoma, 543f, 548, 548f
Mucin 4 (MUC4), 541 in gastric adenocarcinoma, 516t in head and neck lesions, 246t-248t in tumors of soft tissue and bone, 88, 92t Mucin 5 (MUC5), 246t-248t Mucin 5AC (MUC5AC) in colorectal adenocarcinoma, 524t in gastric adenocarcinoma, 516t in GI tract, 509t in head and neck lesions, 273t in intraampullary papillary-tubular neoplasms, 563, 564f in mucinous adenocarcinoma, 521f in pancreatic ductal adenocarcinoma, 543f Mucin 6 (MUC6), 541 in gastric adenocarcinoma, 516t in GI tract, 509t in intracholecystic papillary-tubular neoplasms, 559, 559f Mucinous adenocarcinoma immunophenotypes, 521f metastatic, of rectum, 844f pulmonary, 421-422, 422t of thymus, 371 Mucinous carcinoma in gallbladder, 561-562 intestinal-type, 689, 689f tubular and spindle cell carcinoma, 638-639, 639b Mucinous neoplasms appendiceal, 520-521, 521b, 521f cystic biliary, 560, 582 key diagnostic points, 551b pancreatic, 550-551, 551f intraductal papillary, 549-550 ovarian and fallopian tube, 688-690 Mucinous tumors, endocervical-like (seromucinous), 690, 690f Mucoepidermoid carcinoma, 294-296, 294f-295f key diagnostic points, 296b oncocytic variant of, 294-295 thymic, 371 Mucosa-associated lymphoid tissue, 407-408 Mucosa-associated lymphoid tissue lymphomas, 163 extranodal marginal zone, 156f lymphoepithelial lesions in, 163 mediastinal, 385 Mucosal ganglioneuroma, 536f Mucosal melanoma, 260-262, 260f differential diagnosis of, 262, 262f, 289-290 epithelioid, 261f key diagnostic points, 262b melanocytic markers in, 260-262, 261f sinonasal, 256t Müllerian adenosarcoma, 675-676, 677f Müllerian endometrioid adenocarcinoma, 525, 526f Multifocal leukoencephalopathy, progressive, 773, 773f
Multigene expression assays, 244t Multiinfarct vascular dementia, 772-773 Multiple myeloma, 409 Multiple myeloma oncogene 1 (MUM1) double staining for, 27f in mediastinal hematopoietic tumors, 382-384 in non-Hodgkin lymphoma, 177t Multiplex immunoenzymatic techniques, 25-27 Multiplex immunoperoxidase staining, 25f Multiplex stains, 8-12, 27f Multipotential-subserosal cells, 437-438 Multispectral microscopic imaging, 878 Multistep detection systems, 37t MUM1. See Melanoma associated antigen (mutated) 1; Multiple myeloma oncogene 1 Mural mesenchymal lesions, 532-535, 535b Muramidase (lysozyme), 84 Murine double-minute type 2 (MDM2), 87, 92t Murine typhus, 65 Muscle differentiation markers, 76-78 Muscle markers, 666 Muscle-specific actin in bladder tumors, 625t in breast tumors, 731t, 732f in head and neck lesions, 246t-248t in malignant rhabdoid tumor, 869, 869f-870f in mesothelioma, 444t-446t in rhabdomyosarcoma, 859-861, 859f-860f in sclerosing adenosis, 591-592, 593f in tumors of soft tissue and bone, 77 Mutations, 41. See also specific mutations in cancer of unknown primary site, 242t definition of, 41 deletion and insertion, 41 driver, 41 germline, 41 large-scale, 41 in lung adenocarcinoma, 432, 433f missense, 41 nonsense, 41 passenger, 41 point mutations, 41 silent, 41 small deletions and insertions, 41 small-scale, 41, 49-50 somatic, 41 testing for panels of, 342 MutS protein homolog 2 (MSH2) in gastrointestinal tract, 509, 509t in gynecologic pathology, 654t MutS protein homolog 6 (MSH6) in gastrointestinal tract, 509 in gynecologic pathology, 654t
Index
MYC in B-cell lymphomas, 169 in Burkitt lymphoma, 169, 170f in prostate carcinoma, 607t Mycobacterium avium, 63 Mycobacterium leprae, 70-72 Mycobacterium tuberculosis, 64 Mycoplasma pneumoniae, 58t Mycosis fungoides, 183, 491-492, 491f Myelin-associated glycoproteins, 79 Myelitis, 770 Myelogenous leukemia, extramedullary, 385 Myeloid leukemia cutis, 488, 490f Myeloid sarcoma, 185t Myelopathy, 771 Myeloperoxidase, 833t in dysgerminoma, 701t in mediastinal hematopoietic tumors, 382-384 in myeloid leukemia cutis, 488, 490f MyoD in head and neck lesions, 267-269, 268f in rhabdomyosarcoma, 859-861, 859f in tumors of soft tissue and bone, 78 MyoD1 in head and neck lesions, 246t-248t in rhabdomyosarcoma, 375-376, 376f, 859-861, 861f in tumors of soft tissue and bone, 78 Myoepithelial carcinoma in breast, 731 in soft tissue and bone, 78-79 Myoepithelial cells antibodies to, 714b, 719b in breast lesions, 710-714, 711f, 711t, 715f-717f, 717t, 719t in tumors of soft tissue and bone, 75 Myoepithelial tumors, 123 Myoepithelioma, 293-294, 293f clear cell, 296t key diagnostic points, 294b Myofibroblastic sarcoma, epithelioid inflammatory, 86 Myofibroblastic tumors inflammatory in soft tissue and bone, 106-107, 106f uterine, 678 in lung, 408 Myofibroblastoma, 731, 732f Myogenic neoplasms, 375-376, 376f Myogenic regulatory proteins, 854-855 Myogenin in gynecologic pathology, 654t in head and neck lesions, 246t-248t, 256t, 267-269, 267f-268f in prostatic tumors, 603t in rhabdomyosarcoma, 859-861, 859f, 861f in tumors of soft tissue and bone, 75, 91t Myoglobin, 78, 859f Myoid hamartoma, 742, 745f Myomelanocytoma, 499, 501f
Myometrial invasion, 672 Myosin, 77 smooth muscle, 654t. See also Smooth muscle myosin heavy chain tropomyosin, 77 Myosin II, 77 Myxofibrosarcoma, 107 Myxoid chondrosarcoma, extraskeletal, 78-79, 211t-212t Myxoid liposarcoma, 115-117 Myxoid malignant fibrous histiocytoma, 107 Myxoinflammatory fibroblastic sarcoma, 105-106 Myxopapillary ependymoma, 766t, 785-789, 786f
N
Naegleria fowleri, 66 Nap-A, 420 a-Naphthol pyronin, 24t Napsin, 226 Napsin A, 843 Nasal cavity tumors, 254-278 Nasopharyngeal angiofibroma, 275-277, 280-282, 281f, 282b Nasopharyngeal carcinoma, 185f, 278-282, 278f-280f key diagnostic points, 280b metastatic, 186f nonkeratinizing, 249t Nasopharyngeal papillary adenocarcinoma, 282, 282b, 283f Nasopharyngeal tumors, 278-282 National Cancer Institute (NCI), 14-15 Bethesda panel, 52-54, 53f National Institute of Standards and Technology (NIST), 14-15 Natural killer/T-cell lymphoma extranodal, nasal type, 181-182, 181f frequency of, 178f NB84 in melanocytic neoplasms, 192 in neuroblastoma, 351 in tumors of soft tissue and bone, 86-87 NBF. See Neutral buffered formalin NCI. See National Cancer Institute NDUFA13 gene, 341 NE secretory protein-55 (NESP-55), 324, 510, 532f Neck. See Head and neck Negative controls, 18, 32f Neoplasms. See also Tumors; specific sites, types vs. abscesses, 827-828 vs. dysplasia, 828 Neoplastic liver diseases, 573-583 Nephroblastoma, 871 Nephrogenic adenoma, 623 fibromyxoid variant, 623 key diagnostic points, 624b of urinary bladder, 623, 623f
915
Nerve sheath differentiation markers, 79-80 Nerve sheath myxoma, 501-502 Nerve sheath tumors, 811-812 cutaneous, 500-502 key diagnostic points, 114b in soft tissue and bone, 110-114 Nervous system immunohistochemical stains, 765t immunohistology of, 762-828 Nervous system cysts, 818-819 Nervous system diseases immunohistochemistry of, 762 infectious diseases, 770-772 histopathology of, 770-771 organisms in, 771-772 Nervous system tumors, 775-818 clinical perspective on, 764-766 diagnostic pitfalls, 824-828 differential diagnoses, 766, 766t, 776t-777t, 779t grading malignant potential, 775-778 metastatic tumors, 817-818 primitive neuroectodermal tumors, 803 radiologic perspective on, 764-766 NETs. See Neuroendocrine tumors NeuN, 765t, 822 Neural cell adhesion molecule. See also CD56 in cancer of unknown primary site, 224 in endocrine tumors, 324-325 in neuroendocrine lung neoplasms, 402t in tumors of soft tissue and bone, 79 Neural lesions, 536-537 Neurilemmoma, 811-812 Neurinoma, 811-812 Neuroamines, 401b Neuroblastic tumors classification of, 855 key diagnostic points, 858b pediatric, 855-876 Neuroblastoma, 799 adrenal, 349-351 differential features of, 793t, 855-856 with differentiation, 856f extraadrenal, 349-351, 350f key diagnostic points, 858b key features of, 875t markers in, 349-350, 350f metastatic to lymph nodes, 350f molecular diagnostic applications, 351 olfactory, 255-259, 255f key diagnostic points, 259b staining pattern, 256t pediatric, 855-876 poorly differentiated, 855-856 primitive cells, 255-258 in soft tissue and bone, 74-75, 123 undifferentiated, 855-856
916
Index
Neuroblastoma marker. See NB84 Neurocysticercosis, 771 Neurocytoma, central, 788f, 799 Neuroectodermal tumors, 377-378 Neuroendocrine adenoma of the middle ear, 313-314, 317f, 318b Neuroendocrine antibodies, 224-225 Neuroendocrine carcinoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cervical, 663-664 cytokeratin 7 in, 390t cytokeratin 20 in, 391t cytokeratin profile, 225 endometrial, 671-672 gastric adenocarcinoma, 518 high-grade (poorly differentiated). See also Small cell carcinoma in ampulla, 566 in colon, 532, 532f esophageal, 513f in GI tract, 531-532 key diagnostic points, 532b key differential diagnosis, 559b in pancreas, 558-559 intermediate-grade. See Carcinoid, atypical key diagnostic points, 225b large cell, 356, 400 of cervix, 359, 359f diagnostic criteria for, 400 in lung, 427t markers in, 357f of prostate, 597-598 large cell undifferentiated, 224 low-grade. See Carcinoid markers in, 264f mediastinal, 371-373, 372t small cell of breast, 361, 361f in head and neck, 263f key diagnostic points, 264b metastatic, 487-488, 488f sinonasal, 249t, 263-264 of thymus, 371-373, 372f, 372t well-differentiated, 400, 531 Neuroendocrine markers discriminative values, 402t in melanocytic lesions, 196-197 in neuroendocrine carcinoma, 263f in non-NE neoplasms, 425, 426f in olfactory neuroblastoma, 258259, 258f in ovarian and tubal tumors, 687 in pancreas, 542 in pituitary adenoma, 265-266, 265f used with postfixation, 833t Neuroendocrine neoplasms. See also Neuroendocrine tumors ampullary, 565-566, 566b extrahepatic, 562-563 in gallbladder, 562 in lung, 400-404, 423-425 antibodies in, 401-404 markers in, 403f prostatic, 597-598, 608
Neuroendocrine system diffuse, 401 dispersed, 401 Neuroendocrine tumors, 362 appendiceal, 530, 530b CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t colorectal, 531, 531b duodenal, 529, 529f-530f esophageal, 528, 528b extrahepatic, 562 gastric, 528, 528b gastrointestinal, 527-532, 529f-530f, 537 laryngeal, 287 differential diagnosis of, 289t key diagnostic points, 290b in liver, 583 pancreatic, 555-559 key diagnostic points, 558b key differential diagnosis, 558b rectal, 529-530, 529f-530f of small bowel, 528-530, 530b of soft tissue, 74-75 well-differentiated in gallbladder, 562, 562f in GI tract, 532f Neuroendocrine type small cell carcinoma, 693-694, 694f Neuroepithelial bodies, 356 Neuroepithelial cysts, 818 Neuroepithelial tumors, dysembryoplastic, 797-798, 798f Neurofibroma, 812, 812f anatomic molecular diagnostic applications, 812 differential features of, 776t-777t in soft tissue and bone, 110-111 Neurofibromatosis, 501f Neurofibromatosis 1, 812 Neurofibromin, 812 Neurofibrosarcoma, 113 Neurofilament proteins (NFPs) in brain tumors, 787f, 796f in demyelination, 821, 821f in endocrine tumors, 325 in head and neck lesions, 246t-248t in medullary thyroid carcinoma, 338f in nervous tissue, 765t in olfactory neuroblastoma, 258-259 in pancreatic endocrine tumors, 353f in tumors of soft tissue and bone, 73-75 used with postfixation, 833t Neurogenic sarcoma, 113 Neuroglial tissue, heterotopic, 310 Neurologic diseases biopsies directed toward, 767t confirmatory features of, 767t Neuron-specific enolase, 833t alpha, 323 beta, 323 in cancer of unknown primary site, 224-225 in desmoplastic small round cell tumor, 865-866, 866f
Neuron-specific enolase (Continued) discriminative value, 402t in endocrine tumors, 323, 353, 353f in Ewing sarcoma/PNETs, 863, 863f-864f gamma, 323 in head and neck lesions, 246t-248t, 267-269, 268f, 273t in large cell neuroendocrine carcinoma, 357f in lung neoplasms, 356f, 401, 402t, 403f, 427t in malignant rhabdoid tumor, 869, 869f in mesothelioma, 444t-446t in nervous tissue, 765t in neuroblastoma, 350, 350f, 856-857, 856f in non-NE neoplasms, 425, 426f in olfactory neuroblastoma, 258259, 258f in oligodendroglioma, 793f in pancreatic tumors, 556f in pituitary adenoma, 265-266, 265f, 328f in sinonasal tract tumors, 256t in small cell carcinoma, 356f in solid-pseudopapillary neoplasms, 554f Neuronal loss, 822, 823f-824f Neuronal tumors, 795-799 glioneuronal tumors, 798-799 key diagnostic points, 798b Neuropeptides, 401b Neuropils, 855-856 Neurothekeoma, 501-502 cellular, 199-200, 201f, 501-502, 503f conventional, 501-502 Neurotrophic receptor tyrosine kinase (NTRK1), 341 Neutral buffered formalin, 20 New fuchsin, 24t Next-generation sequencing (NGS), 47-48, 48f NF2 gene, 812 NFPs. See Neurofilament proteins NGS. See Next-generation sequencing Nipah virus, 68, 68f NIST. See National Institute of Standards and Technology NKH1/LEU19, 79. See also CD57 NKI-beteb, 194 NKI/C3, 84, 194 NKX3-1 in cancer of unknown primary site, 233 in prostate carcinoma, 598-599, 600f, 607t, 621t in urothelial carcinoma, 621t NKX4-1, 587 NLPHL. See Nodular lymphocytepredominant Hodgkin lymphoma Nodal marginal zone lymphoma, 163 features of, 155t prognostic and therapeutic studies, 163
Index
Nodular ganglioneuroblastoma, 855-856 Nodular hyperplasia focal, 571-572, 573t thyroid, 334t Nodular lymphocyte-predominant Hodgkin lymphoma, 130, 146f Nodular lymphoid hyperplasia BALT, 408 Nodular lymphoid infiltrates of uncertain nature, 408-409 Nodular melanoma, 189, 190f Nodules placental site, 679 stromal, endometrial, 673-675, 674f Non-Hodgkin lymphoma diagnostic immunohistochemistry, 139-143, 149f immunohistology of, 148-188 large-cell, 202f pulmonary, 409 Nonalcoholic duct-destructive chronic pancreatitis, 567 Nonintestinal-type adenocarcinoma, 272 Noninvasive pancreatobiliary papillary neoplasms, 563 Nonmyogenous-β, 77 Nonmyogenous-γ, 77 Nonsense mutations, 41 Nonspecific enolase. See Neuronspecific enolase NordiQC, 2, 15 Novocastra/Leica, 10 NSE. See Neuron-specific enolase NTRK. See Neurotrophic receptor tyrosine kinase Nuclear envelope proteins, 73 Nuclear factor-kB, 138t NUTM1 antibody in head and neck lesions, 246t-248t, 277-278 in upper aerodigestive tract carcinomas, 249t NUTM1 midline carcinoma, 249t NYBR1, 739
O
Oat cell carcinoma, 393t OBF1, 150 OCA-B47, 150 Occludin, 79-80 OCT Binding Factor 146, 150 OCT2 in Hodgkin lymphoma, 132-133 in non-Hodgkin lymphoma, 150, 177t OCT3/4 in germ cell tumors, 236 in mediastinal tumors, 379-380, 381f, 382t in testicular tumors, 648t OCT4 in dysgerminoma, 700, 701t in embryonal carcinoma, 702 in gynecologic pathology, 654t
OCT4 (Continued) in ovarian and tubal tumors, 686 in seminoma, 647, 647f in testicular tumors, 644 OFMTs. See Ossifying fibromyxoid tumors Olfactory neuroblastoma, 255-259, 255f cell types, 255-258 differential diagnosis of, 256t, 258-259 key diagnostic points, 259b neuroendocrine markers in, 258259, 258f staining pattern, 256t Oligoastrocytoma, 780f, 790-791 anaplastic, 779t, 791 differential features of, 779t Oligodendroglioma, 788f, 789-791 anaplastic, 766t, 790f, 791, 792f anatomic molecular diagnostic applications, 791 differential diagnosis, 766t key diagnostic points, 790b malignant, 791 markers in, 793f with microgemistocytes, 826t mixed oligodendrogliomaastrocytoma, 790-791 theranostic applications, 791 Olympus Cell Imaging Software, 878-879 OMGD. See Otitis media with glandular differentiation Oncocytic type papillae, 549-550 Oncocytoma differential diagnosis of, 296t renal, 635-636, 635b Oncogenes. See also specific genes in follicular tumors, 333 in pancreatic neoplasms, 545 in prostate carcinoma, 609-610 in skin, 506-507 Oncoproteins. See also specific proteins mucin-related, 540-541 Oncotype DX (Genomic Health), 747-748, 752, 756-757, 758t, 759 Open systems, 28 OPSCC. See Oropharyngeal squamous cell carcinoma Optic nerve pilocytic astrocytoma, 812 Oral cavity tumors, 284-287 Oropharyngeal squamous cell carcinoma, 250-252, 251f keratinizing, 250 nonkeratinizing, 250, 251f Ossifying fibromyxoid tumors (OFMTs), 88, 117 Osteoblastic differentiation, 88-89 Osteocalcin, 89 Osteoclast-like giant cells, 546-547 Osteoclast-type giant cell salivary duct carcinoma, 253-254 Osteoclastic giant cell carcinoma, 562 Osteofibrous dysplasia, 128-129
917
Osteonectin, 89 Osteosarcoma, 125, 874-876 fibroblastic, 92t key diagnostic points, 876b key features of, 875t soft tissue, 123 Otitis media with glandular differentiation, 316-318, 317f Ovarian carcinoma BerEP4 in, 223, 223f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t key diagnostic points, 236b key differential diagnosis, 671t molecular diagnostic applications, 709 primary vs. metastatic adenocarcinoma, 688t synchronous with endometrial, tubal, and peritoneal carcinomas, 672-673 Ovarian markers, 848 Ovarian neoplasms, 683-684 Ovarian sex cord tumors, 676 Ovarian tumors carcinoids, 703, 703f desmoplastic small round cell, 694, 695f epithelial, 687-694, 688f genomic applications for, 708 immunohistochemical markers for, 684 lymphoma, 694, 694f metastases, 704-706, 704f, 706b molecular diagnostic applications, 709 mucinous, 521f, 688-690 serous, 687-688, 688f-689f theranostic applications for, 708-709 Ovary, 683-706 Overfixation, 35
P
p16 in adenoid basal carcinoma, 663, 664f in cervical carcinoma, 665f in cervical lesions, 658, 660f in cervicovaginal cytology, 849f in endocervical adenocarcinoma, 661, 662f-663f in female genital tract, 653 in gynecologic pathology, 654t in head and neck tumors, 246t-248t, 850f in melanocytic neoplasms, 196 in upper aerodigestive tract carcinomas, 249t in uterine tumors, 667t, 683 p27, 246t-248t p30/32 glycoprotein, 86 p30/32 mic2 (CD99), 855 p38, 542 p40, 391-393, 843 P50RS. See α-Methylacyl-coenzyme A racemase
918
Index
p53 in astrocytomas, 795, 795f in Barrett esophagus, 510, 511f, 538f in bladder cancer, 628-629 in brain tumors, 785f in dysplastic gallbladder, 560, 560f in endometrial carcinoma, 669-670, 669f-670f, 682 in fibrous tumors, 806, 806f in GI tract, 509t, 510 in gynecologic pathology, 654t in head and neck lesions, 246t-248t, 248-250, 248f in meningiomas, 806f signature lesions, 707 in thyroid cancer, 340t, 341 in upper aerodigestive tract carcinomas, 249t in urothelial carcinoma, 617-618, 618f, 628, 628f in uterine tumors, 667t, 668f, 683 in uterus, 666 in vulvar intraepithelial neoplasia, 656-658, 658f in Wilms tumor, 872-873, 874f p57, 654t p63, 843 in adenoid basal carcinoma, 663, 664f in adenoid cystic carcinoma, 299f, 300 in anal squamous cell carcinoma, 526, 527f in breast, 710-711, 711f, 711t in breast carcinoma, 712f, 713-714, 714f, 716f, 729-730, 730f, 731t in gastric adenocarcinoma, 516t in GI tract, 509t, 510 in gynecologic pathology, 654t in head and neck lesions, 246t-248t, 850f in lung neoplasms, 388t-389t, 391, 393f, 394t, 419-420, 419t, 843 in mesothelioma, 444t-446t in non-Hodgkin lymphoma, 177t in pancreatic carcinoma, 543, 544f in prostate, 586-587, 587t in prostate carcinoma, 587-589, 590f-591f, 596f, 621t in prostatic atrophy, 590, 592f in prostatic intraepithelial neoplasia, 587-589, 589f in sinonasal tract lesions, 256t, 263-264, 264f, 274t in skin carcinomas, 489f in sweat gland tumors, 483, 483f in thyroid tumors, 336, 337f in upper aerodigestive tract carcinomas, 249t in urothelial carcinoma, 598-599, 601f, 617, 621t p120 in breast carcinoma, 724f-726f, 848 and E-cadherin, 722-723, 724f-725f, 848 P501S. See Prostein
Paget disease anal, 526-527 of breast, 732-733 in cancer of unknown primary site, 241-242, 241f extramammary, 241-242, 482-483, 482f key diagnostic points, 242b mammary, 241-242, 733b of nipple, 732, 733f pseudopaget disease, 733, 734f vulvar, 653-655, 655b, 655f PAI-1. See Plasminogen activator inhibitor 1 Paired box gene 2 (Pax-2), 235, 633 in head and neck lesions, 246t-248t in mediastinal tumors, 382t Paired box gene 5 (Pax-5) in Hodgkin lymphoma, 130-132, 141t in lung lymphomas, 410t in mediastinal tumors, 382-384, 384f in non-Hodgkin lymphoma, 150, 177t Paired box gene 8 (Pax-8), 235, 633 in bladder adenoma, 623, 623f in breast carcinoma, 739 in cancer of unknown primary site, 235 in clear cell carcinoma, 692-693 in endometrioid tumors, 690-692 in gynecologic pathology, 654t in head and neck lesions, 246t-248t key diagnostic points, 235b in mediastinal germ cell tumors, 382t in ovarian tumors, 685, 688-690, 688t, 848 PAX8/PPARG gene fusion, 333, 333f, 340t, 341 in peritoneal carcinoma, 841f in renal carcinoma, 616, 634f, 638, 639f in spindle cell thymoma, 367f in thymic carcinoma, 369-370, 369f-370f, 372f in thymoma, 365-366, 366f in thymus, 364, 364f in uterine carcinomas, 666, 671t Paired-end sequencing, 47 PAM50 (ARUP Laboratories), 758t PanCK, 249t Pancreas antigens in, 540-543 autoimmune diseases of, 566-568 exocrine, 540 immunohistology of, 540-559 neuroendocrine, 540 Pancreatic carcinoma acinar cell, 552-553 adenosquamous, 547 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t colloid, 547-548, 548f medullary, 547 poorly differentiated (high-grade) neuroendocrine, 558-559
Pancreatic carcinoma (Continued) undifferentiated, 546 undifferentiated with osteoclast-like giant cells, 546-547, 547f Pancreatic ductal adenocarcinoma, 543-545, 543f-544f anatomic molecular diagnostic applications, 545 genomic applications, 545 key diagnostic points, 545b key differential diagnosis, 546b medullary, 545 novel tumor markers, 545 undifferentiated, 546 undifferentiated with osteoclast-like giant cells, 546-547, 547f Pancreatic ductal carcinoma, 546-548 Pancreatic endocrine tumors, 353-355 markers in, 353, 353f molecular diagnostic applications, 355 nonfunctional, 354, 355f Pancreatic gastrinomas, 354, 354f Pancreatic intraepithelial neoplasia, 548-549 Pancreatic neoplasms exocrine, 543-555 intraductal papillary mucinous neoplasms, 549-550 intraductal tubulopapillary neoplasms, 551 mucinous cystic neoplasms, 550-551 neuroendocrine neoplasms, 555-559 other ductal carcinomas, 546-548 serous cystadenoma, 551-552, 552f solid-pseudopapillary, 554-555 mucinous, 521f Pancreatic neuroendocrine tumors, 556-558 anatomic molecular diagnostic applications, 557-558 antibodies in, 556, 556f functional, 556 grading, 556 key diagnostic points, 558b key differential diagnosis, 558b Pancreatic polypeptide, 352t, 353-354, 355f Pancreaticobiliary-type papillae, 549-550 Pancreatitis autoimmune, 566-567 diagnosis of, 568 immunoglobulin G4–associated, 567 type 1, 568 type 2, 568 chronic, 567 granulocytic epithelial lesion– forming, 568 sclerosing chronic, 567 lymphoplasmacytic, 567 Pancreatobiliary adenocarcinoma, 519, 541, 561
Index
Pancreatobiliary papillary neoplasms, noninvasive, 563 Pancreatoblastoma, 553-554, 554f anatomic molecular diagnostic applications, 554 genomic applications, 554, 554f key diagnostic points, 554b Pancytokeratin in adrenocortical tumors, 346f in head and neck lesions, 246t-248t in medullary thyroid carcinoma, 338f in parathyroid adenomas, 343f in pheochromocytoma, 347f in sinonasal tract tumors, 256t in thymic carcinoma, 369f-370f in thymoma, 365-366, 366f in thymus, 364, 364f Panencephalitis, subacute sclerosing, 772 Panepithelial membrane mucin, 541 PanIN. See Pancreatic intraepithelial neoplasia Pankeratin, 213t PanNETs. See Pancreatic neuroendocrine tumors PAP. See Peroxidase-antiperoxidase; Prostatic acid phosphatase Papillae gastric-type, 549-550, 549f intestinal-type, 549-550, 550f oncocytic type, 549-550 pancreaticobiliary-type, 549-550 Papillary adenocarcinoma, nasopharyngeal, 282b, 283f Papillary ductal carcinoma in situ, 718, 718f Papillary epithelial mesothelioma, well-differentiated, 472-473, 473f Papillary lesions of breast, 719t immunohistochemistry of, 717-719 intracystic (encapsulated) carcinoma, 718-719, 719f myoepithelial cell antibodies in, 719b Papillary meningioma, 809 Papillary neoplasms intracystic, 559 intraductal, 559 intraductal mucinous, 563 pancreatobiliary, noninvasive, 563 Papillary renal cell carcinoma, 637 clear cell, 640-641 differential diagnosis of, 638b immunohistochemistry of, 638b Papillary serous carcinoma, 474 Papillary squamous lesions, vulvar, 656 Papillary thymic carcinoma, 371 Papillary thyroid carcinoma, 240-241, 334f-335f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t distribution of markers in, 330, 330f follicular variant, 332, 332f genetic alterations in, 340t marker expression in, 334t molecular approaches to, 340-341 oncocytic type, 336, 336f
Papillary-tubular neoplasms intraampullary, 563 intracholecystic, 559 Papillary tumors cytokeratins in, 331t of pineal region, 801 Papilloma choroid plexus, 799, 800f intraductal, 718, 718f Schneiderian, inverted type, 274t Parachordoma, 74, 123 Paraffin-embedded tissue sections, 42 Paraffin temperature, 35 Parafibromin, 344-345, 345f Paraganglia, extraadrenal, 348-349 Paraganglioma, 74-75 gangliocytic, duodenal, 566 in head and neck, 312-314, 313f key diagnostic points, 314b of larynx, 289-290, 289t mediastinal, 372t, 373 sinonasal, 256t Paranasal sinuses: tumors of, 254-278 Parathyroid adenomas intrathyroidal, 343, 343f markers in, 343, 343f Parathyroid carcinoma, 345f Parathyroid hormone, 343, 343f in head and neck lesions, 246t-248t in parathyroid adenomas, 343f Parathyroid tumors, 343-358 immunophenotype of, 372t key diagnostic points, 345b of mediastinum, 373 molecular approaches to, 344-345 Paratope, 3 Parvovirus B19, 58t, 60, 60f, 71f Passenger mutations, 41 PATHIAM (Ventana), 883t Pathologists, 68-70 Pathway Her2 test (Roche/Ventana), 30 Pathwork Diagnostics, 243 PCA3 gene, 611, 613f PD-1. See Programmed death 1 PD-L. See Programmed death ligand PDX1, 532f PE10, 226 Pediatric neoplasms. See also specific neoplasms antibodies in, 854-855 immunohistology of, 854-876 PEL. See Primary effusion lymphoma Pelvic tumors, local, 234b Penicillium marneffei, 66 Peptide hormones, 225 Peptidylglycine α-amidating enzyme, 353f PER creatine kinase, 489f Perforin in Hodgkin lymphoma, 134-135 in non-Hodgkin lymphoma, 153 Periductal stromal tumors, 742 Perineurioma cutaneous, 501, 503f hybrid schwannoma/perineurioma, 113
919
Perineurioma (Continued) intraneural, 111-113 reticular, 111-113 sclerosing, 111-113 in soft tissue and bone, 111-113, 112f Peripheral cholangiocarcinoma, 581 Peripheral nerve sheath tumors benign, 110-113 differential diagnosis of, 262, 262f key diagnostic points, 502b malignant, 201f, 812 of dermis, 501f differentiation from CUPS, 211t-212t in soft tissue and bone, 78-79, 113-114, 114f in soft tissue and bone, 79 Peripheral nervous system demyelination, 822 Peripheral primitive neuroectodermal tumors, 803, 803f Peripheral small cell carcinoma, of lung, 400 Peripheral T-cell lymphoma, 178 frequency of, 178f histology of, 179f lymphoepithelioid cell variant, 140-141 not otherwise specified, 141-142 Peritoneal carcinoma, 672-673 Peritoneal carcinomatosis, 241 Peritoneal fluid: carcinoma in, 841f Peritoneal mesothelioma, 706-709, 706t, 707f Perivascular epithelioid cell (PEC) tumors (PEComas), 414, 499, 639-640 differentiation from CUPS, 211t-212t in soft tissue and bone, 76, 109-110, 111f uterine, 676 Perivascular inflammation, 768-769, 769f Perivascular neoplasms, 378-379 Perivenous encephalitis, 768-769 Peroxidase-antiperoxidase (PAP), 1 Peroxidase-antiperoxidase (PAP) method, 7, 7f, 9f, 24t Peroxisome proliferator-activated receptor (PPAR) γ-1, 333, 333f Pheochromocytoma, 74-75 adrenal, 349, 349f markers in, 347-348, 347f Phosphohistone H3, 765t, 775 PHOX2B, 351 Phyllodes tumor, 739-742, 741f-743f malignant, 741, 744f PI. See Proliferation index Pigmented actinic keratosis, 199 Pigmented melanophages, 31f Pigmented villonodular synovitis, 128 PIK3CA/MTOR pathway, 608-609 Pilar tumors, 485-486, 486f key diagnostic points, 487b malignant proliferating, 486
920
Index
Pilocytic astrocytomas, 780-782, 781f differential features of, 776t-777t optic nerve, 812 Pineal cell tumors, 800-801 Pineal cysts, 818, 819t Pineal gland tumors, 329 Pineal parenchymal tumors of intermediate differentiation, 329 Pineal region tumors, papillary, 801 Pineoblastoma, 801, 801f diagnosis of, 764f differential features of, 793t Pineocytoma, 329, 329f, 776t-777t, 800, 800f PiT-1, 246t-248t Pituitary adenoma, 264-266, 265f, 327f-328f classification of, 327, 327f-328f, 327t distribution of markers in, 328f immunohistochemistry of, 327-328 key diagnostic points, 266b neuroendocrine markers in, 265266, 265f staining pattern, 256t Pituitary carcinoma, 328 Pituitary hormone, 256t Pituitary tumors, 328 Pixels (PIcture Elements), 877 Placental alkaline phosphatase in dysgerminoma, 700, 700f, 701t in germ cell tumors, 236 in gynecologic pathology, 654t in intratubular germ cell neoplasia, 646f in mediastinal germ cell tumors, 382t in mediastinal seminoma, 379-380, 381f in melanocytic neoplasms, 191 in ovarian and tubal tumors, 686 in testicular tumors, 644-645, 648t Placental site, exaggerated, 679 Placental site nodules, 679 Placental site trophoblastic tumors, 679 Placental transmogrification of lung, 418 Plague, 70 Plasma cell granuloma of lung, 417 Plasma cell neoplasms, 167 Plasma cell–rich thymoma, 368 Plasmablastic lymphoma, 171-172, 172f Plasmacytoid urothelial carcinoma, 619, 621f Plasmacytoma, 167 extraosseous, 385 isolated, 167 Plasminogen activator inhibitor 1, 760 Plasmodium falciparum, 58t, 66 Plasmodium vivas, 66 Platelet-derived growth factor receptor (PDGF-R), 476t Platelet-endothelial cell adhesion molecule 1 (PECAM-1), 81
PLCH. See Pulmonary Langerhans cell histiocytosis Pleomorphic adenoma, 290-292, 290f, 292f features of, 291t key diagnostic points, 292b Pleomorphic carcinoma, 405 Pleomorphic leiomyosarcoma, 109 Pleomorphic lipoma, 115 Pleomorphic liposarcoma, 115 Pleomorphic lobular carcinoma, 723 Pleomorphic lobular carcinoma in situ, 723, 727f Pleomorphic mesothelioma, 463 Pleomorphic rhabdomyosarcoma, 100, 859 Pleomorphic sarcoma, undifferentiated, 497 Pleomorphic xanthoastrocytoma, 776t-777t, 780f, 784-785, 785f Pleura, normal, 437-438, 437f Pleural injury, 437-438, 437f Pleural neoplasms, 437-468, 437f-438f antigens and antibodies in, 447-460 calcifying fibrous pseudotumors, 466-467 desmoid tumors, 467 diagnostic considerations, 460-463 diagnostic pitfalls, 468-477 epithelioid mesothelioma immunohistochemical markers for, 471t markers for, 448t, 475t fibrous tumors, 465t lymphomas, 467-468 mesothelioma-like tumors, 465 metastatic adenocarcinoma, 450t, 842f metastatic carcinoma, 237, 239b negative markers for, 456-459 positive markers for, 447-456 rare primary, 463-464 solitary fibrous tumors, 464-465 thymomas, 467 Pleuritis, fibrosing, 477t Pleuropulmonary blastoma, 467 PLPS. See Pleomorphic liposarcoma PMEL-17–related monoclonal antibodies, 193-195 PMLBCL. See Primary mediastinal large B-cell lymphoma PMS2, 509, 509t PNETs. See Primitive neuroectodermal tumors Pneumocystis carinii, 58t Pneumocystis jiroveci, 65-66, 66f PNL, 196 Podoplanin. See also D2-40 in angiosarcomas, 96 in mediastinal tumors, 380t, 382t in mesothelioma, 452t in pleural neoplasms, 450t-451t in testicular tumors, 644 in tumors of soft tissue and bone, 83, 94 Point mutations, 41, 45
Polar spongioblastoma, 776t-777t Polyclonal antibodies, 3-4 Polymer-based labeling methods, 9-10, 10f, 37t Polymerase chain reaction (PCR), 42-43, 43f-44f allele-specific (AS-PCR), 45-46 for determining site of origin, 242t emulsion (emPCR), 47 microsatellite instability testing, 681-682, 682f PCR-RFLP analysis, 45, 46f quantitative (qPCR), 45 real-time, 44-45, 46f reverse transcription (RT-PCR), 43-44, 45f, 242t Polymerase chain reaction (PCR) products, 46-47, 47f Polymorphous low-grade adenocarcinoma, 292, 296-298, 297f features of, 291t key diagnostic points, 298b Polyomavirus, 70-72, 72f Polyomaviruses, 61 Polypoid lesions, 536-537 Polyps antrochoanal, 282 benign fibroblastic, 537 colonic, 522 fundic gland, 514 Polyvalent detection systems, 8-12 Poorly differentiated adenocarcinoma, of lung, 852, 853f Poorly differentiated carcinoma extrahepatic, 562 of liver, 582 Poorly differentiated neuroblastoma, 855-856 Poorly differentiated (high-grade) neuroendocrine carcinoma in ampulla, 566 in GI tract, 531-532 key diagnostic points, 532b key differential diagnosis, 559b in pancreas, 558-559 Poorly differentiated synovial sarcoma, 91t Poorly undifferentiated carcinoma extrahepatic, 562 of unknown primary site, 853f Popcorn cells, 130, 150 Positive controls, 18 Postatrophic hyperplasia, 590, 593f Postfixation, 832, 833t Posttransplant lymphoma, 813, 814f Posttransplant lymphoproliferative disease, Hodgkin-like, 143, 143f Posttransplant lymphoproliferative disorders, 813 POU5F1, 686 Prealbumin, 765t Preanalytic effects, 30 Premalignant lesions, 563 PreservCyt (Cytyc), 829-830 Pretreatment, 31t Primary biliary cirrhosis, 570
Index
Primary effusion lymphoma, 173, 410t, 412 Primary mediastinal B-cell lymphoma, 168 Primary mediastinal large B-cell lymphoma, 173, 174f Primitive neuroectodermal tumors, 863f with advanced neuronal differentiation, 803 anatomic molecular diagnostic applications, 803 central nervous (cPNETs), 803 in children and adolescents, 862-865 diagnosis of, 764f differential features of, 793t differentiation from CUPS, 211t-212t extraskeletal, 210f of mediastinum, 378, 378f peripheral (pPNETs), 91, 91t, 803, 803f sinonasal, 256t, 270-272 in skin, 488, 489f in soft tissue and bone, 74 Prion diseases, 58t Prions, 773 Pro-PSA (pPSA), 233 Proconvertase 2 (PC2), 353f Proconvertase 3 (PC3), 353f ProEx C ICC assay (TriPath Oncology), 849-850 Progesterone in cancer of unknown primary site, 228-229 key diagnostic points, 229b Progesterone receptor, 833t in breast tumors, 748 in colorectal adenocarcinoma, 524t in endometrial carcinoma, 682 in endometrial stromal sarcoma, 673-674, 674f in gastric adenocarcinoma, 515-516 in gynecologic pathology, 654t in mucinous cystic neoplasms, 550, 551f in pancreatic neuroendocrine tumors, 556f in prostatic mesenchymal tumors, 603t in skin tumors, 506 in solid-pseudopapillary neoplasms, 554f in thyroid carcinoma, 330f, 338f in uterine tumors, 667t, 671t, 683 Prognostic markers, 833t Programmed death 1 (PD-1), 137 Programmed death ligand (PD-L), 137 Progressive multifocal leukoencephalopathy, 773, 773f Prolactin in head and neck lesions, 246t-248t in pituitary adenoma, 265-266, 266f, 327f Proliferation centers, 160, 161f Proliferation index (PI), 775-777, 884
Proliferation markers, 850 in bladder cancer, 628-629 in brain tumors, 775 in urothelial carcinoma, 628, 628f Proliferative ductal epithelial lesions, 719-721 Prolymphocytic leukemia/lymphoma, T-cell, 182-183 Prospero-related homeobox 1 (Prox1), 82 Prostate adenosis of, 591, 593f antigens and antibodies in, 584-587 basal cell layer, 587t immunohistology of, 584-614 partial atrophy of, 590, 592f sclerosing adenosis of, 591-592, 593f Prostate adenocarcinoma benign mimics, 590-594 colorectal, 602 diagnosis of, 587-589, 590f high-grade, 598-599, 600f key diagnostic points, 82b metastatic, 360, 360f metastatic to head and neck, 320-321 with neuroendocrine cells, 360, 360f, 597 posthormonal therapy histology, 595b posttherapy changes, 594-596, 594f vs. colorectal adenocarcinoma, 526, 526f Prostate carcinoma acinar, 598f AMACR, 586, 586t CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t differential diagnosis of, 619, 621t early detection markers, 611-614 epigenetic changes in, 607t, 608 genetic alterations in, 606f, 607t genomic alterations in, 611, 612f genomic applications, 606-614 integrated genomics for, 610-611 key diagnostic points, 602b large cell neuroendocrine, 597-598 microarray signature profiling, 611, 613f morphometry/karyometry of, 608 oncogenes, 609-610 pathogenesis of, 606f prognostic factors, 606-611 proliferation index, 606-607 small cell, 597, 598f small focus of, 587-589, 588f, 589b theranostic applications, 606-614 therapy targets, 611-614 tumor suppressor genes, 609-610 urothelial, 598-600, 602b, 602f vs. urothelial carcinoma in prostate, 602b Prostate carcinoma antigens, 232-233, 233b Prostate ductal adenocarcinoma, 596-597, 596f Prostate-specific acid phosphatase, 586
921
Prostate-specific antigen, 232-233, 584-585 benign PSA, 585 in extraprostatic tissues and tumors, 585b in lung neoplasms, 389t in mesothelioma, 444t-446t in parathyroid adenomas, 343f pro-PSA, 585, 621t in prostate adenocarcinoma, 595, 595f, 598-599, 600f in prostate basal cell layer, 587t in prostate carcinoma, 597, 598f, 621t in small cell carcinoma, 358t in urothelial carcinoma, 599, 602f, 621t Prostate-specific membrane antigen, 233, 585 in prostate adenocarcinoma, 598-599, 600f in prostate carcinoma, 621t in urinary bladder adenocarcinoma, 622, 622f in urothelial carcinoma, 621t Prostate stem cell antigen, 543f Prostate tumors diagnostic immunohistochemistry of, 587-606 endocrine, 359-360, 360f mesenchymal, 602-606, 603t neuroendocrine neoplasms, 597-598 Prostatic acid phosphatase, 232-233, 586 in endocrine tumors, 353 in lung neoplasms, 389t in mesothelioma, 444t-446t in small cell carcinoma, 358t Prostatic atrophy, 590-591, 592b, 593f Prostatic duct carcinoma, 596-597 Prostatic intraepithelial neoplasia AMACR, 586, 586t high-grade, 587-589, 588f-589f Prostatic lineage-specific markers, 608 Prostatic urethra, 598-600 Prostein (P501S) in cancer of unknown primary site, 233 in prostate tumors, 586, 597-599, 598f, 600f, 621t in urinary bladder adenocarcinoma, 622, 622f in urothelial carcinoma, 621t Protein, 41 Protein A, 11 Protein A conjugate method, 11, 11f Protein A-peroxidase-antiperoxidase (PROA-PAP) method, 11, 11f Protein Atlas (www.proteinatlas.org), 4 Protein gene product 9.5 (PGP9.5), 323, 350, 350f, 357f Protein “spots”, 29 Proteomics, quantitative in situ, 30 Protozoal infections, 66 PrP. See Antiprion protein PSA. See Prostate-specific antigen Psammomatous carcinoids, glandular, 565-566
922
Index
Psammomatous meningioma, 806-807 PSAP. See Prostate-specific acid phosphatase Pseudallescheria boydii, 66 Pseudoepitheliomatous hyperplasia, 284, 284f Pseudofollicles, 160 Pseudolymphoma, 408, 583 Pseudomesotheliomatous adenocarcinoma, 465-466, 465f Pseudomesotheliomatous carcinoma, 465-466 Pseudomesotheliomatous epithelioid hemangioendothelioma, 466, 466f-467f Pseudomyogenic (epithelioid sarcomalike) hemangioendothelioma, 94, 95f Pseudoneoplastic lymphoid lesions key diagnostic points, 493b of skin, 492-493 Pseudopaget disease, 733, 734f Pseudopapillary cells, 554 Pseudorhabdoid tumors, 416 Pseudosarcomatous stroma, 368 Pseudotumors calcifying fibrous, of pleura, 466-467 in GI tract, 534-535 inflammatory, 408, 417 PSMA. See Prostate-specific membrane antigen PSTTs. See Placental site trophoblastic tumors PTEN in prostate carcinoma, 607t in uterine tumors, 666, 668f PTGS2, 607t PU.1, 133 Pulmonary adenocarcinoma cytokeratin profile, 454t immunohistochemical markers for, 471t immunohistochemical profile, 453t immunohistochemical tests in, 470t mucinous, 421-422, 422t TTF-1 in, 225, 226f vs. breast carcinoma, 422 vs. colon cancer, 422 vs. metastatic carcinoma, 422-423 Pulmonary blastoma, 405 Pulmonary endocrine tumors, 356-358 Pulmonary eosinophilic granuloma, 413-414 Pulmonary histiocytosis X, 413-414 Pulmonary Langerhans cell histiocytosis, 413-414 Pulmonary large cell neuroendocrine carcinoma, 356 Pulmonary meningothelial-like nodules, minute, 418 Pulmonary non-Hodgkin lymphoma, 409 Pulmonary small cell carcinoma, 225, 226f Pulmonary tumorlets, 357f Pyloric-type mucin, 541
Pyothorax-associated lymphoma, 410t, 413 Pyrosequencing, 47
Q
Q fever, 65, 70 QIF. See Quantitative immunofluorescence QIRS. See Quantifiable Internal Reference Standard QISP. See Quantitative in situ proteomics qPCR. See Quantitative PCR Quality control, 14-19 daily materials, 16t definition of, 14 false-negative results, 832, 834f, 836 internal controls, 19 internal tissue control, 824, 824f negative controls, 18 positive controls, 18 specimens of limited quality, 835-836 total test, 14, 14t Quantifiable Internal Reference Standard (QIRS), 19 Quantification, 29-30 Quantitative immunofluorescence, 877-878 automated quantitative analysis (AQUA) method, 880-881, 881f clinical applications, 882-884 strengths and limitations, 881-882 Quantitative immunohistochemistry, 877 Quantitative in situ proteomics, 30 Quantitative internal reference standards, 30 Quantitative PCR (qPCR), 45 Quantum-dot cellular automata, 883t
R
Rabies, 62, 62f, 772 Radiation therapy changes, 595-596, 595f RAH. See Regressing atypical histiocytosis Rapid cell blocks, 830 rarB2, 607t RAS, 340t, 341 RASSF1A, 607t Rb. See Retinoblastoma RB1, 344 RCC. See Renal cell carcinoma RCCma, 475t Reaction products, 24t Reagents. See also specific reagents antibodies as, 3-4 incubation of, 24 for soft tissue and bone tumor studies, 90t troubleshooting, 32t validation of, 15-19
Real-time polymerase chain reaction, 44-45, 46f Receptor tyrosine kinases, 627-628 Rectal adenocarcinoma metastatic mucinous, 844f secondarily involving urinary bladder, 619, 620f Rectal neuroendocrine tumors, 529-530 Reference standards, 29-30 Region of interest, 877 Regressing atypical histiocytosis, 493-494 Rehydration, 834 Renal angiomyolipoma, 639-640, 640f Renal cell carcinoma, 631, 634f-635f anatomic molecular diagnostic applications, 641-643 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t chromophobe, 637-638, 638b classification of, 633b clear cell, 236b, 636-637, 637b clear cell papillary, 640-641, 641b differential diagnosis of, 296t, 475t genomic applications, 643-644 key differential diagnosis, 636b metastatic in head and neck, 319-320 vs. pulmonary adenocarcinoma, 422-423 molecular parameters, 642b papillary, 637, 638b prognostic parameters, 642b with prominent leiomyomatous proliferation, 640-641 sarcomatoid, 474-475 theranostic applications, 643-644 Renal cell carcinoma antibody, 632 Renal cell carcinoma antigen, 234 Renal cell carcinoma markers, 234 key diagnostic points, 235b in lung neoplasms, 389t, 452t, 475t Renal oncocytoma, 635-636 Renal tumors angiomyoadenomatous, 640-641 antibodies in, 632-635 epithelial, 633b immunohistology of, 631-644 Respiratory epithelial adenomatoid hamartoma, 274t Respiratory syncytial virus, 58t, 62, 62f Restriction endonucleases, 45 Restriction enzymes, 45 Restriction fragment length polymorphism (RFLP) analysis, 45, 46f Restriction sites, 45 RET/PTC gene, 340-342, 340t Reticulohistiocytosis, congenital self-healing, 493 Retinoblastoma, 87 Retinoblastoma protein, 618 Reverse transcriptase, 43-44 Reverse transcription, 43-44, 45f
Index
Reverse transcription PCR (RT-PCR), 43-44, 45f for determining site of origin, 242t, 244 RT-PCR assay (bioTheranostics), 244 Revised Guidelines of the Clinical and Laboratory Standards Institute, 2 RFLP. See Restriction fragment length polymorphism Rhabdoid malignant melanoma, 200 Rhabdoid meningioma, 809 Rhabdoid thymic carcinoma, 371 Rhabdoid tumors atypical teratoid/rhabdoid tumor CNS tumors, 803, 804f differential features of, 793t in soft tissue and bone, 124 in GI tract, 525 of lung, 416-417, 417f malignant, 868-871 extrarenal, 124 key features of, 875t Rhabdomyoma, 76, 97-98, 284f, 285 Rhabdomyomatous thymoma, 368 Rhabdomyosarcoma, 858-862 alveolar, 858, 859f-861f differentiation from CUPS, 211t-212t gene-expression analysis of, 861-862 immunomarkers for, 91t, 859-861 in sinonasal tract, 266-267, 267f in soft tissue and bone, 74, 100, 100f botryoid, 858 cerebral, 811 diagnostic profile, 859-861, 859f differential diagnosis, 862 differential features of, 793t differentiation from CUPS, 211t-212t embryonal, 858, 858f, 860f-861f differentiation from CUPS, 211t-212t extrahepatic, 563 gene-expression analysis of, 861-862 immunomarkers for, 859-861 in sinonasal tract, 267f in soft tissue and bone, 98-99, 99f epithelioid, 101, 859 gene-expression analysis of, 861-862 genetic aberrations in, 862 key diagnostic points, 101b, 270b, 862b key features of, 875t mediastinal, 375, 376f pleomorphic, 100, 211t-212t, 858-859 prognostic markers for, 862 prostatic, 603t sclerosing, 101, 859-861 sinonasal, 266-269 markers in, 268f staining pattern, 256t in soft tissue and bone, 76
Rhabdomyosarcoma (Continued) spindle-cell/sclerosing, 859, 860f immunomarkers for, 859-861 in soft tissue and bone, 92t, 100-101, 101f of urinary bladder, 625t Rhodococcus equi, 63 Ribonucleic acid (RNA), 40-41 heterogeneous, 40-41 micro-RNA (miRNA), 39-40 ribosomal (rRNA), 39-41 small nuclear (snRNA), 39-41 transfer RNA (tRNA), 39-41 Ribosomal RNA (rRNA), 39-40 Rickettsia africae, 68 Rickettsia conorii, 63 Rickettsia parkeri, 63, 68 Rickettsia rickettsii, 63, 63f Rickettsial pox, 65 Rickettsioses, 70 Rituximab therapy, 145 RMS. See Rhabdomyosarcoma RNA. See Ribonucleic acid Roche/Ventana, 8, 10, 30 Rocky Mountain spotted fever (RMSF), 63, 63f, 70 Rosai-Dorfman disease, 79, 815t Rosenthal fibers, 781-782, 781f Rosettes of Homer Wright, 255-258 Rotterdam assay, 756 Rotterdam Assay, 758t Round cell liposarcoma, 115-117 Round cell mesothelioma, 463 Round cell tumors, desmoplastic, 269, 270f RTU reagents, 28
S
S-100 protein, 34f, 833t in adenoid cystic carcinoma, 299f, 300 alpha-alpha, 192 alpha-beta, 192 in astrocytomas, 782 beta-beta, 192 in breast, 710, 711t in breast carcinoma, 731t in ceruminous adenoma, 311, 312f in clear cell sarcoma, 121, 121f in CNS tumors, 789f, 815t in colorectal adenocarcinoma, 524t diagnostic pitfalls with, 825, 825f in dysgerminoma, 701t in extraadrenal neuroblastoma, 350, 350f false-negative or decreased staining, 832, 834f in gastric adenocarcinoma, 516t in glial heterotopia, 310, 310f in granular cell tumors, 284-285, 284f, 501, 502f in gynecologic pathology, 654t in head and neck lesions, 246t-248t, 308, 308f immunoreactivity of, 429, 429f in Langerhans cells, 413, 413f in lung neoplasms, 388t-389t, 427t, 429, 429f
923
S-100 protein (Continued) in mediastinal tumors, 377, 377f, 382-384 in melanocytic neoplasms, 192, 192f-193f, 199-200, 201f in meningioma, 806, 806f in mesothelioma, 444t-446t, 462f in mucosal melanoma, 260-262, 261f in myoepithelioma, 293, 293f in nasopharyngeal carcinoma, 279-280, 280f in nervous tissue, 765t in olfactory neuroblastoma, 258259, 258f in ovarian and tubal tumors, 687 in pancreatic ductal adenocarcinoma, 543f in peripheral nerve sheath tumors, 113, 114f, 262, 262f, 500, 501f in pheochromocytoma, 348, 349f in pleural neoplasms, 465t in schwannoma, 110-111, 112f in sinonasal tract lesions, 256t, 273t-274t in skin tumors, 489f, 498, 500-501, 500f-502f in thyroid tumors, 336 in tumors of soft tissue and bone, 78-79, 91t-92t in upper aerodigestive tract carcinomas, 249t in uterine tumors, 673t in vulvar granular cell tumors, 656, 656f S-100A protein, 598-599, 601f S-100A4 protein, 570 S-100A6 protein, 587t Salivary duct carcinoma, 304-307, 305f-307f key diagnostic points, 307b low-grade, 307 micropapillary, 253-254 mucin-rich, 253-254 osteoclast-type giant cell, 253-254 sarcomatoid, 253-254 variants, 253-254 Salivary gland tumors, 290-310 anatomic molecular diagnostic approaches to, 310 basaloid, 291t CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t clear cell, 296t in lung, 417 theranostics, 310 Salivary gland-type adenocarcinoma, 272 Salivary gland-type carcinomas, 371 SALL4 in germ cell tumors, 236 in gynecologic pathology, 654t in mediastinal tumors, 379-380, 381f, 382t in ovarian and tubal tumors, 686 in testicular tumors, 644, 648t in yolk sac tumors, 701-702
924
Index
Salmonella, 65 Sample preparation, 14-15, 19 Sandwich (indirect-conjugate) method, 6, 6f Sanger sequencing, 46-47, 47f Sarcoma. See also specific sites, types differentiation from CUPS, 208, 211t-212t immunohistochemical features of, 454t monstrocellular, 793f TTF-1 expression in, 393t undifferentiated or embryonal of liver, 583 of uterus, 676 undifferentiated pleomorphic, 497 Sarcomatoid carcinoma cytokeratin/vimentin coexpression in, 219, 220f in gallbladder, 562 gastric, 517 in head and neck, 252-253 immunohistochemical features of, 454t in lung, 404-405, 405f renal cell carcinoma, 474-475 salivary duct carcinoma, 253-254 urothelial, 625t Sarcomatoid cholangiocarcinoma, 581-582 Sarcomatoid melanoma, 201f-202f Sarcomatoid mesothelioma diagnostic considerations, 460-461, 461f immunohistochemical features of, 453, 453f, 454t, 462f, 465t markers in, 452t Sarcomeric contractile proteins, 77 SARS. See Severe acute respiratory syndrome SATB2, 88-89 Sausage sign, 567 SCA. See Serous cystadenoma Scaffolding proteins, 79-80 ScanScope XT (Aperio), 883t SCBs. See Cytoscrape cell blocks SCC. See Squamous cell carcinoma Scedosporium, 66 Schistosomiasis, 771 Schneiderian papilloma, inverted type, 274t Schwann cells, 75 Schwannoma, 811-812 differential features of, 776t-777t of GI tract, 534 hybrid schwannoma/perineurioma, 113 malignant, 113 mediastinal, 377, 377f melanotic, 196-197, 197f of peripheral nerve, 789f in soft tissue and bone, 78-79, 110-111, 112f Sclerosing adenosis in breast, 715f differential diagnosis, 717t key diagnostic points, 592b of prostate, 591-592, 593f
Sclerosing cholangitis, 570 Sclerosing epithelioid fibrosarcoma in skin, 494-495 in soft tissue and bone, 88, 105, 105f Sclerosing hemangioma, 415, 415f416f, 416t Sclerosing mediastinitis, 373-374 Sclerosing pancreatitis chronic, 567 lymphoplasmacytic, 567 Sclerosing papillary lesions, 716f Sclerosing rhabdomyosarcoma (RMS), 101 Sclerosing (ancient) thymoma, 368 SCNEC. See Small cell neuroendocrine carcinoma Scrapie, 773 Screening immunohistochemistry, 207-208 Scrub typhus, 65 SCSCC. See Spindle cell “sarcomatoid” squamous cell carcinoma SDRPL. See Splenic diffuse red-pulp small B-cell lymphoma Sebaceous carcinoma, 484, 484f Sebaceous tumors, 484, 484b Secondary tumors of kidney, 641 of testis, 649 Secretogranins, 323-324 Secretory meningiomas, 807, 807f SEF. See Sclerosing epithelioid fibrosarcoma Self-healing reticulohistiocytosis, congenital, 493 Seminoma chromosomal alterations in, 649, 650t classic, 646-647, 646f-647f immunohistochemistry of, 648t immunohistology of, 648b key diagnostic points, 380b markers for, 237b mediastinal, 379-380, 381f spermatocytic, 648, 648t Sensitivity, 3-4 Sentinel lymph nodes biopsies of in breast carcinoma, 734-738, 736f-737f in gynecologic pathology, 672 intraoperative molecular testing, 737-738, 738f for metastatic melanoma, 197-198 in breast carcinoma, 734-735, 736f examination of, 734-738 identification of, 734-735 immunohistochemistry of, 736-737 intraoperative molecular testing of, 737-738, 738f key diagnostic points, 738b in metastatic melanoma, 197-198 micrometastatic disease in, 738b Sequence polymorphisms, 45 Seromucinous (endocervical-like) tumors, 690, 690f
Serosa: papillary serous carcinoma of, 474 Serotonin, 352, 352t Serous carcinoma endometrial, 668-670, 669f-670f key diagnostic points, 671b key differential diagnosis, 667t, 671t endometrioid, 668f metastatic high-grade, in peritoneal fluid, 841f ovarian and fallopian tube neoplasms, 687-688, 688f-689f papillary, of serosa, 474 vs. peritoneal mesothelioma, 706t Serous cystadenoma, 551-552, 552b, 552f Sertoli cell tumors, 648b, 649, 698-699 Sertoli-Leydig cell tumors, 697-698, 698f Serum amyloid adenocarcinoma/Creactive protein, 573t 17-1 antigen. See Epithelial celladhesion molecule 76-gene profile/assay, 756 Severe acute respiratory syndrome, 68, 69f, 71f Sex cord tumors, 699 Sex cord–stromal tumors, 694-695 key diagnostic points, 695b vs. endometrioid carcinoma, 691t SF-1. See Steroid factor 1 SFT. See Solitary fibrous tumor SHD, 202f Short tandem repeats (STRs), 39-40 Signal transducer and activator of transcription STAT3, 137 Signal transducer and activator of transcription STAT5, 137 Signal transducer and activator of transcription STAT6, 137 Signet-ring cell adenocarcinoma, 525 Signet-ring cell carcinoma, 393t Signet-ring cell–like features: thymoma with, 368 Silent mutations, 41 Silver enhancement, 24t Silver in situ hybridization (SISH), 751-752, 751t Single-nucleotide polymorphisms (SNPs), 41, 45 Single-strand conformation polymorphism (SSCP) analysis, 45 Sinonasal tract adenocarcinoma, 272 immunohistochemistry of, 273t intestinal-type immunohistochemistry of, 274t key diagnostic points, 275b nonintestinal-type, 274t Sinonasal tract lesions immunohistochemistry of, 274t small blue round cell tumors, 256t Sinonasal undifferentiated carcinoma, 249t, 259-260, 259f key diagnostic points, 260b staining pattern, 256t
Index
Sinusitis, chronic, 274t Sinusoid-like microvessels, 576 SISH. See Silver in situ hybridization Site-specific markers, 843 Skeletal muscle differentiation markers, 78-79 Skeletal muscle tumors, 97-101 benign, 97-101 Skeletal muscle-α, 77 Skin, 479 Skin carcinoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t immunoreactivity patterns, 489f Merkel cell (NE) carcinoma, 360-361, 360f prognostic markers for, 506-507 Skin tumors endocrine tumors, 360-361, 487-488 epithelial, 479-488 fibroblastic/myofibroblastic neoplasms, 494-495 immunohistology of, 479-507 mesenchymal, 494-506 pseudoneoplastic lymphoid lesions, 492-493 small cell neoplasms, 506, 506f with smooth muscle differentiation, 499 vascular neoplasms, 502-505 Slides hematoxylin and eosin (H&E), 42 mounting, 24-25 whole-slide imaging, 877-878 SLNs. See Sentinel lymph nodes SMAD4 (mothers against decapentaplegic homolog 4) in cancer of unknown primary site, 232 in intraductal tubulopapillary neoplasms, 551, 551f key diagnostic points, 232 in ovarian and tubal tumors, 688t in pancreatic neoplasms, 545 Small B-cell lymphoma diagnosis of, 158-159 evaluation of, 157-167 prognostic issues, 159-160 therapeutic issues, 159-160 Small blue round cell tumors, 256t Small bowel neuroendocrine tumors, 528-530, 530b Small-cell blastic tumors, 185t Small cell carcinoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t of cervix, 663-664, 665f combined, 400 cytokeratin 7 in, 390t cytokeratin 20 in, 391t definition of, 400 differential features of, 793t hypercalcemic type, 694 immunohistochemical profiles, 358t immunomarkers for, 91t laryngeal, 288-290, 288f markers for, 356f
Small cell carcinoma (Continued) of nervous system, 793t peripheral, in lung, resembling carcinoid tumor, 400 of prostate, 598f squamous, in lung, 419t TTF-1 expression in, 393t undifferentiated classification of, 207 in skin and subcutis, 506f of urinary bladder, 622-623 Small cell lung carcinoma, 356, 400, 403f-404f immunohistochemical reactivities, 419t metastasis to ovary, 693-694 peripheral, resembling carcinoid tumor, 400 squamous, 419t vs. Merkel cell carcinoma, 425-426 Small cell lymphoma, 492 Small cell mesothelioma, 463 Small cell neoplasms lymphoid, 168b of skin and subcutis, 506, 506f Small cell neuroendocrine carcinoma in breast, 361, 361f in head and neck, 263f key diagnostic points, 264b in lung, 419t metastatic, 487-488, 488f in ovary, 693-694, 694f sinonasal tract, 256t, 263-264 upper aerodigestive tract, 249t Small deletions and insertions, 41 Small intestinal adenocarcinoma, 519-520, 520b, 520f Small intestine epithelial lesions of, 519-520 neuroendocrine tumors in, 528-530, 530b Small lymphocytic leukemia, 154, 155t, 160-161 Small nuclear (snRNA), 39-41 Small round cell tumors blue round cell tumors, 256t desmoplastic, 117, 118f immunomarkers for, 91t in lung, 417 malignant, of soft tissue and bone, 91t mesenchymal, 506 Small-scale mutations, 41, 49-50 Small vessel disease, 767t, 774-775 SMARCB1 gene in malignant rhabdoid tumor, 869, 872f in pediatric neoplasms, 855 in tumors of soft tissue and bone, 124 Smears air-dried, 832, 834 aspirate, 830f Smooth muscle actin, 833t in head and neck lesions, 246t-248t, 267-269, 268f in sinonasal tract lesions, 253-254, 254f, 274t, 275, 276f
925
Smooth muscle actin (Continued) in spindle cell tumors, 92t in upper aerodigestive tract carcinomas, 249t in vascular proliferations, 775 α-isoform in breast, 711t in gynecologic pathology, 654t in malignant rhabdoid tumor, 869 in mediastinal leiomyosarcoma, 375, 376f in prostatic mesenchymal tumors, 603t Smooth muscle differentiation, 499 Smooth muscle myosin, 654t Smooth muscle myosin heavy chain in breast tumors, 710-711, 711f, 711t, 713-714, 714f-716f, 742, 745f in fibroadenoma, 711f in head and neck lesions, 246t-248t Smooth muscle tumors atypical intradermal, 108 benign, 107-110 EBV-associated, 109, 110f in female genital tract, 674-675, 675f in gastrointestinal tract, 535, 537 prostatic, 603-604 in soft tissue and bone, 107-110 Smooth muscle-α, 77 Smooth muscle-γ, 77 SNPs. See Single-nucleotide polymorphisms SNUC. See Sinonasal undifferentiated carcinoma Soft-part sarcoma alveolar, 285 differentiation from CUPS, 211t-212t in oral cavity, 285f Soft tissue antigens and antibodies, 85b Soft tissue myoepitheliomas, 78-79 Soft tissue osteosarcoma, 123 Soft tissue sarcomas differential diagnosis of, 202 immunohistochemical features of, 465t Soft tissue tumors, 89-129 antibody reagents for study of, 90t antigens and antibodies in, 73-89 immunohistology of, 73-129 malignant small round cell tumors, 91t markers of, 85-88 primary neoplasms, 117-125 spindle cell tumors, 92t Software algorithms for image analysis, 878-881 Solid-pseudopapillary neoplasms, 554-555, 555f antibodies in, 554, 554f genomic applications, 555 key diagnostic points, 555b key differential diagnosis, 555b
926
Index
Solitary fibrous tumors of CNS, 809-810 in GI tract, 534-535 in head and neck, 275-277, 277f lipomatous or fat-forming, 103-104 mediastinal, 373-374, 374f of pleura, 464-465 pleuropulmonary, 464b prostatic, 603t, 604-605, 604f in soft tissue and bone, 103-104, 103f Soluble adenylyl cyclase (sAC), 196 Somatic mutations, 41 Somatostatin in endocrine tumors, 325 in gastrointestinal endocrine tumors, 352t in pancreas, 353-354 Somatostatinoma, 354, 556 ampullary, 565-566, 566f Sommer sector, 823f SOX2, 382t SOX9, 196, 196f SOX10, 80, 196 SOX17, 382t SP-A. See Surfactant protein A Specificity, 17-18 Specimen collection from cancer of unknown primary site, 206-207 recommendations for, 762 requirements for, 42 specimens of limited quality, 835-836 techniques, 829-832 ways to maximize tissue for immunostaining, 835b Spermatocytic seminoma, 648, 648t Spheroids, 821f Spinal cord lesions, 764-766 Spinal masses, 814-816 Spindle cell angiosarcoma, 92t, 97f Spindle cell carcinoma in breast, 731t classification of, 207 in gallbladder, 562 hepatocellular, 579 metaplastic, 729-730, 730f mucinous, 638-639 pulmonary, 404-405 thymic, 371 Spindle cell cholangiocarcinoma, 581-582 Spindle cell differentiation in esophageal carcinoma, 513 in gastric adenocarcinoma, 517 Spindle cell lipoma, 115 Spindle cell melanoma, 261f Spindle cell neoplasms in breast, 731 in soft tissue and bone, 92t in urinary bladder, 625t Spindle cell rhabdomyosarcoma, 859, 860f immunomarkers for, 92t, 859-861 in soft tissue and bone, 100-101, 101f
Spindle cell sarcoma in breast, 731t synovial, monophasic, 92t Spindle cell sarcomatoid squamous cell carcinoma, 252-253 Spindle cell squamous cell carcinoma in head and neck, 252-254, 253f key diagnostic points, 254b markers in, 253-254, 253f-254f Spindle cell thymoma, 366, 367f adenomatoid, 368 desmoplastic, 368-369 immunohistochemical phenotype, 365t with papillary or pseudopapillary features, 368 Splenic diffuse red-pulp small B-cell lymphoma (SDRPL), 167 Splenic marginal zone lymphoma, 155t, 166-167 SPNs. See Solid-pseudopapillary neoplasms Spongiform encephalopathy, 773-774 bovine, 774 Sporothrix schenckii, 66 Squamoid corpuscles, 553-554 Squamoproliferative lesions in head and neck, 245-254 reactive, 245 Squamous cell carcinoma anal, 526, 527f basaloid, 249t, 250, 512 key diagnostic points, 252b staining patterns, 513f CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t esophageal, 511-512, 512f cytokeratin reactivity in, 511, 511f key diagnostic points, 512b in gastrointestinal tract, 512-513, 513b in head and neck, 245-250, 850f immunohistochemical reactivities, 419t key diagnostic points, 250b in lung, 419-420, 454t markers in, 452t metastatic in head and neck, 850f in lung, 420 nasopharyngeal, 278 oropharyngeal, 250-252, 251f pleomorphic, 479 sarcomatoid, of skin, 479, 480f of skin, 481f, 507, 507f small cell, 479 spindle cell in head and neck, 252-254 of skin, 479 subtypes, 479 TTF-1 expression in, 393t upper aerodigestive tract, 249t variants, 512-513, 513b
Squamous intraepithelial lesions cervical, 658-659, 659b, 659f-660f vulvar, 656-658 Squamous lesions, papillary, 656 SRMS. See Spindle cell rhabdomyosarcoma SSCP analysis. See Single-strand conformation polymorphism analysis Staining. See also Immunostaining absent or weak, 32t, 33f with appropriate positive staining of positive control, 33-35 with appropriate staining of positive control, 35 of both specimen and control, 31-33 with processing problems, 35 accuracy of, 17-18 artifactual, 36, 36t automated, 27-30 background, 35-36, 36t counterstaining, 24-25 double, 26f double (multiplex) stains, 8-12 establishing new stains, 13-14 home brewing, 13, 13t “in section” variation, 20 nonspecific, 4-5, 5f, 23 nonspecific background, 32f positive, 33-35 ready-to-use approaches, 13-14, 13t specificity of, 17-18 troubleshooting, 30-31, 32t validation of results, 15-19 weak or absent, 32t, 33f Staining reagents. See Reagents Standardization, 14-19, 832-834 internal reference standards, 17, 19 for sample preparation, 19 sample recommendations for, 15b Staphylococcus aureus, 58t, 65 Steatohepatitis, 569-570 Steroid cell tumors, 699 Steroid factor 1 (SF-1) in adrenocortical tumors, 346 in cancer of unknown primary site, 236 in endometrioid carcinoma, 691t in ovarian and tubal tumors, 685 STK11/Lkb1 Peutz-Jeghers gene, 545 Stomach lesions. See also under Gastric epithelial, 513-519 neuroendocrine tumors, 528 nonneoplastic conditions, 513-514, 515b Storage, 834 Streptavidin, 8. See also Biotin-streptavidin Streptococcus pneumoniae, 65 Streptococcus pyogenes, 65 Stromal invasion assessment of, 710-714 myoepithelial cell antibodies for, 714b Stromal nodules, endometrial, 673675, 674f
Index
Stromal sarcoma endometrial, 211t-212t, 673-675, 674f differentiation from CUPS, 211t-212t low-grade, 673-675 high-grade tumors, 603 immunohistochemistry of, 603t of prostate, 603 spindle cell features of, 603 Stromal tumors endometrial, 675f key diagnostic points, 675b mixed, 674-675 periductal, 742 Stromal tumors of uncertain malignant potential, 603, 603t Stromomyoma, 674-675 STRs. See Short tandem repeats Struma ovarii, 703, 703f STUMPs. See Stromal tumors of uncertain malignant potential Subcutaneous panniculitic type T-cell lymphoma, 183 Subcutaneous panniculitis-like lymphoma, 178f Subependymal giant cell astrocytomas, 784 Subependymoma, 787 Substance P, 352t Substrate, 24 Sugar tumors, 414 Superficial hemangiopericytoma, congenital, 494-495 Superficial spreading melanoma, 189, 190f Surfactant antibodies, 429-430, 430f Surfactant protein A (SP-A) in lung neoplasms, 416t, 422t in mesothelioma, 444t-446t in nonneuroendocrine lung neoplasms, 388t-389t in pulmonary neoplasms, 396t Surgery. See Intraoperative molecular testing SUZ12 translocation, 683 Sweat duct tumors, 481-483, 482f Sweat gland tumors, 484b SYBR Green 1, 44 Synaptic vesicle proteins, 324 Synaptobrevin, 324 Synaptophysin in adrenocortical tumors, 346f in atypical carcinoid, 471-472, 472f in brain tumors, 787f-788f, 797f in cancer of unknown primary site, 224 in cervical cancer, 665f in colon cancer, 524t, 532, 532f discriminative value, 402t in endocrine tumors, 324, 353f in esophageal carcinomas, 513f in Ewing sarcoma/PNETs, 863, 863f in GI tract, 509t, 510 in granulosa cell tumors, 696t in gynecologic pathology, 654t in head and neck lesions, 246t-248t
Synaptophysin (Continued) in lung tumors, 356f, 357-358, 402t, 403f, 419t, 421t, 427t in malignant rhabdoid tumor, 869, 869f-870f in medulloblastoma, 802, 802f in mesothelioma, 444t-446t in nervous tissue, 765t in neuroblastoma, 258-259, 258f, 350f, 856-857, 857f in neuroendocrine carcinoma, 263-264, 263f, 357f, 402t, 403f, 532, 532f, 556f in non-NE neoplasms, 425, 426f in olfactory neuroblastoma, 258259, 258f in oligodendroglioma, 791, 793f in ovarian tumors, 693-694 in pancreatic tumors, 556f in pheochromocytoma, 347f in pineoblastoma, 801, 801f in pineocytoma, 329, 329f in pituitary adenoma, 265-266, 265f, 328f in sinonasal tract tumors, 256t in skin carcinomas, 489f in small cell carcinoma, 356f, 665f in solid-pseudopapillary neoplasms, 554f in spindle cell thymoma, 367f in thyroid carcinoma, 338f in tumors of soft tissue and bone, 91t in upper aerodigestive tract carcinomas, 249t Synaptosomal associated protein 25, 338f Synaptogramins, 324 Syncytial cell masses, 805t Syncytial Hodgkin lymphoma, 188t Syncytial meningiomas, 806 Syndecan-1, 151 Synovial sarcoma, 78-79, 117-119 biphasic, 117 differentiation of, 208, 211t-212t key diagnostic points, 120b monophasic, 92t, 117, 119f, 373-374, 375f PCR-RFLP analysis of, 46f in pleura, 467 poorly differentiated, 91t, 117-119 SYT/SSX1 and SYT/SSX2 rearrangements in, 46f TTF-1 expression in, 393t Synovitis, pigmented villonodular, 128 g-Synuclein, 380t Syphilis, 64, 64f, 771 SYT gene, 50-51, 51f SYT/SSX fusion, 46f, 50-51, 51f SYTO9, 44
T
T-cell antigens, 134-135, 135f, 139 T-cell/histiocyte-rich large B-cell lymphoma, 172, 173f
927
T-cell large granular lymphocytic leukemia/lymphoma, 183-184 T-cell lymphomas, 177-184 adult T-cell leukemia/lymphoma, 184 angioimmunoblastic, 142, 178f, 180, 181f diagnostic pitfalls, 184 enteropathy-type, 178f, 182, 182f frequency of, 178f hepatosplenic, 178f, 183 key diagnostic points, 184b peripheral, 178 frequency of, 178f histology of, 179f not otherwise specified, 141-142 subcutaneous panniculitic type, 183 T-cell prolymphocytic leukemia/ lymphoma, 182-183 T-cell–associated antigens, 152-153 T-cell–rich B-cell lymphoma, 141t, 142, 142f T cells in CIDP, 822, 822f in Guillain-Barré syndrome, 822, 822f immature, 366, 366f thymic, 364f, 365 T-LGLL. See T-cell large granular lymphocytic leukemia/ lymphoma T3, 329-331 T4, 329-331 TAG. See Tumor-associated glycoprotein Tanycytic ependymoma, 776t-777t, 787 TargetPrint assay, 755-756 TCL1, 382t TdT. See Terminal deoxynucleotidyl transferase Technical artifacts, 836-837, 837f Technical issues, 30-36 Techniques, 1-38 Telomerase, 606f, 607t Telomere dysfunction, 606f, 607t Temperature, melting (Tm), 44 Temporal bone tumors, 310-321, 320f Temporal lobe epilepsy, 822 Tendon sheath: giant cell tumor of, 128 Tendons: clear cell sarcoma of, 120-121 Teratocarcinoma, 404-405 Teratocarcinosarcoma, 269, 269f Teratoid/rhabdoid tumor, atypical CNS tumors, 803, 804f differential features of, 793t in soft tissue and bone, 124 Teratoma, 702-703 benign cystic, 699 monodermal, 699 Terminal deoxynucleotidyl transferase (TdT), 151, 382-384 Test-battery approach, 22, 22t Testicular sex cord tumors, 648b, 649
928
Index
Testicular tumors anatomic molecular diagnostic applications, 649-652 antigens/antibodies in, 644-645 diagnostic immunohistochemistry of, 645-649 immunohistology of, 644-652 secondary, 649 3,3′5,5′-Tetramethylbenzidine, 24t Tetranitroblue tetrazolium, 24t Tetrazolium, 24t TFE3. See Transcription factor E3 TGB. See Thyroglobulin Thecoma, 695-699 Thermo/Lab Vision, 10 Theros H/I (bioTheranostics), 758t Theros MGI (bioTheranostics), 758t ThinPrep (Hologic), 829, 830f, 832, 834f Third ventricle: colloid cyst of, 818-819 3DHistech, 878-879 Thrombomodulin in cancer of unknown primary site, 233-234 in mesothelioma, 444t-446t, 452t, 459t, 468t-470t in pleural neoplasms, 448t in prostate carcinoma, 621t in tumors of soft tissue and bone, 83 in urothelial carcinoma, 598-599, 601f, 617, 621t in urothelial differentiation, 233, 234f Thymic carcinoids, 372f Thymic carcinoma, 365, 369-370, 369f-370f clear cell, 371 differential diagnosis of, 431 hepatoid, 371 key diagnostic points, 370b micronodular, with lymphoid hyperplasia, 370-371 neuroendocrine, 371-373, 372f papillary, 371 primary, 369-371 with rhabdoid features, 371 spindle cell, 371 variants, 370-371, 371b Thymic epithelial neoplasms, 365-371, 365t Thymine (T), 39 Thymocytes normal, 364f, 365 in thymoma, 366, 366f Thymoma, 365-369, 366f ancient (sclerosing), 368 atypical, 365 CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t conventional, 365t cytokeratin 7 in, 390t cytokeratin 20 in, 391t intrapulmonary, 417-418 lymphoma mimicry, 184f, 186, 186f-187f markers, 185t
Thymoma (Continued) micronodular, with lymphoid hyperplasia, 368 plasma cell–rich, 368 pleural, 467 with pseudosarcomatous stroma, 368 rhabdomyomatous, 368 with signet-ring cell–like features, 368 spindle cell, 366, 367f adenomatoid, 368 immunohistochemical phenotype, 365t with papillary or pseudopapillary features, 368 variants, 367-369, 369b Thymus endocrine tumors of, 362 mucinous adenocarcinoma of, 371 neuroendocrine carcinoma of, 372t normal, 363-365, 364f salivary gland–type carcinomas of, 371 Thyroglobulin, 329-331 in cancer of unknown primary site, 225 in head and neck lesions, 246t-248t key diagnostic points, 225b in lung neoplasms, 389t in mesothelioma, 444t-446t in parathyroid adenomas, 343f in thyroid tumors, 330, 330f, 339, 339f Thyroid carcinoma anaplastic (undifferentiated), 240-241, 332, 333f genetic alterations in, 340t marker expression in, 334t molecular approach to, 341 follicular, 240-241 genetic alterations in, 340t Hürthle cell, 240-241 key diagnostic points, 337b markers in, 334t, 335-336 medullary, 240-241, 337-342 key diagnostic points, 340b markers in, 338f metastatic to larynx, 289-290, 289t molecular approach to, 342 microarray gene profiling in, 342 molecular aspects of, 342b papillary, 240-241, 334f, 334t poorly differentiated, 240-241 genetic alterations in, 340t marker expression in, 334t molecular approach to, 341 testing for panels of mutations in, 342 theranostics of, 342 types of, 240-241 Thyroid gland, normal, 326, 326f Thyroid peroxidase, 329-331 Thyroid-stimulating hormone, 246t-248t
Thyroid transcription factor 1 (TTF-1), 33f, 329-331, 843 in atypical carcinoid, 471-472, 473f in cancer of unknown primary site, 225-226, 853f in colorectal adenocarcinoma, 524t, 525f in endocrine tumors, 356-357 in gastric adenocarcinoma, 516t in head and neck lesions, 246t-249t, 263-264, 264f in hepatocellular carcinoma, 576, 576f-577f immunoreactivity of, 427-429 key diagnostic points, 226b in lung tumors, 225, 226f, 356-357, 388t-389t, 391, 393t, 395f, 396t, 398-400, 399t, 402, 402t, 403f, 416f, 416t, 419-420, 419t, 421t-422t, 843, 844f, 847f in mesothelioma, 444t-446t, 448f, 452t-453t, 468t-469t, 471t in parathyroid adenomas, 343f in pleural neoplasms, 448t, 450t-451t in sclerosing hemangioma, 415, 416f in sinonasal tract tumors, 256t in skin tumors, 487-488, 488f in small cell carcinoma, 225, 226f, 358t, 402, 403f in spindle cell thymoma, 366, 367f in thyroid carcinoma, 330f, 338f, 339 in thyroid gland, 326, 326f in uterine carcinomas, 671t in well-differentiated neuroendocrine tumors, 532f Thyroid transcription factor 2 (TTF-2), 329-331 Thyroid tumors CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t hyalinizing trabecular, 329, 330f metastic lung adenocarcinoma, 319-320, 319f TIA-1, 134-135, 135f, 153 TIA-2, 83 Tissue fixation double, 20 techniques for, 19-22 Tissue identity testing, DNA-based, 54-55, 54f Tissue inhibitor of metalloproteinases, 136-137 Tissue microarrays, 17-18, 144-145 Tissue of origin determination molecular assays, 242-244, 242t multigene expression assays, 244t Tissue of Origin test (Pathwork Diagnostics), 243 Tissue samples preparation of, 19 specimen requirements, 42 techniques for processing, 19-22 troubleshooting variables, 31t ways to maximize, 835b
Index
TissueGnostics, 878-879 TissuemorphDP cell-analysis software (Visiopharm), 878-879 TissueStudio (Definiens), 878-879 Titration checkerboard, 12, 14t of primary antibody and detection system, 12-13 TLE1. See Transducin-like enhancer 1 TMAs. See Tissue microarrays TNBT. See Tetranitroblue tetrazolium Tonofibrils, 73 Tonofilaments, 73 Total test, 14, 14t Touch preps, 42 Toxoplasma, 66, 67f, 765t Toxoplasma gondii, 58t Toxoplasma pallidum, 58t Toxoplasmosis, 767t TP53 gene mutations in breast carcinoma, 758-759 detection of, 50, 50f in prostate carcinoma, 607t Trabecular carcinoma, 487 TRAF2, 137 TRAF5, 137 Transcription, 40, 40f reverse, 43-44, 45f Transcription factor E3 (TFE3), 87, 246t-248t Transcription factors in endocrine tumors, 325 in Hodgkin lymphoma, 132-134 Transducin-like enhancer 1 (TLE1) in head and neck lesions, 246t-248t in spindle cell tumors, 92t in synovial sarcoma, 118, 119f in tumors of soft tissue and bone, 88, 91t Transfer RNA (tRNA), 39-40 Transforming growth factor-beta, 587t Transitional cell carcinoma CD2, 3, 4, 5, 7, 8, 56, and 138 expression in, 428t cytokeratin 7 in, 390t cytokeratin 20 in, 391t of kidney, 214f Transitional meningioma, 779t, 806 Transitional mesothelioma, 462 Translation, 40, 40f Transplantation, liver, 572-573 Transthyretin, 799, 800f TRAP, 155t Trastuzumab (Herceptin), 749, 851 Treponema pallidum, 64, 64f Trichoepithelioma, desmoplastic, 485, 486f TriPath Imaging, 883t TriPath Oncology, 849-850 Triton tumor, 269, 269f malignant, 76, 113 Tropheryma whipplei, 63-64, 771 Trophoblastic disease differential diagnosis of, 679f gestational, 678-680, 683
Trophoblastic tumors epithelioid, 679-680 placental site, 679 Tropomyosin, 77 Troponin, 77 Troponin C, 77 Troponin I, 77 Troponin T, 77 Troubleshooting, 30-36 variables to check, 31t weak or absent staining, 32t Trypanosoma cruzi, 66 Trypsin, 553f in acinar cell carcinoma, 552-553 TSA. See Tyramine signal amplification TTF-1. See Thyroid transcription factor 1 TTF-2. See Thyroid transcription factor 2 Tubal carcinoma, 672-673 adenocarcinoma, 688t intraepithelial, 707, 707f Tubal neoplasms, 684 Tuberculosis, 771 Tuberous sclerosis, 639-640 Tubular adenosis, 717t Tubular carcinoma, 717t Tubulolobular carcinoma, 725, 728f Tubulopapillary neoplasms, intraductal, 551 Tularemia, 70, 70f Tumor-associated glycoprotein, 388t-389t, 444t-446t Tumor-associated glycoprotein-72 (TAG-72), 840 in skin carcinomas, 489f in sweat duct tumors, 481-482, 482f Tumor-associated glycoprotein-72 (TAG-72/CA72-4), 191 Tumor differentiation, 224-237 neuroendocrine, 225b Tumor infiltration, 775, 778, 780f Tumor necrosis factor, 134 Tumor suppressor genes in follicular tumors, 333 in pancreatic neoplasms, 545 in prostate carcinoma, 609-610 Tumoral intraepithelial neoplasms (intraductal and intracholecystic neoplasms), 559-560 Tumorlets, pulmonary, 357f Tumors. See also Neoplasms; specific sites, types of unknown primary site, 852-853 Typhus, 70 epidemic, 65 murine, 65 scrub, 65 Tyramine (Tyr), 37t biotinylated (tyramide), 10 Tyramine (Tyr) amplification, 10, 11f Tyramine signal amplification (TSA), 10-11 Tyrosinase in head and neck lesions, 246t-248t in mucosal melanoma, 260-262, 261f in tumors of soft tissue and bone, 85
929
Tyrosinase-related antibodies, 195, 195f
U
UEA1. See Ulex europaeus I agglutinin UESL. See Undifferentiated or embryonal sarcoma of the liver UK NEQAS, 2, 15, 17t, 20 Ulcerative colitis, 59f Ulex europaeus, 775 Ulex europaeus I agglutinin (UEA1), 83 Undifferentiated carcinoma classification of, 207 endometrial, 672 in GI tract, 525 large cell classification of, 207 of lung, 427t neuroendocrine, 224 of liver, 582 with osteoclast-like giant cells, 546-547, 547f pancreatic, 546-547, 547f sinonasal, 259-260, 259f key diagnostic points, 260b staining pattern, 256t small cell classification of, 207 in skin and subcutis, 506f Undifferentiated large cell neoplasms, 426-427, 427t Undifferentiated neuroblastoma, 855-856 Undifferentiated or embryonal sarcoma of the liver, 583 Undifferentiated sarcoma pleomorphic, 497 of uterus, 676 United Kingdom, 28 uPA. See Urokinase plasminogen activator Upper aerodigestive tract tumors, mucosal, 249t, 250 Urachal cysts, 230, 230f Urethra, prostatic, 598-600 Urinary bladder immunohistology of, 615-618 inflammatory myofibroblastic tumor of, 624, 624f-625f nephrogenic adenoma of, 623, 623f rectal adenocarcinoma secondarily involving, 619, 620f small cell carcinoma of, 622-623 spindle cell neoplasms of, 625t Urinary bladder adenocarcinoma, 622-624, 622f Urokinase plasminogen activator, 760 Uroplakin, 616-617 Uroplakin III in cancer of unknown primary site, 233-234 in lung neoplasms, 389t in urothelial carcinoma, 621t
930
Index
Urothelial carcinoma, 619-622 antigens/antibodies in, 616-618 cell-cycle regulators in, 626f, 628-629 chromosomal numerical alterations in, 627 differential diagnosis of, 616t, 619, 621t genetic alterations in, 626f high-grade, 601f immunohistochemical results, 621t incidence of, 615 muscle invasive, 615-616, 632t non–muscle invasive, 615-616 oncogenesis of, 625, 626f pathogenesis of, 625, 626f plasmacytoid, 619, 621f prognostic parameters, 632t proliferation index markers in, 628, 628f in prostate, 598-600, 602b, 602f sarcomatoid, 625t superficial, 632t vs. colon adenocarcinoma, 526 vs. colorectal adenocarcinoma, 526f vs. prostate carcinoma, 619, 621t Urothelial differentiation, 233, 234f Uterine tumors adenocarcinoma, 671t anatomic molecular diagnostic applications, 683 carcinoma, 667t epithelioid, 673t genomic applications, 680-682 GISTs, 676-678 immunohistochemical markers for, 664-667 mesenchymal, 673-678, 673t PEComas, 676 sarcoma, 683 that resemble ovarian sex cord tumors, 676 theranostic applications, 682-683 Uterus, 664-683
V
Vagina, 653-658 Validation of protocols, 15-19 Validation studies, 17 Variable fixation artifacts, 32f-33f Varicella zoster virus, 57, 59-60 Vascular dementia, multiinfarct, 772-773 Vascular endothelial growth factor receptor 3, 83, 94 Vascular malformations, 775, 775t Vascular markers, 729-731 Vascular proliferations in glioblastoma, 791-792 identification of, 775 Vascular tumors, 91-97 benign, 91-94 differentiation from CUPS, 211t-212t key diagnostic points, 94b, 504b locally aggressive and rarely metastasizing, 94
Vascular tumors (Continued) malignant, 94-97 mediastinal neoplasms, 378-379, 380t of skin, 502-505 VEGFR3. See Vascular endothelial growth factor receptor 3 Venezuelan hemorrhagic fever, 61 Venous malformations, 775t Ventana, 883t Vesicle-associated membrane proteins, 324 Vesicular monoamine transporters, 324 VHL gene, 815-816 VIAS (TriPath Imaging and Ventana), 883t Villin in adenocarcinomas, 229t in cancer of unknown primary site, 229 in colon carcinoma, 230, 231f in colorectal adenocarcinoma, 523, 523f, 524t in gastric adenocarcinoma, 515, 516t in gastrointestinal tract, 509t, 510 in head and neck lesions, 246t-248t, 273t key diagnostic points, 230b in lung neoplasms, 389t Villonodular synovitis, pigmented, 128 Vimentin, 833t in adrenocortical tumors, 346f in bladder tumors, 625t coexpression in carcinomas, 219, 219b-220b, 220f in desmoplastic small round cell tumor, 865-866, 866f in Ewing sarcoma/PNETs, 863, 863f in fibrosarcoma, 495, 496f in gastric adenocarcinoma, 516t in gynecologic pathology, 654t in head and neck lesions, 246t-248t, 253-254, 253f, 267-269, 268f in lung neoplasms, 388t-389t, 390-391, 395f, 403f, 404-405, 405f, 416-417, 416t, 417f, 427t in melanocytic neoplasms, 189, 202f in meningioma, 805, 805f in mesothelioma, 444t-446t, 453t, 462f, 468t-470t in nervous tissue, 765t in neuroblastoma, 350f in oligodendroglioma, 791, 793f in pancreatic endocrine tumors, 353f in pheochromocytoma, 347f in pleural neoplasms, 448t, 465t in rhabdoid tumors, 416-417, 417f, 869f in rhabdomyosarcoma, 859-861, 859f in sinonasal tract tumors, 256t
Vimentin (Continued) in skin tumors, 479, 480f in solid-pseudopapillary neoplasms, 554f in thyroid carcinoma, 330f, 338f in tumors of soft tissue and bone, 73, 75 in upper aerodigestive tract carcinomas, 249t in uterus, 666 in vascular proliferations, 775 Vina green, 24t VIPoma, 556 Viral hemorrhagic fevers, 60-61, 70 Viral infections, 56-62. See also specific infections of liver, 571 Visiopharm, 878-879 von Hippel-Lindau disease, 815 von Recklinghausen disease, 812 von Willebrand factor, 80 von Willebrand syndrome, 80 Vulva, 653-658 Vulvar intraepithelial neoplasia, 656-658, 658f Vulvar lesions exophytic, 656, 657f granular cell tumors, 656, 656f papillary squamous, 656 squamous intraepithelial, 656-658 Vulvar Paget disease, 653-655, 655f diagnostic issues, 653-655 key diagnostic points, 655b Vulvovaginal mesenchymal lesions, 655-656, 656b vWF. See von Willebrand factor VZV. See Varicella zoster virus
W
Waldenström macroglobulinemia/LPL, 162 Wall of syrinx, 818, 819t Washing, 23 WDLPS. See Well-differentiated liposarcoma Weibel-Palade bodies, 80, 406, 407f, 466, 467f Well-differentiated neuroendocrine tumors, 531 Well-differentiated liposarcoma, 115, 116f West Nile virus, 58t, 67, 67f West Nile virus encephalitis, 67, 771-772 Wet fixation in alcohol, 832 Whipple disease, 63, 771 Whole-slide imaging, 877-878 Wilms tumor, 871-874, 872f-873f anaplastic, 872-873, 874f epithelial differentiation in, 872873, 873f key diagnostic points, 874b key features of, 875t morphologic features of, 871-872, 872f skeletal muscle differentiation in, 872-873, 873f
Index
Wilms tumor gene 1 (WT1), 838 in Brenner tumors, 693 in cancer of unknown primary site, 226-227, 227f in clear cell carcinoma, 692-693 in desmoplastic small round cell tumor, 865-866, 866f-867f in gynecologic pathology, 654t in head and neck lesions, 246t-248t key diagnostic points, 227b in lung neoplasms, 389t in mesothelioma, 444t-446t, 448f, 452t-453t, 462f, 468t-471t, 475t, 839f in metanephric adenoma, 636, 636f in neuroblastoma, 350f in ovarian tumors, 685-688, 689f, 693-694, 848 in pediatric neoplasms, 855 in pleural neoplasms, 448t, 450t-451t
Wilms tumor gene 1 (WT1) (Continued) in tumors of soft tissue and bone, 83 in uterine carcinomas, 667, 671t WM. See Waldenström macroglobulinemia WNV. See West Nile virus World Health Organization (WHO) classification of renal epithelial tumors, 633b criteria for grading of meningioma, 770t grading of brain tumors, 775 Wound response gene set, 756 Wound response model, 758t WPBs. See Weibel-Palade bodies WSI. See Whole-slide imaging WT1. See Wilms tumor
X
X-hapten, 132 X-linked inhibitor of apoptosis protein (XIAP), 476t
931
Xanthoastrocytoma, pleomorphic, 776t-777t, 780f, 784-785, 785f Xanthogranuloma, 766t Xanthoma, 592-594, 594f
Y
Yellow fever, 61, 61f Yersinia pestis, 70, 70f Yolk sac differentiation, 517-518 Yolk sac tumors, 701-702, 701f immunohistochemistry of, 648t immunohistology of, 648b markers for, 237b vs. clear cell carcinoma, 693t
Z
ZO-1, 79-80 ZO-2, 79-80 ZO-3, 79-80 Zygomycetes, 70-72
